CHAPTER

The Eukaryotic
Cell Cycle

A two-cell C. elegans embryo stained with ant ibodies against tubulin

(red) and CeBUB-1, a spindle checkpoint protein (green). DNA is stained
with DAPI (blue). CeBUB-1 is localized on the chromosomes and
kinetochore-attached spindle microtubules during metaphase in the
smaller, posterior cell (right). It is presumed to monitor chromosome
attachment and tension. The larger, anterior cell (left) has already entered
anaphase, and CeBUB-1 is no longer detectable on the chromosomes or
spindle microtubules. Thus asynchrony of this second cell cycle in the C.
e/egans embryo allows the observation of both the presence of a
functional spindle checkpoint protein during metaphase and its absence
after initiation of anaphase. [Encanada et al., 2005, Mol. Bioi. Ce/1 16:1 056.]

roper control of cell division is vital to all organisms. In molecular mechanisms regulating eukaryotic cell division dis-

P unicellular organisms, cell division must be balanced

with cell growth so that cell size is properly maintained.
If several divisions occur before parental cells have reached
cussed in this chapter have gone a long way in explaining
how replication control goes awry in cancer cells. Appropri-
ately, Leland Hartwell, Tim Hunt, and Paul Nurse were
the proper size, daughter cells eventually become roo small to awarded the Nobel Prize in Physiology or Medicine in 2001
be viable. If cells grow too large before cell division, the cells for the initial experiments that elucidated the master regula-
function improperly and the number of cells increases slowly. tors of cell division in all eukaryotes.
In developing mu lticellular organisms, the replication of each The term cell cycle refers to the ordered series of events
cell must be precisely controlled and timed to faithfully and that lead to cell division and the production of two daughter
reproducibly complete the developmental program in every cells, each containing chromosomes identical to those of the
individual. Each type of cell in every tissue must control its parental cell. Two main molecular processes take place dur-
replication precisely for normal development of complex or- ing the cell cycle, with resting intervals in between: during
gans such as the brain or the kidney. In a normal adult, cells the S phase of the cycle, each parental chromosome is dupli-
divide only when and wliere they are needed. However, loss cated to form two identical sister chromatids; in mitosis (M
of normal controls on cell replication is the fundamenta l de- phase), the resulting sister chromatids are distributed to each
fect in cancer, an all-too-familiar disease that kills one in daughter cell (Figure 19-1). Chromosome replication and
every six people in the developed world (see Chapter 24 ). The segregation to daughter cells must occur in the proper order

OUTLINE

19.1 Overview of the Cell Cycle and Its Control 875 19.5 Entry into Mitosis 897

19.2 Model Organisms and Methods to Study the 19.6 Completion of Mitosis: Chromosome Segregation
Cell Cycle 877 and Exit from Mitosis 903

19.3 Regulation of CDK Activity 883 19.7 Surveillance Mechanisms in Cell Cycle
Regulation 906
19.4 Commitment to the Cell Cycle and DNA
Replication 890 19.8 Meiosis: A Special Type of Cell Division 913
(;} OVERVIEW ANIMATION: Cell Cycle Control
FIGURE 19-1 The fate of a single parental chromosome through-
out the eukaryotic cell cycle. Following mitosis (M), daughter cells
contain 2n chromosomes in diploid organisms and ln chromosomes in
haploid organisms. In proliferating cells, G 1 is the period between the
"bi rth" of a cell following mitosis and the initiation of DNA synthesis,
which marks the beginning of the 5 phase. At the end of the 5 phase,
cells enter G2 containing twice the number of chromosomes as G1 cells
(4n in diploid organisms, 2n m haploid organisms). The end of G2 is
marked by the onset of mitosis, during which numerous events leading
to cell division occur. The G~< 5, and G2 phases are collectively referred
to as interphase, the period between one mitosis and the next. Most
nonproliferating cells in vertebrates leave the cell cycle in G~< entering
the G0 state. Although chromosomes condense only during mitosis,
here they are shown in condensed form throughout the cell cycle to
emphasize the number of chromosomes at each stage. For simplicity,
the nuclear envelope is not depicted.

in every cell division. If a cell undergoes chromosome segrega- and protein synthesis, control of cell division appears to be
tion before the replication of all chromosomes has been com- a fundamental cellular process that evolved and was largely
pleted, at least one daughter cell will lose generic information. optimized early in eukaryotic evolu tion. Because of this
Likewise, if a second round of replication occurs in one region similarity, research with diverse organisms, each with its
of a chromosome before cell division occurs, the genes encoded own particular experimental advantages, has contributed
in that region are increased in number out of proportion to to a growing understanding of how cell cycle events are
other genes, a phenomenon that often leads to an imbalance of coordinated and controlled. Biochemical, genetic, imaging,
gene expression that is incompatible with viability. and micromanipulation techniques a ll have been employed
High acc uracy a nd fidelity are required to ensure that in studying various aspects of t he eukaryotic cell cycle.
DNA replication is carried out correctly and that each These studies have revea led that cel l division is controlled
daughter cell inherits the correct number of each chromo- primarily by regulating the timing of entry into the cell di -
some. To achieve this, cell division is controlled by surveil - vision cycle, nuclear DNA replication, and mitosis.
lance mechanisms known as checkpoint pathways that The master controllers of the cell cycle are a small number
prevent initiation of each step in cell division until earlier of heterodimeric protein kinases that contain a regulatory sub-
steps on which it depends have been completed and mistakes unit (cyclin) and a cata lytic subunit (cyclin-dependent kinase,
that occurred during the process have been corrected. Muta- or CDK). These heterodimeric kinases regulate the activities of
tions that inactivate or alter the no rmal operation of these multiple proteins involved in entry into the cell cycle, DNA
checkpoint pathways contribute to the generation of ca ncer replication, and mitosis by p hosphorylating them at specific
cells because they result in chromosomal rearrangements regulatory sites, activating some and inhibiting others to coor-
and abnormal numbers of chromosomes, which lead to fur- dinate their activities. Regulated degradation of proteins also
ther mutations and changes in gene expression level that plays a prominent role in important cell cycle transitions. Since
cause uncontrolled cell growth (see Chapter 24 ). protein degradation is irreversible, this ensures that the pro-
In the late 1980s, it became clear that the molecular cesses move in only one di rection through the cell cycle.
processes regulating the two key events in the cell cycle- In this chapter, we first present an overview of th e cel l
chromosome replication and segregation-are fundamen- cycle and then describe the various experimental systems that
tally similar in all eukaryotic cells. Initially, it was surprising contributed to our current understanding of it. We will then
to many researchers that cells as diverse as budding yeast discuss cyclin-dependent kinases (CDKs) and the many dif-
and developing human neurons use nearly identical pro- ferent ways these key cell cycle controllers can be regulated.
teins to regulate their division. However, like transcription Next, we' ll examine each cell cycle phase in greater detail

874 CHAPTER 19 The Eukaryotic Cell Cycle

with an emphasis on how control of CDK activity governs The Cell Cycle Is an Ordered Series of Events
the events that take place in each phase. We will then discuss leading to Cell Replication
the system of checkpoint pathways that establish the order of
the cell cycle and ensure that each cell cycle phase occurs with As illustrated in Figure 19-1, the cell cycle is divided into four
accuracy. In our discussion we will emphasize the general major phases. Cycling (replicating) mammalian somatic cells
principles governing cell cycle progression and will use a spe- grow in size and synthesize RNAs and proteins required for
cies-spanning nomenclature when discussing the factors con- DNA synthesis during the G 1 (first gap) phase. When cells
trolling each cell cycle phase. The chapter concludes with a have reached the appropriate size and have synthesized the
discussion of meiosis, a special type of cell division that gen- required proteins, they enter the cell cycle by traversing a
erates haploid germ cells (egg and sperm), and the molecular point in G 1 known as START. Once this point has been
mechanisms that distinguish it from mitosis. crossed, cells are committed to cell division. The first step to-
ward successful cell division is entry into the S (synthesis)
phase, the period in which cells actively replicate their chro-
19.1 Overview of the Cell Cycle mosomes. After progressing through a second gap phase, the
G 2 phase, cells begin the complicated process of mitosis, also
and Its Control
called the M (mitotic) phase, which is divided into several
We begin our discussion by reviewing the stages of the eu- stages (Figure 19-2).
karyotic cell cycle, presenting a summary of the current In discussing mitosis, we commonly use the term chro-
model of how the cycle is regulated. We will see how DNA mosome for the replicated structures that condense and be-
replication leads to the creation of two identical DNA mol- come visible in the light microscope during the early stages
ecules during the DNA synthesis phase and how these DNA of mitosis. Thus each chromosome is composed of two iden-
molecules are compacted and structured for their segrega- tical DNA molecules resulting from DNA replication, plus
tion into daughter cells. We will then introduce the master the histones and other chromosome-associated proteins (see
regulators of the cell cycle, the cyclin-dependent kinases, be- Figure 6-39). The two identical DNA molecules and associ-
fore concluding with an overview of the principles that gov- ated chromosomal proteins that form one chromosome are
ern the cell cycle to ensure that the process occurs in the called sister chromatids. Sister chromatids are attached to
correct temporal fashion and without mistakes. each other by protein cross-links along their lengths.

~ FOCUS ANIMATION: Mitosis

VIDEO: Visualizing Mitosis with Fluorescent Probes

FIGURE 19- 2 The stages of mitosis.

During prophase, the nuclear envelope Spindle Sister
poles
breaks down, microtubules form the mitotic
spindle apparatus, and chromosomes
condense. At metaphase, attachment of
chromosomes to inicrotubules via their
kinetochores is complete. During anaphase,
microtubule motors and shortening of
spindle microtubules pull the sister chroma- Prophase Met aphase
tids toward opposite spindle poles. After
chromosome movement to the spindle poles,
chromosomes decondense, and cells
reassemble nuclear membra~es around the
daughter-cell nuclei and undergo cytokinesis.

G,

19.1 Overview of the Cell Cycle and Its Cont rol 875
 During interphase, the part of the cell cycle between the specificity of the complex, that is, which proteins it phosphory-
end of one M phase and the beginning of the next, the outer lates. Each cyclin is only present and active during the cell cycle
nuclear membrane is continuous with the endoplasmic reticu- stage it promotes and hence restricts the kinase activity of the
lum. With the onset of mitosis in prophase, the nuclear enve- CDKs it binds to just that cell cycle stage. Cyclin-CDK com-
lope retracts into the endoplasmic reticulum in most cells plexes activate or inhibit hundreds of proteins involved in cell
from higher eukaryotes, and Golgi membranes break down cycle progression by phosphorylating them at specific regula-
into vesicles. This is necessary so that the microtubules, nu- tory sites. Thus proper progression through the cell cycle is
cleated by the centrosomes, can interact with the chromo- governed by activation of the appropriate cyclin-CDK com-
somes to form the mitotic spindle, consisting of a plex at the appropriate time. As we will see, restricting cyclin
football-shaped bundle of microtubules with a star-shaped expression to the appropriate cell cycle stage is one of the
cluster of microtubules radiating from each end, or spindle many mechanisms cells employ to regulate the activities of
pole. A multiprotein complex, the kinetochore, assembles at each cyclin-CDK heterodimer.
each centromere. After nuclear envelope breakdown, the ki-
netochores of sister chromatids associate with microtubules
Several Key Principles Govern the Cell Cycle
coming from opposite spindle poles (see Figure 18-37), and
chromosomes align in a plane in the center of the cell at meta- The goal of each cell division is to generate two daughter cells
phase. During the anaphase period of mitosis, sister chroma- of identical genetic makeup. To achieve this, cell cycle events
tids separate. They initially are pulled by microtubules must occur in the proper order. DNA replication must always
toward the spindle poles and then are further separated as the precede chromosome segregation. Today we know that the ac-
spindle poles move away from each other (see Figure 19-2). tivity of the key proteins that promote cell cycle progression,
Once chromosome separation is complete, the mitotic the CDKs, fluctuates during the cell cycle. For example, CDKs
spindle disassembles and chromosomes decondense during that promoteS phase are active during S phase but are inactive
telophase. The nuclear envelope re-forms around the segre- during mitosis. CDKs that promote mitosis are only active dur-
gated chromosomes as they decondense. The physical divi- ing mitosis. These oscillations in CDK activity "are a fundamen-
sion of the cytoplasm, called cytokinesis, yields two daughter tal aspect of eukaryotic cell cycle control, and we have gained
cells. Following mitosis, cycling cells enter the G 1 phase, em- some understanding over the last few years as to how these
barking on another turn of the cycle. oscillations are generated. Oscillations are generated by posi-
The progression of cell cycle stages is the same for all tive feedback mechanisms, where specific CDKs promote their
eukaryotes, though the time it takes to complete one turn of own activation. These positive feedback loops are coupled to
the cycle varies considerably between organisms. Rapidly subsequent negative feedback mechanisms where, indirectly or
replicating human cells progress through the full cell cycle in with a built-in delay, CDKs promote their own inactivation.
about 24 hours: G 1 takes 9 hours; the S phase, 10 hours; G 2, Oscillators not only propel the cell cycle forward but also cre-
4.5 hours; and mitosis, 30 minutes. In contrast, the full cycle ate abrupt transitions between different cell cycle states, which
takes only 90 minutes in rapidly growing yeast cells. The cell is essential to bring about distinct cell cycle states.
divisions that take place during early embryonic develop- Laid on the cell cycle oscillator machinery is a system of
ment of the fruit fly Drosophila melanogaster are completed surveillance mechanisms that further ensures that the next
in as little as 8 minutes! cell cycle event is not activated before the preceding one has
In multicellular organisms, most differentiated cells been completed or before errors that occurred during the
'exit" the cell cycle and survive for days, weeks, or in some preceding step are corrected. These surveillance mechanisms
cases (e.g., nerve cells and cells of the eye lens) even the life- are called checkpoint pathways, and their job is it to ensure
time of the organism without dividing again. Such postmi- accuracy of the chromosome replication and segregation
totic cells generally exit the cell cycle in Gh entering a phase processes. The system that ensures that chromosomes are
called G 0 (see Figure 19-1 ). Some G 0 cells can return to the segregated accurately is so efficient, a mis-segregation event
cell cycle and resume replicating; this re-entry is regulated, occurs only once in 10 4-10 1 divisions! These multiple layers
thereby providing control of cell proliferation. of control put on the cell cycle control machinery ensure that
the cell cycle is robust and error free.
Cyclin-Dependent Kinases Control
the Eukaryotic Cell Cycle
KEY CONCEPTS of Section 19.1
As mentioned in the chapter introduction, passage through the
cell cycle is controlled by heterodimeric protein kinases that Overview of the Cell Cycle and Its Control
comprise a catalytic subunit and a regulatory subunit. The The eukaryotic cell cycle is divided into four phases: G 1
concentrations of the catalytic subunits, the cyclin-dependent (the period between mitosis and the initiation of nuclear
kinases (CDKs), are constant throughout the cell cycle. How- DNA replication), S (the period of nuclear DNA replica-
ever, they have no kinase activity unless they arc associated tion), G 2 (the period between the completion of nuclear
with a regulatory cyclin subunit. Each CDK can associate with DNA replication and mitosis), and M (mitosis).
a small number of different cyclins that determine the substrate

876 CHAPTER 19 The Eukaryotic Cell Cycle .

 system would require an inactivating mutation in each of the
Cells commit to a new cell division at a specific point in G 1 two copies of the gene to render its activity nonfunctional).
.. known as START. Haploid yeast can be easily employed to screen or select for
Cyclin-CDK complexes, composed of a regulatory cyclin mutants with specific defects, such as defects in cell prolif-
subunit and a catalytic cyclin-dependent kinase (CDK) sub- eration. Additional advantages of the two systems are the
unit, drive progression of a cell through the cell cycle. relative ease with which one can manipulate the expression
of individual genes, their fully sequenced genomes, and the
Cyclins activate CDKs and are present only in the cell cy-
ease with which they can be cultivated and manipulated so
cle stage that they promote.
that cultures of yeast cells progress through the cell cycle in
CDK activities oscillate during the cell cycle. Positive and a synchronous manner.
negative feedback loops drive these oscillations. Budding yeast cells are ovoid in shape and divide by bud-
Surveillance mechanisms, called checkpoint pathways, ding (Figure 19-3a). The bud is the future daughter cell and
guarantee that each cell cycle step is completed correctly be- begins to form concomitant with the initiation of D A repli-
fore the next one is initiated. cation and continues to grow throughout the cell cycle (Fig-
ure 19-3b). Cell cycle stage can therefore be inferred from the
size of the bud, which makes S. cerevisiae a useful system for
identifying mutants that are blocked at specific steps in the
. 19. 2 Model Organisms and Methods cell cycle. Indeed, it was in this organism that Lee Hartwell
and colleagues first identified mutants that were defective in
to Study the Cell Cycle progressing through specific cell cycle stages. Like mamma-
The unraveling of the molecular mechanisms governing cell lian cells, the budding yeast cell cycle has a long G 1 phase,
cycle progression in eukaryotes was remarkably speedy and and the study of the budding yeast cell cycle shaped our un-
fueled by the powerful combination of genetic and biochem- derstanding of how the G 1-S phase transition is controlled.
ical approaches. In this section we will discuss several model Fission yeast cells are rod shaped and grow entirely by
systems and their contribution to the discovery of the mo- elongation at their ends (Figure 19-4a). After the completion
lecular mechanisms of cell division. The three most impor- of mitosis, cytokinesis occurs by the formation of a septum
tant systems employed to study the cell cycle are the (Figure 19-4b). The molecular mechanisms governing G 2 and
single-celled yeasts Saccharomyces cerevisiae (budding yeast) entry into mitosis are very similar between fission yeast and
and Schizosaccharomyces pombe (fission yeast) and oocytes metazoan cells, and studies with this organism revealed the
and early embryos of the frog Xenopus laevis. We will also molecular events surrounding the GrM phase transition.
discuss the fruit fly Drosophila melanogaster, which proved Budding and fission yeast are both useful for the isola-
extremely powerful in the study of the interplay between cell tion of mutants that are blocked at specific steps in the cell
division and development as well as how the study of mam- cycle or that exhibit altered regulation of the cycle. Since cell
malian tissue culture cells led to the characterization of cell cycle progression is essential for viability, scientists isolated
cycle control in mammals. conditional mutants that encode proteins that are functional
The studies of the cell divisiOn cycle in many different at one temperature but become inactive at a different, often
experimental systems also led to two remarkable discoveries elevated, temperature (e.g., due to protein misfolding at the
about the general control of the cell cycle. First, complex non-permissive temperature). Mutants arrested at a particu-
molecular processes such as initiation of DNA replication lar cell cycle stage are easily distinguished from normal di-
and entry into mitosis are all regulated and coordinated by a viding cells by microscopic examination. Thus, in both of
small number of master cell cycle regulatory proteins. Sec- these yeasts, cells with temperature-sensitive mutations caus-
ond, these master regulators and the proteins that control ing defects in specific proteins required to progress through
them are highly conserved, so that cell cycle studies in fungi, the cell cycle were readily isolated (see Figure 5-6). Such cells
sea urchins, insects, frogs, and other species are directly ap- are called cdc (cell division cycle) mutants.
plicable to all eukaryotic cells, including human cells. How can one identify which gene is defective in a given
cdc mutant? The wild-type alleles of recessive temperature-
sensitive cdc mutant alleles can be isolated readily by trans-
Budding and Fission Yeast Are Powerful Systems
forming haploid mutant cells with a plasmid library prepared
for Genetic Analysis of the Cell Cycle from wild-type cells and then plating the transformed cells at
Budding and fission yeast have proved to be valuable sys- the non-permissive temperature (Figure 19-5). Haploid mu-
tems for the study of the cell cycle. Although they both be- tant cells cannot form colonies at the non-permissive tem-
long to the kingdom of fungi, they are only distantly related. perature. However, a transformed mutant cell can grow into
Both organisms can exist in the haploid state, carrying only a colony if it also contains a plasmid that carries the wild-
one copy of each chromosome. The fact that these yeasts can type allele that complements the recessive mutation; the plas-
exist as haploid cells makes them powerful genetic systems. m ids bearing the wild-type allele can then be recovered from
It is easy to generate mutations that inactivate genes in hap- those cells, allowing the identification of the complementing
loids because there is only one copy of each gene (a diploid gene. Because many of the proteins that regulate the cell

19. 2 Model Organisms and Methods to Study the Cell Cycle 877
G) VIDEO: Mitosis and Budding in 5. cerevisiae

(a) (b)
( ' ; ; \ Mother
D cell

Cytokinesis

Daughter
cell

Chromosome
segregation; Growth
nuclear
division

START

Spindle pole
body
Spindle duplication
formation;
nuclear Bud
migration
emergence

DNA
replication

FIGURE 19-3 The budding yeastS. cerevisiae. (a) Scanning cells are irreversibly committed to undergoing a cell cycle. G2 is not
electron micrograph of 5. cerevisiae cells at va rious stages of the cell well defined in budd ing yeast and is therefore denoted in parentheses.
cycle. The larger the bud, which emerges at the end of the G1 phase, Note that the nuclear envelope does not disassemble during mitosis
the farther along in the cycle the cell is. (b) Main events in the 5. in 5. cerevisiae and other yeasts. The small 5. cerevisiae chromosomes
cerevisiae cell cycle: Daughter cells are born smaller than mother cells do not condense sufficiently to be visible by light microscopy.
and must grow to a greater extent in G1 before they are large enough [Part (a) courtesy of E. Schachtbach and I. Herskowitz.]
to enter the S phase. START is the point in the cell cycle after which

cycle are highly conserved, human cDNAs cloned into yeast cally have large eggs, and fertilization is followed by multiple
expression vectors often can complement yeast cell cycle mu- synchronous cell cycles. By isolating large numbers of eggs
tants, leading to the rapid isolation of human genes encoding from fema les and fertilizing them simultaneously by add ition
cell cycle control proteins. In fact, it was the ability of the of sperm (or treating them in ways that mimic fertilization ),
human gene encoding CDKJ to complement the growth de- researchers can obtain extracts from cells at specific points in
fects caused by inactivation of fission yeast CDKl that led to the cell cycle for analysis of proteins and enzymatic activities.
rhe appreciation of the high degree of conservation that ex- To understand how X. laevis oocytes and eggs can be
ists among eukaryotic cell cycle regulators. used for the analysis of cell cycle progression, we must first
lay out the events of oocyte maturation, w hich can be reca-
Frog Oocytes and Early Embryos Facilitate pitulated in vitro. So far, we discussed mitotic division. Oo-
cyres, however, undergo a meiotic division (see Figure 19-38
Biochemical Characterization
fo r an overview of meiosis). As oocytes develop in the frog
of the Cell Cycle Engine ovary, they replicate their DNA and become arrested in G 2
Biochemical studies require the preparation of cell extracts for 8 months, during which time they grow in size to a diam-
from many cells. For biochemical studies of the cell cycle, the eter of 1 mm, stockpiling all the materials needed for the
eggs and early embryos of amphibians and marine mu ltiple cell divisions of the early embryo. When stimulated
invertebrates are particularly suitable. These organisms typi- by a male, an adu lt fema le's ovarian cells secrete the steroid

878 CHAPTER 19 The Eukaryotic Cell Cycle

g VIDEO: Mitosis and Cell Division in 5. pombe

(a) (b) Cytokinesis

Nuclear division
~
Chromosome
segregation
0 START

DNA
replication
Spindle
formation

Chromosome
condensation

~
Spindle pole
body duplication

Cell growth
FIGURE 19-4 The fission yeastS. pombe. (a) Scanning electron point in the cell cycle after which cells are irreversibly committed to
micrograph of 5. pombe cells at various stages of the cell cycle. Long undergoing a cell cycle. As in 5. cerevisiae, the nuclear envelope does
cells are about to enter mitosis; short cells have just passed through not break down during mitosis. [Part (a) courtesy of N. Hajibagheri.)
cytokinesis. (b) Main events in the 5. pombe cell cycle. START is the

Transform with
plasmid library EXPERIMENTAL FIGURE 19-5 Wild-type cell division
15
of wi ld-type cycle (COO genes can be isolated from aS. cerevisiae
cdc28 S. cerevisiae DNA
cells grown Transformed genomic library by functional complementation of cdc
at 25 C form Gene X cdc28 15 cells mutants. Mutant cells with a temperature-sensitive mutation in a
colonies grown at 37 C

J?@\) 0 @
CDC gene are transformed with a genomic library prepared from
wild-type cells and plated on nutrient agar at the non-permissive
temperature (37 (). Each transformed cell takes up a single
Gene Y No colony plasmid containing one genomic DNA fragment. Most such

0 formation fragments include genes (e.g., X and Y) that do not encode the

@~
defective Cdc protein; transformed cells that take up such
@ fragments do not form colonies at the non-permissive temperature.
CDC28 The rare cell that takes up a plasmid containing the wild-type

0 Q@@ version of the mutant gene (in this case CDC28, a cyclin-dependent
kin asP) is complemented, allowing the cell to replicate and form a

@~~
Isolate CDC2B
---------+ ~ plasmid
(?520
<=@
0 colony at the non-permissive temperature. Plasmid DNA isolated
from this colony carries the wild-type CDC gene corresponding to
the gene that is defective in the mutant cells. The same procedure
is used to isolate wild-type cdc genes in 5. pombe. See Figures
Cells in colony at
various cell cycle 5-17 and 5-18 for more detailed illustrations of the construction
stages and screening of a yeast genomic library.

19. 2 Model Organisms and Methods to Study the Cell Cycle 879
(a) First polar body Second polar body 11 synchronous
Progesterone divisions

l
--.... !
D
Oocyte Meiosis I Egg arrested Male Female First cleavage Blastula

-.
arrested in G 2 in metaphase pronucleus pronucleus
of meiosis II

(b)
~
"""'
, ....
:

,;,.,:::.;~;:.~t,;., ---~

FIGURE 19-6 Progesterone sti mulates meiotic maturation of schematically to represent egg cells arrested in metaphase of meiosis II.
Xenopus oocytes. (a) Step D : Progesterone treatment of G2-arrested Step 11: Fertilization by sperm releases eggs from their metaphase
Xenopus oocytes surgically removed from the ovary of an adult female arrest, allowing them to proceed through anaphase o.f meiosis II and
causes the oocytes to enter meiosis I. Two pairs of synapsed homolo- undergo a second highly asymmetrical cell division that eliminates one
gous chromosomes (blue) connected to meiotic spindle microtubules chromatid of each chromosome in a second polar body. The resulting
(green) are shown schematically to represent cells in metaphase of haploid female pronucleus fuses with the haploid sperm pronucleus
meiosis I. Step fJ : Segregation of homologous chromosomes and a to produce a diploid zygote. Step B :The zygote undergoes DNA
highly asymmetrical cell division expels half the chromosomes into replication and the first mitosis. Step 111: The first mitosis is followed by
a small cell called the first polar body. The oocyte immediately com- 11 more synchronous divisions to form a blastula. (b) Micrograph of
mences meiosis II and arrests in metaphase II to yield an egg. Two Xenopus eggs. [Part (b) copyright e ISM/Phototake.]
chromosomes connected to spindle microtubules are shown

hormone progesterone, which induces the G 2-arrested oo- covered. This activity was called maturatio n-promoting
cytes to enter meiosis. As we will see in Section 19.8, meiosis factor (MPF) because of its abi lity to induce entry into meio-
consists of two consecutive chromosome segregation phases sis when injected into Grresting oocytes.
known as meiosis I and meiosis II. Progesterone triggers oo-
cytes to undergo meiosis I and progress to the second meiotic
Fruit Flies Reveal the Interplay Between
metaphase, where they arrest and await fertilization (Figure
19-6). At this stage the cells are called eggs. When fertilized Development and the Cell Cycle
by sperm, the egg nucleus is released from its metaphase II The development of complex tissues often requires specific
arrest and completes meiosis. The resulting haploid egg nu- modifications to the cell cycle. Understanding the interplay
cleus then fuses with the haploid sperm nucleus, producing a between development and cell division is thus crucial if we ..'
diploid zygote nucleus. DNA replication follows, and the want to understand how complex organisms are built. Dro-
first mitotic division of embryogenesis begins. The resulting sophila melanogaster has established itself as the premier
embryonic cells then proceed through 11 more rapid, syn- model system for studying the interplay between develop-
chronous cell cycles, generating a hollow sphere of cells ment and the cell cycle. Not only does the development of
called the blastula. Cell division then slows, and subsequent this organism involve several highly unusual cell cycles, the
divisions are non-synchronous, with cells at different posi- powerful genetic techniques that can be applied to fruit flies
tions in the blastula dividing at different times. facilitated the discovery of genes involved in the developmen-
The advantage of using X. laevis to study factors in- tal control of the cell cycle. The fi rst 13 nuclear divisions of
volved in mitosis is that large numbers of oocytes and eggs the fertilized Drosophila embryo all occur in ::t common cyto-
\:an be prepared that are all proceed ing synchronously plasm and are rapid cycles of DNA replication and mitosis
through the cell cycle events that follow progesterone treat- (with no gap phases), fueled by key cell cycle regulators that
ment and fertilization. This makes it possible to prepare suf- were stockpiled in the egg cytoplasm as it matured. These
ficient amounts of extract for biochemical experiments from divisions are called the syncytial divisions and occur in uni-
cells that were all at the same point in the cell cycle. It was in son (Figure 19-7). As maternal stockpiles run out, gap phases
this system that the cyclin-CDK complexes that trigger mito- are introduced, first G 2 , followed by G 1 Most cells in the
sis and the oscillatory nature of their activity was first dis- embryo cease to divide at this point, form plasma membranes,

880 CHAPTER 19 The Eukaryotic Cell Cycle

 0 VIDEO: Syncytial Divisions of the Drosophila Embryo

Mitotic divisions
in the syncytium
Mitosis
Endocycles in Endocycles in (stem cells)
differentiating larval tissues
larval tissues Meiosis
Increase in (egg and sperm)
Mitotic divisions cel l size Division and
in the d1fferent1ation Endocycles
nervous system of imaginal disks (ovary)
1st 2nd 3rd
in star instar instar
~ :
~~
Embryo Larval stages Pupa
T_~
Adult
FIGURE 19-7 Cell division patterns during the life cycle of hence larval growth. In the pupa, during a process called metamorpho-
Drosophila melanogaster. After fertilization, nuclei in the embryo sis, imaginal disks, the tissues that give rise to the adult organs,
undergo 13 rapidS phase-M phase cycles. These are followed by three undergo mitotic divisions and then differentiate to form adult
divisions that include a G2 phase. All these nuclear divisions occur structures. Several types of divisions are seen in the adult fly. Stem cells
. within a common cytoplasm and are therefore called the syncytial undergo mitotic divisions, meiosis gives rise to sperm and egg, and
divisions. During late stages of embryogenesis and t hroughout larval endocycles create polyploid cells in the ovary. [Adapted from Lee and
development (with the exception of the nervous system), cells undergo Orr-Weaver, 2003, Ann. Rev. Genet. 37:545- 578.)
endocycles. This leads to an increase in cellular ploidy and size and

and utilize a specialized cell cycle known as the endocycle. In organization and developmental signals governing cell cycle
the endocycle, cells replicate their DNA but do not undergo control--<:ell culture systems nevertheless provide critical in-
mitosis. This leads to an increase in gene dosage and fuels sights into the mammalian cell's intrinsic mechanisms govern-
increased macromolecule biosynthesis, which allows individ- ing cell division. Researchers also work toward establishing
ual cells to grow in size. Thus the embryo, which has now culture systems that more closely resemble the cell architec-
developed into a crawling larva, grows simply by an increase ture in tissues. For example, polymers are currently being de-
in cell size and not through cell multiplication. A select num- veloped that allow scientists to grow cells in 3-D culture.
ber of cells do not share this fate. These cells are in the ima- As we wi ll see in Chapter 21, primary human cells and
ginal disks, the organs that will give rise to the adu lt fly other mammalian cells have a finite life span when cultured
tissues during metamorphosis. Metamorphosis occurs during in vitro. Normal human cells, for example, divide 25-50
the pupa stage and transforms larvae into adult flies. The di- times, but thereafter proliferation slows and eventually
visions that give rise to the adult fly are canonical cell cycles stops. This process is called replicative senescence. Cells can
leading to the adult fly being a diploid organism. escape this process and become immortalized, allowing re-
searchers to establish cell lines. Although these cell lines har-
bor genetic alterations that affect some aspects of cell
The Study of Tissue Culture Cells Uncovers
proliferation, they are nevertheless a useful tool to study cell
Cell Cycle Regulation in Mammals cycle progression in human cells. These cell lines provide an
Cell cycle regulation in hllman cells is more complex than in inexhaustible supply of cells that, as we will see next, can be
other non-mammalian systems. To understand this increased manipulated to progress through the cell cycle in a synchro-
level of complexity and to understand the cell cycle alterations nous manner, allowing for the analysis of protein levels and
that are the cause of cancer, it is important to study the cell enzymatic activity at different stages of the cell cycle.
cycle not only in model organisms but also in human cells.
Researchers use normal or tumor cells grown in plastic dishes
Researchers Use Multiple Tools
to study the properties of the human cell cycle, a method
called tissue culture or cell culture. It is, however, important to Study the Cell Cycle
to note that many of the cell types used to study the human The experimental analysis of cell cycle properties requires that
<.:dl cycle themselves have altered cell cycle properties due to we are able to determine the cell cycle stage of individual cells.
genetic alterati ons that occurred during their culturing o r be- Light microscopy provides some estimate of cell cycle progres-
cause they were isolated from human tumors. Furthermore, in sion. For example, light microscopy allows a researcher to
vitro culture conditions do not resemble those found in the determine whether cultured mammalian cells are in interphase
organism and could lead to altered behavior of cells. Although (Gh S phase, and G2 ) or in mitosis. Mammalian tissue culture
some aspects of mammalian cell division are not recapitulated cells are flat and adhere to the plastic dish during interphase
in cell culture conditions-such as the importance of tissue but round up and form spherical structures as they undergo

19. 2 Model Organisms and Methods to Study the Cell Cycle 881
FIGURE 19-8 Human cells undergoing
mitosis. He La Kyoto cells were filmed as they
underwent mitosis. The images shown were
taken every 20 minutes. Cells are flat during
interphase, but as cells undergo mitosis, they
round up and divide. Subsequently, they flatten
out again. [Courtesy of Sejal Vyas and Paul Chang, MIT.]

mitOSIS (Figure 19-8). Fluorescence microscopy of cellular a particular cell cycle stage. This cell cycle arrest is usually ac-
structures or the analysis of specific cell cycle markers, that is, complished by restricting nutrients or adding anti-growth fac-
proteins that are only present in certain cell cycle stages, al- tors, which cause cells to arrest in G 1 In budding yeast, for
lows for a more accurate determination of cell cycle stage. example, cells treated with a mating pheromone arrest in G 1
In addition to microscopic tools, cell cycle researchers When the pheromone is removed from cells (usually by wash-
use flow cytometry to determine the D~A content of a cell ing them extensively), cells exit the G 1 arrest and progress
population (Figure 19-9; see also Figure 9-2). Cells are through the cell cycle in a synchronous manner. In mammalian
treated with a DNA-binding fluorescent dye and the amount cells, removal of growth factors by removing serum from the
of dye that incorporates into the DNA of cells can then be culture medium (serum starvation) arrests cells in G 0 Re-
quantitatively assessed using a flow cytometer. Cells are then add ition of serum allows cells to re-enter the cell cycle. Other
sorted by their DNA content, and the percentage of cells in methods involve blocking a certain cell cycle.step with chemi-
GI> S phase, and G 2 or mitosis can be assessed in this man- cals. Hydroxyurea inhibits DNA replication, leading to an S
ner. Cells in G 1 will have half as much DNA as cells in G2 or phase arrest. On removal of the drug, cells will resume DNA
mitosis. Cells undergoing DNA synthesis in S phase will synthesis in unison. Nocodazole disrupts the mitotic spindle
have an intermediate amount of DNA. and halts the cell cycle in mitosis. Once the drug is washed
To characterize different cell cycle events, it is essential to away, cells will resume progression through mitosis in a syn-
examine cell populations that progress through the cell cycle in chronous manner. In budding and fission yeast, the conditional
unison. Researchers achieve this by reversibly arresting cells in cell division cycle (cdc) mutants introduced ea rlier have proved
a powerful tool for creating synchronous cultures. Tempera-
ture-sensitive cdc mutants, when incubated at the non-permis-
1200 sive temperature, arrest in a particular cell cycle stage because
they are defective in a certain key cell cycle protein. Returning
cells to the permissive temperature allows cells to continue with
900 the cell division cycle in a synchronous fas hion.
Unreplicated
.!!!.
Qi

-(.)

0
Cii 600
.0
E KEY CONCEPTS of Section 19.2
:::;)
z Model Organisms and Methods to Study the Cell Cycle
Replicated
300
The ability to isolate mutants and the powerful genetic
tools of budding and fission yeast allowed for the isolation
of key factors important for cell cycle regulation.

1C 2C
Frog eggs and early embryos from synchronously fertil-
ized eggs provide sources of extracts fo r biochemical studies
DNA content
of cell cycle events and identified the oscillatory nature of
: XPE. MEN AL FIGURE 19 9 Analysis of DNA content by cyclin-CDK complexes.
flow cytometry. Haploid yeast cells were grown in <.ulture and stained
Fruit flies are a powerful system to investigate the inter-
with propidium iodide, a fluorescent dye that incorporates into DNA.
The x axis shows DNA content, they axis the number of cells. The DNA
play between cell division and the developmental programs
content ana lysis shows two predominant populations of cells: cells responsible for building multicellular organisms.
with unreplicated DNA (lC) and with replicated DNA (2C). The cells Human tissue culture cells are used to study the properties
between the two peaks represent cells that are in the process of of the mammalian cell cycle.
undergoing DNA replication. [Courtesy of Heid1 Blank.]

882 CHAPTER 19 The Eukaryotic Cell Cycle

 transition points in the cell cycle. A key discovery in cell cycle
The generation of synchronized cell populations through studies was that cyclin-dependent kinases govern progression
reversibly arresting cells in a particular cell cycle stage allows through the cell cycle. Three key features about these kinases
the researcher to examine the behavior of proteins and cel- are important to keep in mind throughout this chapter:
lular processes during the cell cycle.
Cyclin-dependent kinases (CDKs) are only active when
bound to a regulatory cyclin subunit.
19.3 Regulation of CDK Activity Different types of cyclin-CDK complexes initiate different
event~. G 1 CDKs and G 1 /S phase CDKs promote entry into
In the following seuiuns we describe the current model of eu-
the cell cycle, S phase CDKs trigger S phase, and mitotic
karyotic cell cycle regulation summarized in Figure 19-10 and
CDKs initiate the events of mitosis (Figure 19-11 ).
present some of the experiments that led to this understanding.
As we will see, results obtained with different experimental Multiple mechanisms are in place to ensure that the different
systems and approaches have provided insights into each of the CDKs are only active in the stages of the cell cycle they trigger.

(i) VIDEO: Dynamic Behavior of Mitotic Cyclin in Hela Cells

APC/C and phosphatases

induce late steps in mitosis

APC/C ubiquitin-protein
ligase induces
anaphase

Prophase Metaphase

M
Mitotic CDKs G 1 CDKs and
induce mitosis G 1/S phase CDKs
prepare cells
for S phase

SCF
ubiquitin-protein
ligase induces
S phase
s
S phase CDKs activate
DNA replication
FIGURE 19-1 0 Regulation of cell cycle transitions. Cell cycle down and chromosomes align on the mitotic spindle but they cannot
transitions are regulated by cyclin-CDK protein kinases, protein separate until the anaphase-promoting complex (APC/C), a ubiquitin-
phosphatases, and u biquitin-protein ligases. Here the cell cycle is protein ligase, ubiquitinylates the anaphase inhibitor protein securin,
diagrammed, with the major stages of mitosis shown at the top. In early marking it for degradation by proteasomes. This results in degradat ion
G1, no cyclin-CDKs are active.ln mid-G1, G1/S phase CDKs activate of protein complexes linking the sister chromatids and the onset of
transcription of genes required for DNA replication. S ph11se is initiated anaphase as sister chromatids separate. After chromosome movement
by the SCF ubiquitin-protein ligase that ubiquitinylates inhibitors of S to the spindle poles, the APC/C ubiquitinylates mitotic cyclins, causing
phase CDKs, marking them for deg radation by proteasomes. The S phase their degradation by proteasomes. The resulting drop in mitotic CDK
CDKs then activate DNA replication and DNA synthesis commences. activity, along with the action of protein phosphatases, results in
Once DNA replication is complete, cells enter G2 In late G2, mitotic CDKs chromosome decondensation, reassembly of nuclear membranes
trigger entry into mitosis. During prophase, the nuclear envelope b reaks around the daughter-cell nuclei, and cytokinesis.

19.3 Regulation of CDK Act ivity 883

FIGURE 19-11 An overview of how CDKs regulate cell cycle G 1/S phase S phase Mitotic APC/C
progression. Cells harbor different types of CDKs that initiate different CDKs CDKs CDKs activity
events of the cell cycle. Importantly, the CDKs are only active in the
stages of the cell cycle they trigger. G/S phase CDKs are active at the
G1-5 phase transition to trigger entry into the cell cycle. 5 phase CDKs
-~
are active during 5 phase and trigger 5 phase. Mitotic CDKs are active
~
u
during mitosis and trigger mitosis. A ubiquitin ligase known as the <(
anaphase-promoting complex or cyclosome (APC/C) catalyzes two key
cell cycle transitions by ubiquitinylating proteins, hence targeting them
for degradation. The APC/C initiates anaphase and exit from mitosis.

Metaphase-anaphase
transition

In thts section we will first discuss the properties of CDKs They bind to different types of cycljns and together promote
and investigate the structural basis of their activation and different cell cycle transitions. CDK4 a nd CDK6 are G 1
regulation. We will then see how cyclins activate CDKs and CDKs and promote entry into the cell cycle, CDK2 functions
investigate the multiple regulatory mechanisms that restrict as a G/S phase and S phase CDK, and CDK1 is the mitotic
the different cyclins to the appropriate cell cycle stage. We CDK. For historical reasons, the names of various cyclin-
will see that protein degradation plays an essential part in dependent kinases from yeasts and vertebrates differ. When-
this process. In addition, we will discuss how post-transla- ever possible, we will use the general terms G 1, G /S phase,
tional modifications to CDKs an d inhibitory proteins that S phase, and mitotic CDKs ro describe CDKs instead of the
directly bind to cyclin-CDK complexes are essential addi- species-specific terminology. Table 19-1 lists the different
tional control mechanisms in restricting different cycl in- names of the various CDKs and indicates when in the cell .
CDK activities to the appropriate cell cycle stage. cycle they are active.
CDKs are not only regulated by cyclin binding but also
Cyclin-Dependent Kinases Are Small Protein by both activating and inhibitory phosphorylation. To-
gether, these regulatory events ensure that CDKs are only
Kinases That Requ ire a Regulatory Cyclin
active at the appropriate cell cycle stage. The three-dimensional
Subunit for Their Activity structure of CDKs provides insight into how the activity of
Cyclin-dependent kinases are a family of small (30-40 kD) these protein kinases is regulated. Unphosphorylated, inac-
serine/threonine kinases. They are not active in the monomeric tive CDK contains a flexible region, called the T loop, that
form but, as mentioned previously, require an activating blocks access of protein substrates to the active site where
subunit to be active as a protein kinase. In budding and fis- ATP is bound (Figure 19-12a). Steric blocking by the T loop
sion yeast, a single CDK controls progression through the largely explains why free CDK, unbound to cyclin, has little
cell cycle. Its activity is specified by cell-cycle-stage-specific protein kinase activity. Unphosphorylatecl CDK bound to
cyclin subunits. Mammalian cells contain as many as nine one of its cyclin partners has minimal but detectable protein
CDKs, wtth four of them, CDKl, CDK2, CDK4, and CDK6, kinase activity in vitro, although it may be essentially inac-
having clearly been shown to regulate cell cycle progression. tive in vivo. Extensive interactions between the cyclin and

TABLE 19-1 Cyclins and CDKs: Nomenclature and Their Roles in the Mammalian Cell Cycle

CDK Cyclin Function General Name

- ~

- -- - ~ ~

-- ~ - - - - - -
CDKl Cyclin A, cyclin B Mitosis Mitotic CDKs

CDK2 Cyclin E, cyclin A Entry mto the cell cycle G/S phase CDKs
S phase S phase CDKs

CDK4 Cyclin D c. G, CDKs

Entry mto the cell cycle

CDK6 Cycli n D G, G 1 CDKs

Entry into the cell cycle

884 CHAPTER 19 The Eukaryotic Cell Cycle

 (a) Free CDK2 (b) Low-activity cyclin A-CDK2 (c) High-activity cyclin A-CDK2

FIGURE 19-12 Structural models of human CDK2. (a) Free, extensively with cyclin A, moves several angstroms into the catalytic
inactive CDK2 unbound to its cyclin subunit, cyclin A. In free CDK2, the cleft, repositioning key catalytic side chains required for the phos-
T loop blocks access of protein substrates to the 'Y phosphate of the photransfer reaction. The red ball marks the position of the threonine
bound ATP, shown as a ball-and-stick model. The conformations of the (Thr-160) whose phosphorylation activates CDKs. (c) Phosphorylated,
regions highlighted in yellow are altered when CDK is bound to cyclin high-activity cyclin A-CDK2 complex. The conformational changes
A. (b) Unphosphorylated, low-activity cyclin A-CDK2 complex. induced by phosphorylation of the activating threonine (red ball) alter
Conformational changes induced by binding of a domain of cyclin A the shape of the substrate-binding surface, greatly increasing the
(blue) cause the T loop to pull away from the active site of CDK2 so that affinity for protein substrates. [Courtesy of P. D. Jeffrey. See A. A. Russo et al.,
substrate proteins can bind. The cxl helix in CDK2, which interacts 1996, Nature Struct. Bioi. 3:696.]

the T loop cause a dramatic shift in the position of the T The G 1 cyclins are the lynchpin in coordinating the cell
loop, thereby exposing the CDK active site (Figure 19-llb). cycle with extracellular events. Their activity is subject to
As we will see shortly, high activity of the cyclin-CDK com- regulation by signal transduction pathways that sense the
plex requires phosphorylation of the activating threonine, in presence of growth factors or cell proliferation inhibitory
the T loop, causing additional conformational changes in the signals. In metazoans, G 1 cyclins are known as cyclin Ds,
cyclin-CDK complex that greatly increase irs affinity for pro- and they bind to CDK4 and CDK6. G 1 cyclins are unusual in
tein substrates (Figure 19-12c). As a result, the kinase activ- that their levels do not fluctuate in a specific pattern during
ity of the phosphorylated complex is a hundredfold greater the cell cycle. Instead, in response to macromolecule biosyn-
than that of the unphosphorylated complex. thesis and extracellular signals, their levels gradually increase
throughout the cell cycle.
The G 1/S cyclins accumulate during late Gh reach peak
.. Cyclins Determine the Activity of CDKs levels when cells enter S phase, and decline during S phase
Cyclins are so named because their levels change during the (see Figure 19-11). They are known as cyclin E in metazoans
cell cycle. They form a family of proteins that is defined by and bind to CDK2. The main function for cyclin t-CDK2
three key features: complexes, together with cyclin D-CDK4/6, is to trigger the
G 1-S phase transition. This transition is known as START
Cyclins bind to and activate CDKs. The activity and sub-
and is defined as the point at which cells are irreversibly
strate specificity of any given CDK is primarily defined by
committed to cell division and can no longer return to the G 1
the particular cyclin to which it is bound.
' state. In molecular terms, this means that cells initiate DNA
Cyclins are only present during the cell cycle stage that replication as well as duplicate their centrosomes, which is
they trigger and are absent in other cell cycle stages. the first step in the formation of the mitotic spindle that will
be used during mitosis.
Cyclins not only regulate a particular cell cycle stage but
S phase cyclins are synthesized concomitantly with G 1 cy-
also set in motion a series of events in preparation for the
clins, but levels remain high throughout S phase and do not
next cell cycle stage. In this way, they propel the cell cycle
decline until early mitosis. Two types of S phase cyclins trigger
forward.
S phase in metazoans: cyclin E, which can also promote entry
Cyclins are divided into four classes defined by their into the cell cycle and is therefore also a G 1/S cyclin, and cyclin
. presence and activity during the cell cycle: G 1 cyclins, G 1/S A. Both cyclins bind CDK2 (see Table 19-1) and are directly
cyclins, S phase cyclins, and mitotic cyclins (see Table 19-1 ). responsible for DNA synthesis. As we will see in Section 19.4,
The different types of cyclins are quite distinct from each these protein kinases phosphorylate proteins that activate
other in protein sequence, but all of them contain a con- DNA helicases and load polymerases onto DNA.
served 100 amino acid region known as the cyclin box and Mitotic cyclins bind CDK l to promote entry into and
possess similar three-dimensional structures. progression through mitosis. The metazoan mitotic cyclins

19.3 Regulation of CDK Activity 885

are cyclin A and cyclin B (note cyclin A can also trigger S extract system. Using egg extracts from frogs, researchers
phase when bound to CDK2). Mitotic cyclin-CDK com- showed that mitotic cyclins are sufficient to induce mitosis.
plexes are synthesized during S phase and G 2 , but as we will Cytoplasmic extracts prepared from unfertilized Xenopus
see shortly, their activities are held in check until DNA syn- eggs contain all the materials (mRNAs and proteins) re-
thesis is completed. In Section 19.5, we will see that once quired for multiple cell cycles. When nuclei prepared from
activated, mitotic CDKs promote entry into mitosis by phos- Xenopus sperm (sperm nuclei are used in this experiment
phorylating and activating hundreds of proteins to promote because they are readily isolated in large numbers) are added
chromosome segregation and other aspects of mitosis. Their to such an egg extract, the nucleus and DNA inside it are
inactivation dunng anaphase prompts cells to exit mitosis, induced to behave a~ if progressing through the cell cycle;
which involves the disassembly of the mitotic spindle, chro- that is, they replicate their DNA and then undergo mitosis in
mosome decondensation, the re-formation of the nuclear the extract. Concomitant with the final stages of mitosis, cy-
envelope, and eventually cytokinesis. clin levels decline (Figure 19-13a). Researchers wanted to
Mitotic cyclins were the first cyclins to be discovered, determine whether this protein with its fluctuating levels in
and it was their characterization that led to the discovery of fact had the ability to induce mitosis. Because mitotic cyclins
the oscillatory nature of the activities that govern cell cycle appeared to be unstable at the end of mitosis, the researchers
progression. The experiment that led to their discovery is reasoned that removing the mRNA from the egg extract
described at the end of this chapter as a classic experiment in would prevent the new synthesis .of all unstable proteins in
cell biology (see Classic Experiment 19.1 , Figure 1). the next cell cycle, including that of the mitotic cyclins (all
The fact that cyclins are the rate-limiting proteins in trigger- other stable proteins would still be present in the extract). To
ing cell cycle transitions was discovered using the embryonic do this, they digested all mRNAs with a low concentration of

(a) Untreated extract (b) RNase-treated extract

Addition of Addition of
j s~erm nuclei sperm nuclei

i ~

Time~ Time~

(c) RNase-treated extract+ wild-type mitotic cyclin mRNA (d) RNase-treated extract + nondegradable mitotic cyclin mRNA

Addition of Addition of
sperm nuclei sperm nuclei

i ~ i ~

Time~ "'1<'------ Mitotic arrest -----~3>1

Time~

- =Mitotic CDK activity =Early mitotic events

- =Mitotic cyclin concentration = Late mitotic events

EXPERIMENTAL FIGURE 19-13 Mitotic cyclins are rate and nuclear envelope disassembly, and of late events {orange shading),
limiting for mitosis. In all cases, mitotic CDK activity and mitotic cyclin including chromosome decondensation and nuclear envelope
concentration were determined at various times after addition of reassembly. See text for discussion. [See A. W. Murray et al.. 1989, Nature
sperm nuclei to a Xenopus egg extract treated as indicated in each 339:275; adapted from A. Murray and T. Hunt, 1993, The Cell Cycle: An Introduction,
panel. Microscopic observations determined the occurrence of early W. H. Freeman and Company.)
mitotic events {blue shading), including chromosome condensation

886 CHAPTER 19 The Eukaryotic Cell Cycle

RNase, which then was inactivated by addition of a specific appropriate cell cycle stage and to keep them at the right
inhibitor. This tr eatment destroys mRNAs without affecting concentration.
the tRNAs and rRNAs required for protein synthesis. When
sperm nuclei were added to the RNase-treated extracts, the
nuclei replicated their DNA, but they did not undergo mito-
Cyclin Levels Are Primarily Regulated
sis. Furthermore, mitotic cyclin protein was not detected by Protein Degradation
(Figure 19-13b). Addition of mitotic cyclin mRNA, pro- Multiple mechanisms ensure that CDKs are active in the right
duced in vitro from cloned mitotic cyclin eDNA, to the stage of the cell cycle. Table 19-2 lists these key regulators of
RNase-treated egg extract induced mitosis as observed with CDK!>. In this section we will discuss how the regulation of
the untreated egg extract (Figure l9-13c). Since mitotic cy- cyclin levels is brought about. The timely activation of CDKs
clin is the only newly synthesized protein under these condi- depends, in part, on the presence of the appropriate cyclins in
tions, these results demonstrate that the protein is the only the cell cycle stage where they are needed. Transcriptional
one limiting for entering mitosis. Subsequent studies showed control of the cyclin subunits is one mechanism that ensures
that G 1/S phase cyclins have similar properties. Their ex- proper temporal expression of tbe cyclins. In somatic cells and
pression is sufficient to promote entry into the cell cycle, yeast, waves of transcription factor activities help establish
and therefore all the other proteins needed for cell cycle waves of cyclin activity. A general principle here is that an
entry are present in unl imited amounts. It is thus clear that earlier wave of transcriptional activity helps produce the fac-
the regu lation of cyclin levels is an essential aspect of the tors essential to generate a subsequent transcriptional wave.
cukaryotic cell cycle. As we will see in the following section, As we will see in Section 19.4, transcription of the G 1/S phase
cells utilize mu ltiple mechanisms to restrict cyclins to the cyclins is promoted by the E2F transcription factor complex.

TABLE 19-2 Regulators of Cyclin -CDK Activity

Type of Regulator Function

Kinases and Phosphatases

CAK kinase Activates CDKs

Weel kinase Inhibits CDKs

Cdc25 phosphatase Activates CDKs

Cdc14 phosphatase Activates Cdh l to degrade mitotic cyclms

Cdc25A phosphatase Activates vertebrate S phase CDKs

Cdc25C phosphatase Activates vertebrate mitotic CDKs

Inhibitory Proteins

Sicl Binds and inhibits S phase CDKs

CKis p27KIPI, p57KII'2 , and p21 Cll' Bind and inhibit CDKs

INK4 Binds and inhibits G 1 CDKs

Rb Binds E2Fs, preventing transcription of multiple cell cycle genes

Ubiquitin-Protein Ligases

SCF Degradation of phosphorylated ~1cl or p27"JP 1 to activateS phase CDKs

APC/C + Cdc20 Degradation of securin, initiating anaphase. Induces degradation of B-type cyclins

APC/C + Cdhl Degradation of B-type cyclins in G 1 and geminin in metazoans to allow loading of replicative
helicases on DNA replication origins

19.3 Regulation of CDK Activity 887

Among the many other genes that E2F transcribes are the cyclins not only coincided with entry into mitosis, but its dis-
transcription factors that will promote the synthesis of mitotic appearance occurred concomitantly with exit from mitosis.
cyclins. This finding raised the possibility that mitotic cyclin degrada-
The most important regulatory control that restricts cyclins tion was necessary for cells to exit from mitosis. To test this,
to the appropriate cell cycle stage is ubiquitin-mediated, protea- researchers added an mRNA encoding a nondegradable mi-
some-dependent protein degradation. Because protein degra- totic cyclin (lacking the destruction box) to a mixture of
dation is an irreversible process, in the sense that the protein RNase-treated Xenopus egg extract and sperm nuclei. As
can only be replenished through de novo protein synthesis, this shown in Figure 19-13d, entry into mitosis occurred on sched-
regulatory mechani~m i~ ideal to ensure that the cell cycle en- ule, but exit trom mitosis did not. This experiment demon-
gine is driven forward and cells cannot "go backward" in the strates that mitotic exit requires the degradation of mitotic
cell cycle. In other words, once a particular cyclin is degraded, cyclin. Later studies showed that inhibiting the degradation of
the processes that it activated can no longer take place. other cyclins also severely affected cell cycle progression, indi-
Recall that during ubiquitin-mediated protein degrada- cating that ubiquitin-mediated degradation of cyclins is an es-
tion, ubiquitin-protein ligases polyubiquitinylate substrate sential aspect of the eukaryotic cell cycle.
proteins, marking them for degradation by the proteasomc
(see Figure 3-29). Cyclins are degraded through the action of
two different ubiquitin-protein ligases, SCF (named after the
CDKs Are Regulated by Activ,ating
first letters of its constituents, Skp 1, Cullin, f-box proteins) and Inhibitory Phosphorylation
and the anaphase-promoting complex or cydosome (abbrevi- Regulating the levels of cyclins is not the only mechanism
ated as APC/C in this chapter). SCF controls the G 1-S phase that controls CDK activity. Activating and inhibitory phos-
transition by degrading G/S phase cyclins and, as we will see phorylation events on the CDK subunit itself are essential to
m detail shortly, CDK inhibitory proteins. The APC/C de- control cyclin-CDK activity. Phosphorylation of a threonine
grades S phase and mitotic cyclins, thereby promoting the exit residue near the active site of the enzyme is required for CDK
from mitosis. APC/C also controls the onset of chromosome activity. This phosphorylation is mediated by the CDK-acti-
segregation at the metaphase-anaphase transition by degrad- vating kinase (CAK). In some organisms cyclin binding is a
ing an anaphase inhibitory protein (discussed in Section 19.6). prerequisite for CAK phosphorylation, whereas in others this
The SCF and APC/C are multisubunit ubiquitin ligases phosphorylation event occurs prior to cyclin binding. Al-
that belong to the RING finger family of ubiquitin ligases. though the sequence of assembling active CDKs differs be-
Despite the fact that SCF and APC/C belong to the same tween organisms, it is clear that CAK phosphorylation of
ubiquitin ligase family, their regulation is quite different. CDK is not a rate-limiting step in CDK activation. CAK ac-
The SCF recognizes its substrates only when they are phos- tivity is constant throughout the cell cycle and phosphory-
phorylated. The SCF is continuously active throughout the lates the CDK as soon as a cyclin-CDK complex is formed.
cell cycle, and cell-cycle-regulated phosphorylation of its Two inhibitory phosphorylations on CDK play a critical
substrates ensur~s that they are only degraded at certain role in controlling CDK activity. In contrast to the CAK-in-
stages of the cell cycle. In the case of APC/C-dependent pro- duced activating phosphorylation, these inhibitory phos-
tein degradation, the regulation is reversed. Substrates are phorylations are regulated. A highly conserved tyrosine (Y15
recognizable throughout the cell cycle, but the activity of the in human CDKs) and an adjacent threonine (T14 in humans)
APC/C is cell cycle regulated. APC/C is activated by phos- are subject to regulated phosphorylation. Both residues are
phorylation at the metaphase-anaphase transition, through situated in the ATP binding pocket of the CDK, and their
the action of mitotic CDKs and other protein kinases. The phosphorylation most likely interferes with positioning of
APC/C is then active throughout the rest of mitosis and dur- ATP in the pocket. Changes in the phosphorylation of these
ing G 1 to promote the degradation of cyclins and other mi- sites are essential for the regulation of mitotic CDKs and
totic regulators (see Figure 19-11 ). Substrate specificity of have also been implicated in the control of G/S and S phase
active, phosphorylated APC/C is determined in part by its CDKs. As we will see in Section 19.5, a highly conserved
association with one of two related substrate-targeting fac- kinase called Weel brings about this inhibitory phosphory-
tors called Cdc20 and Cdh I. During anaphase APC/C bound lation, and a highly conserved phosphatase called Cdc25
to Cdc20 ubiquitinylates proteins that lead to chromosome mediates dephosphorylation.
segregation, while during telophase and Gh APC/C-Cdh 1
targets different substrates for degradation. The substrates
of the APC/C conrain recognition motifs. The first to be CDK Inhibitors Control Cyclin-CDK Activity
identified is the destruction box. It is found in most S phase So far, we have discussed the impvrtam:e of regulating cyclin
and mitotic cyclins. This destruction box is both necessary protein levels and of CDK phosphorylation in controlling
and sufficient to target proteins for degradation. CDK activity. The final layer of control that is of critical
Today we know that cyclin degradation at the appropriate importance in the regulation of CDKs is a family of proteins
cell cycle transition is essential for cell cycle progression. The known as CDK inhibitors, or CKis, that directly bind to the
importance of cyclin degradation was again first demonstrated cyclin-CDK complex and inhibit its activity. As we will see
in frog egg extracts. Recall that the accumulation of mitotic in Section 19.4, these proteins play an especially important

888 CHAPTER 19 The Eukaryotic Cell Cycle

role in the regulation of the G 1-S phase transition and its initiate a certain cell cycle stage, CDKs phosphorylate a myr-
integration with extracellular signals. It thus comes as no iad of substrates, thereby directly initiating all aspects of a
surprise that the genes encoding these CKls are often found given cell cycle phase. Analysis of a small number of sub-
mutated in human cancers (discussed in Chapter 24). strates has provided examples that show how phosphoryla-
All eukaryotes harbor CKls involved in regulating S tion by mitotic CDKs mediates many of the early events of
phase and mitotic CDKs. Although these inhibitors display mitosis: chromosome condensation, formation of the mitotic
little sequence simi larity, they are all essential to prevent pre- spindle, and disassembly of the nuclear envelope. We will
mature activation of S phase and M phase CDKs. Inhibitors discuss these events in detail in the sections that follow.
of(; 1 C.J1Ks play an essential role in mediating a G 1 arrest i11 In recent years systematic efforts to identify all CDK sub-
response to proliferation inhibitory signals. A class of CKis strates have been initiated. The challenge in identifying sub-
called INK4s (inhibitors of kinase 4 ) includes several small, strates of a particular kinase is how to distinguish that kinase's
closely related proteins that interact only with the G 1 CDKs. phosphorylation events from those carried out by other ki-
Binding of INK4s to CDK4 and CDK6 blocks their interac- nases. A breakthrough in understanding which proteins are
tion with cyclin D and hence their protein kinase activity. A targets of CDKs was facilitated by the engineering of a CDK
second class of CKis found in metazoan cells consists of mutant in budding yeast that can utilize an analog of ATP that
three proteins-p21 ur, p27K 1P\ and p57K 1P2 . These CKis in- is not bound by other kinases (Figure 19-14). This ATP ana-
hibit G 1/S phase CDKs and S phase CDKs and must be de- log has a bulky benzyl group attached to N 6 of the adenine,
graded before DNA replication can begin. As we will discuss which makes the analog too large to fit into the ATP-binding
in Section 19.7, p21 CIP plays an important role in the response pocket of wild-type protein kinases. However, the ATP-bind-
of metazoan cells to DNA damage. CKis regulating G 1 CDKs ing pocket of the mutant CDK was modified to accommodate
play a critical role in preventing tumor formation. For exam- this large ATP analog. Consequently, only the mutant CDK
ple, both copies of the INK4 gene, which encodes p16, are can utilize this ATP analog as a substrate for transferring its 'Y
found inactivated in a large fraction of human cancers. phosphate to a protein side chain. When the N 6 -benzyl ATP
analog with a labeled 'Y phosphate was incubated with yeast
cell extracts containing a recombinant mitotic CDK contain-
Special CDK Alleles Led to the Discovery
ing the altered ATP-binding pocket, multiple proteins were
of CDK Functions labeled. True yeast mitotic CDK substrates could be verified
Different CDKs initiate different cell cycle phases by phos- among these potential substrates by treatment of cells express-
phorylating specific proteins. It is now clear that rather than ing the mutant CDK with a similar derivative of another ATP
phosphorylating a small number of proteins that in turn analog that binds the mutant CDK in the same way as the

(a) (b)

FIGURE 19-14 ATP analog- dependent CDK mutant. (a) Represen- uttltzed by them. In the 5. cerevisiae CDK mutant, the phenylalanine at
tation of the ATP-binding and catalytic sites of wild-type 5. cerevisiae position 88 is changed to glycine, which lacks a large side chain. The
CDKl (called Cdc28 in budding yeast). Bound ATP and a phenylalanine mutant exhibits high protein kinase activity using N6-(benzyi)ATP.
side chain (purple) in the vicinity of the binding pocket are shown in These models of 5. cerevisiae CDK are based on crystal structures of the
stick format. (b) Bulky ATP analogs such as those containing a benzyl PKA kinase domain, which shares extensive homology with the kinase
group bound to the N6 amino nitrogen are too large to fit into the domain of 5. cerevisiae CDK. [See J. A. Ubersax et al., 2003, Nature 425:859;
ATP-binding pocket of wild-type protein kinases and thus cannot be K. Shah et al., 1997, Proc. Nat'/. Acad. Sci. USA 94:3565.]

19.3 Regulation of CDK Activity 889

N 6 -benzyl ATP analog but that cannot be used for substrate budding yeast, and it was in this organism that the molecular
phosphorylation. Because of the bulky benzyl group, it is ste- mechanisms underlying this cell cycle transition were initially
rically blocked from binding to all other kinases, consequently elucidated. We will therefore first examine the molecular
inhibiting only the mutant CDK. When cells expressing the events governing cell cycle entry in budding yeast. We will
mutant CDK were treated with this specific CDK inhibitor, then investigate the striking similarities between the pathways
most of the putative mitotic CDK targets identified initially that govern cell cycle entry in yeast and metazoan cells and
were found in a dephosphorylaryed state, indicating that these discuss the realization that many genes involved in this deci-
proteins are indeed phosphorylated by the CDK in vivo as sion are frequently found mutated in cancer. Next we will see
well as in vitro. In yeast, th1s procedure identitied most of the that the decision to enter the cell cycle is influenced by extra-
known CDK substrates plus more than 150 additional yeast cellular events and learn about the signaling mechanisms that
proteins. Similar approaches have also been used in mamma- convey these environmental cues to the cell cycle machinery.
lian cells to identify CDK substrates. For example, a hunt for Finally, we will discuss the molecular mechanisms that gov-
substrates of the S phase CDK cyclin A-CDK2 revealed 180 ern initiation of DNA replication. We will describe how deg-
potential substrates. These are currently being analyzed for radation of an S phase CKI is essential for this process and
their functions in cell cycle processes. discover how CDKs ensure that DNA replication occurs only
during 5 phase and then only once.
,

KEY CONCEPTS of Section 19.3 Cells Are Irreversibly Committed to Cell Division
Regulation of CDK Activity
at a Cell Cycle Point Called START
In most eukaryotic cells, the key decision of whether or not
Cyclin-dependent kinases are activated by cyclin subunits.
a cell will divide is made at the point of whether or not to
Their activity is controlled at multiple levels.
enter 5 phase. In most cases, once a cell has. become commit-
Different cyclin subunits activate CDKs at different cell ted to entering the cell cycle, it must complete it. The bud-
cycle stages. Cyclins are present only in the cell cycle stages ding yeast Saccharomyces cerevisiae regulates its proliferation
that they promote. in this manner, and much of our current understanding of
Protein degradation is the key mechanism responsible for the molecular mechanisms controlling entry into the cell
restricting cyclins to the appropriate cell cycle stage. This cycle originated with genetic studies of S. cerevisiae.
degradation is mediated by the u biquitin-proteasome system When S. cerevisiae cells in G 1 have grown sufficiently in
and the ubiquitin ligases APC/C and SCF. size, they begin a program of gene expression that leads to
entry into the cell cycle. If G 1 cells are shifted from a rich
Activating and inhibitory phosphorylation on the CDK
medium to a medium low in nutrients before they reach a
subunit contributes to the regulation of CDK activity.
critical size, they remain in G 1 and grow slowly until they are
CDK inhibit9rs (CKls) inhibit CDK activity by directly large enough to enter the cell cycle. However, once G 1 cells
binding to the cyclin-CDK complex. reach the critical size, they become committed to completing
CDKs initiate every aspect of each cell cycle stage by phos- the cell cycle, entering 5 phase and proceeding through G 2
phorylating many different target proteins. Systematic efforts and mitosis, even if they are shifted to a medium low in nu-
using protein kinases engineered to bind only modified forms of trients. The point in late G 1 when S. cerevisiae cells become
ATP have led to the identification of many of these substrates. irrevocably committed to entering and traversing the entire
cell cycle is called START.
CDK activity is essential for entry into 5 phase. This was
first realized in budding yeast, where temperature-sensitive
19.4 Commitment to the Cell Cycle mutants in the gene encoding CDKl arrest in Gl> failing to
form a bud and to initiate DNA replication (CDKl is the
and DNA Replication only CDK in the budding yeast genome and is known as
The previous section described the multiple mechanisms that CDC28). We now know that a CDK cascade triggers entry
control the different cyclin-CDK complexes. In this and the into the cell cycle. G 1 CDKs stimulate the formation of GtfS
following two sections we will now examine each cell cycle phase CDKs, which then initiate bud formation, centrosome
stage carefully and discuss how a particular cell cycle stage is duplication, and DNA replication. In yeast, the G 1 cyclin
induced and controlled. We will examine how cells initiate gene is called CLN3 (Figure 1 9-15a). Its mRNA is produced
DNA replication and mitosis and how chromosomes are seg- at a nearly constant level throughout the cell cycle, but ItS
regated. We will focus on how cyclin-CDK complexes and translation is regulated in response to nutrient levels and, as
other key cell cycle regulators impact each cell cycle phase we will see shortly, CLN3 is a lynchpin in coupling cell cycle
and examine the mechanisms that coordinate their activities. entry to nutrient signals. Once sufficient Cln3 is synthesized
This section investigates how cells decide whether or not from its mRNA, Cln3-CDK complexes phosphorylate and
to undergo cell division and how DNA replication is initi- inactivate the transcriptional repressor Whi5. Phosphoryla-
ated. The process of cell cycle entry is well understood in tion of Whi5 promotes its export out of the nucleus, allowing

890 CHAPTER 19 The Eukaryotic Cell Cycle

.
,

(a) S. cerevisiae (b) Metazoans

...

- (Cin3-CDKs) - Nutrients - Growth factors

mRNA mRNA

CLN1 or CLN2 gene Cycl in E or cycl in A g en e

START START

Budding/
S phase
1~Spindle pole body
S phase
/ Centrosome
duplication
duplication
FIGURE 19-15 Control of the G,-S phase transition (a) In budding bud formation, and spindle pole body duplication. (b) In vertebrates,
yeast, Cln3-CDK activity rises during G, and is controlled by nutrient G1-CDK activity rises during G1 and is stimulated by the presence of
availability. Once sufficiently active, the kinase phosphorylates the growth factors. When signaling from mitogens is sustained, the resulting
transcriptional inhibitor WhiS, promoting its export from the nucleus. cyclin D-CDK4/6 complexes begin phosphorylating Rb, releasing some
This causes the transcription factor complex SBF to induce the transcrip- E2F, which stimulates transcription of the genes encoding cyclin E, CDK2,
tion of the G1/S phase cyclins CLN 1and CLN2 and of other genes whose and E2F itself. The cyclin E-CDK2 complexes further phosphorylate Rb,
products are needed for DNA replication. G1/S phase CDKs further resulting in a positive feedback loop that leads to a rapid rise in the
phosphorylate WhiS, promoting further CLN1 and CLN2 transcription. expression and activity of both E2F and cyclin E-CDK2. Once G1/ S phase
Once sufficiently high levels of G/S phase CDKs have been produced, CDK is sufficiently high, cells traverse START. They commence DNA
START is traversed. Cells enter the cell cycle: they initiate DNA replication, replication and centrosome duplication.

the transcription factor complex SBF to induce transcription CDKs activate members of a small family of related tran-
of the G 1/S phase cyclin genes CLNl and CLN2 as well as scription factors, referred to collectively as E2F transcription
other genes important for DNA replication. Once produced, factors (E2Fs). During Gt. E2Fs are held mactive through
Cln1/2-CDKs contribute to further Whi5 phosphorylation. their association with the retinoblastoma protein (Rb), and
This positive feedback loop ensures the rapid accumulation G 1 CDKs activate E2Fs by phosphorylating and inactivating
of G 1/S phase CDKs. Once a critical level of Clnl/2-CDKs is Rb. E2Fs then activate genes encoding many of the proteins
reached, these G/S phase CDKs promote bud formation, entry involved in DNA synthesis. They also stimulate transcription
into S phase, and the duplication of the centrosome (also of genes encoding the G 1/S phase cyclins and the S phase cy-
known as the spindle pole body, which later in the cell cycle clins. Thus the E2Fs function in late G 1 similarly to the S.
will organize the mitotic spindle). This state of G /S phase cerevisiae transcription factor complex SBF.
CDK activity, sufficient to initiateS phase, bud formation, and Key to the regulation of E2F function is the Rb protein.
centrosome duplication, is the molecular definition of START. When E2Fs are bound to Rb, they function as transcriptional
repressors. This is because Rb recruits chromatin-modifying
The E2F Transcription Factor and Its enzymes that promote deacetylation and methylation of spe-
cific histone lysines, causing chromatin to assume a condensed,
Regulator Rb Control the G1-S Phase
transcriptionally in;~ctive form. Rb was initially identified as
Transition in Metazoans the gene mutated in retinoblastoma, a childhood cancer of the
The molecular events governing entry into S phase in mam- retina. Subsequent studies found RB to be inactivated in al-
malian-and in fact all metazoan--cells are remarkably simi- most all cancer cells either by mutations in both alleles of RB
lar to those of budding yeast (Figure l9-15b). G 1 cyclins arc or by abnormal regulation of Rb phosphorylation.
present throughout G 1 and are often found to be expressed at Rb protein regulation by G 1 CDKs in mammalian cells is
increased levels in response to growth factors. In turn, the G 1 analogous to that of Cln3-CDK regulation of Whi5 in yeast.

19.4 Commitment to the Cell Cycle and DNA Replication 891

Phosphorylation on multiple sites by G 1 CDKs prevents Rb cascades that ultimately influence transcription and cell cycle
association with E2Fs and promotes its export out of the control. They do so in multiple ways.
nucleus. This allows E2Fs to activate transcription of genes Mitogens activate the transcription of multiple genes. Most
required for entry into S phase. Once the expression of genes of these genes fall into one of two classes-early-response or
coding for G 1/S cyclins and CDK are induced by phosphoryla- delayed-response genes-depending on how soon their en-
tion of some of the Rb molecules, the resulting G 1/S phase coded mRNAs appear. Transcription of early response genes is
CDK complexes further phosphorylate Rb in late G 1 This is induced within a few minutes after addition of growth factors
one of the principal biochemical events responsible for passage by signal transduction cascades that activate preexisting tran-
through START. Since E2F also stimulates its own expression scription factors in the cytosol or nucleus (see Chapter 16).
and that of the G 1/S cyclin-CDKs, positive cross-regulation of Many of the early-response genes encode transcription factors,
E2F and G 1/S cyclin-CDKs produces a rapid rise of both such as c-Fos and c-Jun, that stimulate transcription of the de-
activities in late G 1 layed response genes. The early response transcription factors
As they accumulate, S phase CDKs and mitotic CDKs AP-1 and Myc induce the transcription of genes encoding G 1
maintain Rb protein in the phosphorylated state throughout cyclins and CDKs. In addition to being controlled by transcrip-
the S, G 2, and early M phases. After cells complete anaphase tion of the genes encoding the G 1 cyclins, G 1 CDKs are regu-
and enter early G 1 or G 0, a fall in all cyclin-CDK activities lated by CKis. The CKI p15INK4b is a potent CDK inhibitor.
leads to dephosphorylation of Rb. As a consequence, hypo- In some tissues mitogens inhibit the production of this CKI by
phosphorylated Rb is available to inhibit E2F activity during inhibiting its transcription.
early G 1 of the next cycle and in G 0 -arrested cells. Thus G/S Cell proliferation in many tissues is not only regulated by
phase CDK activity remains low until cells decide to enter a proliferation promoting mitogens but also by anti-mitogens,
new cell cycle and G 1 CDKs break the inhibitory grasp of Rb which prevent entry into the cell cycle. Similarly, during dif-
over E2F. ferentiation, cells cease to divide and enter G 0 . Some differenti-
ated cells (e.g., fibroblasts and lymphocytes) cttn be stimulated
to re-enter the cycle and replicate. Many postmitotic differenti-
Extracellular Signals Govern Cell Cycle Entry ated cells, however, never re-enter the cell cycle to replicate
Whether or not cells enter the cell cycle is influenced by extra- again. Anti-mitogens and differentiation pathways prevent the
cellular as well as intracellular signals. Unicellular organisms accumulation of G 1 CDKs. They antagonize the production of
such as yeast, for example, enter the cell cycle only when they G 1 cyclins and induce the production of CKis. Transforming
have reached an appropriate size, known as the critical cell growth factor 13 (TGF-13) is an important anti-mitogen. This
size. This critical size, in turn, is controlled by nutrients avail- hormone induces a signaling cascade that brings about G 1 ar-
able in the environment. This coordination between cell size rest by inducing the expression of p15INK4b. As we will see in
and cell cycle entry will be discussed in Section 19. 7. Here we Chapter 24, the signaling pathways that regulate G 1 CDKs are
restrict our discussion to the fact that G 1 cyclin synthesis is found mutated in most human cancers.
responsive ro protein synthesis rate, which is in turn con-
trolled by pathways that are regulated by nutrients in the en-
Degradation of an S Phase CDK Inhibitor
vironment. This link between the macromolecule biosynthesis
and cell cycle machinery is best understood in budding yeast. Triggers DNA Replication
In this organism, the G 1 cyclin transcript CLNJ contains a Entry into S phase is defined by the unwinding of origins of
short upstream open-reading frame that inhibits translation DNA replication. The molecular events leading to this event
initiation at the Cln3 open reading frame when nutrients are are best understood in S. cerevisiae. G/S phase CDKs play
limited. This inhibition is diminished when nutrients are in an essential role in this process. They turn off the degrada-
abundance. In the presence of sufficient nutrients, the TOR tion machinery that degrades S phase cyclins during exit
pathway, which senses nutrients and growth factor signals, is from mitosis and G 1 and induce the degradation of a CKI
active and the pathway stimulates translational activity (see that inhibits S phase CDKs.
Figure 8-30). Since Cln3 is a highly unstable protein, its con- One of the important substrates of the G 1/S phase cyclin-
centration fluctuates with the translation rate of its mRNA. CDK complexes is Cdh l. During late anaphase, this substrate
Consequently, the amount and activity of Cln3-CDK com- targeting factor directs the APC/C to ubiquitinylate substrate
plexes, which depend on the concentration of Cln3 protein, is proteins including S phase and mitotic cyclins, marking them
largely regulated by nutrient levels. for proteolysis by proteasomes. The APC/C-Cdh 1 remains ac-
In multicellular organisms, cells are surrounded by nutri- tive throughout GI> preventing the premature accumulation of
ents, and as such, usually nutrients are not rate limiting for S phase and mitotiL cydin~. Phosphorylation of Cdhl by G 1/S
proliferation. Rather, cell proliferation is controlled by the cyclin-CDKs causes it to dissociate from the APC/C complex,
presence of growth-promoting factors (mitogens) and growth inhibiting further ubiquitinylation of S phase and mitotic cy-
inhibitory factors (anti-mitogens) in the cell surroundings. Ad- clins during late G 1 (Figure 19-16). This, combined with the
dition of mitogens to G 0-arrested mammalian cells induces- induced transcription of S phase cyclins during late Gl> allows
as we discussed in Chapter 16-receptor tyrosine-kinase-linked S phase cyclin proteins to accumulate as G 1/S cyclin-CDKs
signal transduction pathways that initiate signal transduction levels rise. Later in the cell cycle, S phase and mitotic CDKs

892 CHAPTER 19 The Eukaryotic Cell Cycle

Exit from mitosis S phase and mitosis and G 1/S phase CDK complexes, it functions as an S phase
and G 1 inhibitor. Initiation of DNA replication occurs when the
Sicl inhibitor is precipitously degraded following its ubiqui-
tinylation by the SCF ubiquitin-protein ligase.
G 1/S phase CDKs
Degradation of Sicl is induced by its phosphorylation by
G/S phase CDKs (see Figure 19-17). It must be phosphory-
Cdc14 phosphatase
lated at at least six sites, which are relatively poor substrates
for the G/S phase CDKs, before it is bound sufficiently well
by SCF to be ubiquitinylated. Multiple, puur G 1/S phase CDK
phosphorylation sites lead to an ultrasensitive, switch-like re-
S phase, mitotic S phase, mitotic sponse in Sicl degradation and hence precipitous activation
cyclins unstable cyclins stable
of S phase CDKs (Figure 19-18). If Sicl were inactivated fol-
FIGURE 19-16 Regulation of S phase and mitotic cyclin levels in lowing the phosphorylation of a single site, Sicl molecules
budding yeast. In late anaphase, t he anaphase-promoting complex would start to get phosphorylated as soon as the levels of
(APC/C) ubiquitinylates S phase and mitotic cyclins. APC/C activity is
G/5 phase CDK activity begin to rise, leading to a gradual
directed toward mitotic cyclins by a specificity factor, called Cdh 1. Cdh 1
decrease in Sicl levels. In contrast, when several sites need to
activity is regulated by phosphorylation. During exit from mitosis and
be phosphorylated, at low levels of G/S phase CDK activity
G1, the specificity factor is dephosphorylated and active; during S
phase and mitosis, Cdhl is phosphorylated and dissociates from the
only a few sites are phosphorylated and Sicl is not destroyed.
APC/C. and the ubiquitin ligase is inactive. The G1/S-phase CDKs, which Only when G 1/S phase CDK levels are high is Sicl sufficiently
themselves are not APC/C-Cdhl substrates, phosphorylate Cdhl at the phosphorylated at multiple sites to target it for degradation.
G1-S phase transition. A specific phosphatase called Cdc74 removes the Thus Sicl degradation occurs only when G 1/S phase CDK
regulatory phosphate from the specificity factor late in anaphase. activity has reached its peak and virtually all other G tiS phase
CDK substrates have been phosphorylated.
Once Sicl is degraded, the S phase cyclin-CDK complexes
take over to maintain Cdhl in the phosphorylated and hence induce DNA replication by, as we will see shortly, phosphor-
inactive state . Only as mitotic CDKs decline and a protein ylating several proteins involved in activating the replicative
phosphatase known as Cdc14 is activated are these inhibitory helicases. This mechanism for activating the S phase cyclin-
phosphates removed from Cdh 1, leading to its reactivation. In CDK complexes-that is, inhibiting them as the cyclins are
mammalian cells similar mechanisms are responsible for sta- synthesized and then precipitously degrading the inhibitor-
bilizing S phase and mitotic cyclins, but the phosphatase in- permits the sudden initiation of replication at large numbers
volved in dephosphorylation of Cdhl has not been identified. of replication origins. An obvious advantage of proteolysis
In S. cerevisiae, as the S phase cyclin-CDK heterodimers for controlling passage through this critical point in the cell
accumulate in late G 1 following the inactivation of APC/C- cycle is that protein degradation is an irreversible process,
Cdhl, they are immediately inactivated by binding of a CKI ensuring that cells proceed in o ne direction through the cycle.
called Sicl that is expressed late in mitosis and in early G 1 The dependence of this event on phosphorylation of multiple
(Figure 19-17). Because Sicl specifically inhibits S phase and poor phosphorylation sites makes the degradation of Sicl,
M phase CDK complexes but has no effect on the G 1 CDK and hence the activation of DNA replication, abrupt.

START

Polyubiquitinylation of
phosphorylated Sic1;
proteasomal

!
degradation DNA
-----~
0
replication
fJ

~
S phase

FIGURE 1917 Control of S phase onset inS. cerevisiae by ubiquitinylation by the SCF ubiquitin ligase and subsequent protea-
regulated proteolysis of the S phase inhibitor, Sicl. The S phase soma l degradation (step fJ). The activeS phase CDKs then trigger
cyclin-CDK complexes begin to accumulate in G 1 but are inhibited by initiation of DNA synthesis (step lll by phosphorylating and recruiting
Sicl. This inhibition prevents initiation of DNA replication until the cells MCM helicase activators to DNA replication origins. [Adapted from R. W.
have completed all G, events. G1/S phase CDKs assembled in late G1 King et at., 1996, Science 274:1652.]
phosphorylate Sicl at multiple sites (step 0 ), marking it for

19.4 Commitment to the Cell Cycle and DNA Replication 893

(a) One optimal G1/S phase CDK site in Sic1 then initiate S phase by phosphorylating proteins important
Sic1 G 1/S phase S phase for the initiation of DNA replication.
protein CDK activity CDK activity

Replication at Each Origin Is Initiated Once

~

0 and Only Once During the Cell Cycle

"'
Qi
> As discussed in Chapter 4, eukaryotic chromosomes are rep-
~ licated from multiple replication origins. Initiation of repli-
c
cation from Lhe~e origins occurs th rougho ut S phase.
0"' However, no eu karyotic o rigin initiates more than once per
a':
S phase. Moreover, S phase continues until replication from
G, multiple origins along the length of each chromosome results
in complete replication of the entire chromosome. These two
(b) Six sub-optimal G 1/S phase CDK site in Sic1
factors ensure that the correct gene copy number is main-
Sic1 G 1/S phase S phase tained as cells proliferate.
protein CDK activity CDK activity
S phase CDKs play an essential role in the regula tion of
DNA replication. The kinases initiate DNA replication only
~
0
at the G 1-S phase transition and prevent re-initiation from
"' origins that have already fired. We will first discuss how ini-
Qi
~
> tiation of DNA replication is controlled and the role of S
c phase CDKs in the process before turning to the mechanisms
'iii
0 whereby these kinases prevent re-initiation.
a': The mechanisms underlying the initiation of DNA replica-
tion are best understood in budding yeast; so we will focus
G 1 ~ S phase our discussion on this organism. H owever, it is important to
FIGURE 19-18 Six suboptimal G1 /S phase CDK phosphorylation
note that the proteins and mechanisms controlling the initia-
sites in Sic1 create a switch-like cell cycle entry. (a) A single optimal
tion of DNA synthesis are essentially the same in all eukary-
G/S phase CDK site in Sicl would result in a sluggish G1-S phase
transition. As G1/S phase CDKs accumulate during G1, Sicl would get
otic species. A protein complex known as the origin-recognition
progressively degraded. As a result, S phase CDKs would slowly rise.
complex (ORC) is associated with all DNA replication
Instead of an abrupt rise in S phase CDK activity, initiation of S phase origins. In budding yeast, replication origins contain an
would be a drawn-out event. (b) Six suboptimal phosphorylation sites 11-base-pair conserved core sequence to w hich ORC binds. In
ensure that Sicl is only fully phosphorylated and hence recognized by multicellular o rganisms, origins of DNA replication lack a rec-
the SCF when G/S phase CDKs have reached high levels. This also ognizable consensus sequence. Instead, chromatin-associated
ensures that Sicl degradation occurs rapidly and when G/S phase factors target ORCs to the DNA. ORC and two add iti onal
CDKs have accomplished all their other G1 tasks. [Adapted from Nash replication initiation factors, Cdc6 and Cdtl , associate with
et al., 2001 , Nature 414:514-521, and D. 0. Morgan, 2006.] the ORC at origins during G 1 to load the replicative helicases
known as the MCM helicase complex onto DNA (Figure
Entry into S phase in metazoan cells is regulated in a 19-19, step 0 ). The MCM helicases fu nction to unwind the
similar manner as in budding yeast. Like Sicl, the CKI p2 7 DNA during the initiation of DNA replication.
prevents the premature activation of S phase CDKs during To ensure that origins fire only once at the beginning of S
G 1. Unlike Sicl, however, this CKI inhibits both S phase phase, MCM helicase complex loading and its activation occur
CDKs and G 1/S phase CDKs and has additi onal cell cycle at two opposing phosphorylation states. The MCM helicases
functions. For example, while inhibiting G /S phase CDKs can only load onto the DNA in a state of low CDK activity
and S phase CDKs, p27 helps assemble and hence activate G 1 that occurs when CDKs are inactivated during exit from mito-
.. '
CDKs. Similar to the situation in yeast, however, p27 is re- sis and during early G 1 In other words, MCM helicases load
moved from cyclin-CDK complexes by ubiquitin-dependent onto DNA when they are unphosphorylated . In contrast, acti-
protein degradation. Two pathways contribute to p27 degra- vation of MCM helicases and the recruitment of DNA poly-
dation. On stimulation with mitogens, mitogen-activated merases to the unwound origin DNA are triggered by S phase
protein kinases phosphorylate p27, promoting its export CDKs. Recall that S phase CDKs only become active when
from the nucleus into the cytoplasm, where one of the cellu- G 1/S phase CDK levels reach their peak and CKis of S phase
lar ubiquitin ligases, KPC, is found. A second pathway, anal- CDKs are destroyed. It is then that phosphorylation by S phase
ogous to the one operating on Sicl, targets p2 7 for CDKs and a second heterodimeric protein kinase, DDK, acti-
degradation at the G 1-S phase transition. As G 1/S phase vates the MCM helicase and recruits the polymcrases to the
CDKs and S phase CDKs reach high levels during late G 1 and sites of replication initiation (Figure 19-19, steps fJ and 10).
early S phase, they begin to phosphorylate p27, targeting the So how do S phase CDKs and DDK collaborate to initi-
CKI for ubiquitinylation by SCF. Degradation of p27 causes ate DNA replication? ORC and the two other initiation fac-
activation of G /S phase and S phase CDKs. These kinases tors, Cdc6 and Cdt1, recruit the MCM helicases to sites of

894 CHAPTER 19 The Eukaryotic Cell Cycle

 - ~~ -- ----~==----===

~~; "'ff~..<'.<'.'<'.ff..'f'.<'...'<:
[ .

state
MCM helicase

MCM helicase S phase CDK

activat ion
DD~ oJ~
~~~

.
Polymerase loading ~
and replication

<!)

FIGURE 19-1 9 The molecular mechanisms governing the DNA. S phase CDKs also prevent reloading of MCM helicases by
initiation of DNA replication. Step 0 : During exit from mitosis and phosphorylating the pre-RC components Cdc6 and Cdtl (shown as
early G~< when CDK activity is low, the MCM loading factors ORC, Cdc6, yellow phosphorylation events), promoting their release from origins
and Cdtl load the replicative helicase, the MCM complex, onto origin and degradation by the SCF. S phase CDKs also phosphorylate MCM
DNA. Step f) : Activation of S phase CDKs and DDK mark the onset of helicases, which leads to their export from the nucleus when the
S phase. They pho5phorylate the MCM helicase, Sld2 and Sld3 helicases disengage from the DNA when replication is complete.
(depicted as green phosphorylation events), to facilitate the loading of Step il: DNA polymerases are recruited to origins, which leads to the
MCM helicase activators-the Cdc45-Sid3 and the GINS complexes- initiation of DNA synthesis (see Figure 4-31 ).
onto sites of replication initiation. This leads MCM helicases to unwind

replication initiation during Gh when CDK activity is low (see S phase CDKs are not only essential to initiate DNA repli-
Figure 19-19, step 0 ). When DDK and S phase CDKs are ac- cation, they are also responsible fo r ensuring that each origin
tivated in late G" DDK phosphorylates two subunits of the fires only once during S phase. Preventing re-firing of origins
MCM helicase. The S phase CDKs phosphorylate two proteins during S phase is brought about by phosphorylation of several
called Sld2 and Sld3. T hese phosphorylation events have an components of the MCM helicase loading machinery and the
activating effect, promoting the recruitment of MCM helicase MCM helicase itself. To distinguish these phosphorylation
activators to sites of replication initiation (the phosphorylation events from the ones required for the initiation of DNA replica-
events shown in green in Figure 19-19, steps 111 and II). The tion, they are depicted in yellow in Figure 19-19. Concomitant
helicase activators are called the Cdc45-Sld3 complex and the with activation of the MCM helicase, Cdc6 and Cdtl dissoci-
GINS com plex. Exactly how they promote activation of the ate from the sites of DNA replication initiation. Once they dis-
MCM helicases is not yet clear. In addition to activating the sociate, their phosphorylation leads to their degradation by the
MCM helicase to unwind DNA, the Cdc45-Sld3 complex and SCF ubiquitin ligase. Phosphorylation of the MCM helicase
the GINS complex recruit polymerases to the DNA, poly- leads to the export of these proteins from the nucleus after they
merase to synthesize the leading strand and polymerase 8 to dissociate from the DNA on completion of DNA replication.
synthesize the lagging strand (see Figure 19-19, step II). The Thus only after CDK activity is lowered by APC/C-Cdhl dur-
replication machinery then initiates DNA synthesis. ing exit from mitosis can the MCM helicases be reloaded onto

19.4 Commitment to the Cell Cycle and DNA Replication 895

DNA. As a result, hclicasc loading is restricted to late stages of likely this occurs as replication forks replicate the DNA (fig-
mitosis and early G 1 (sec figure 19-19, step 0). ure 19-20, step f)). Converting DNA-bound G 1 cohesins into
The general mechanisms governing the initiation of DNA cohesive complexes requires several cohesin loading fac-
replication in metazoan cells parallel those inS. cereL'isiae, tors-including a protein complex related to proteins that
although small differences are found in vertebrates. Phosphor- load the sliding clamp at the replication fork-and the acety-
ylation of MCM helicase activators by G 1/S phase CDKs and lation of the Smc3 subunit. As we will see in Section 19.6,
S phase CDKs likely promotes their recruitment to sites of cohesins are essential to accurately attach the replicated sister
DNA replication initiation. As in yeast, phosphorylation of chromatids onto the mitotic spindle and for their segregation
MCM loadmg factors likely prevent~ reloading of MCM heli- during mitosis. Cells lacking cohesins or the factors that load
cases until the cell passes through mitosis, thereby ensuring them onto chromosomes segregate chromosomes randomly.
that replication from each origin occurs only once during each
cell cycle. In metazoans, a second small protein, geminin, con-
tributes to the inhibition of re-initiation at origins until cells KEY CONCEPTS of Section 19.4
complete a full cell cycle. Geminin is expressed in late G 1; it
Commitment to the Cell Cycle and DNA Replication
binds to and inhibits MCM helicase loading factors as they
are released from origins once DNA replication is initiated START defines a stage in G 1 after which cells are irrevers-
during S phase (see Figure 19-19, step f)), contributing to the ibly committed to the cell cycle. Molecularly, it is defined as
inhibition of re-initiation at an origin. Geminin contains a de- the point when GtfS phase CDK activity reaches levels suf-
struction box at its N-terminus that is recognized by the APC/ ficient to initiate S phase.
C-Cdh 1, causing it to be ubiquitinylated in late anaphase and The molecular events promoting entry into the cell cycle
degraded by proteasomes. This frees the MCM helicase load- are conserved across species. G 1 CDKs inhibit a transcrip-
ing factors, which are also dephosphorylated as CDK activity tional inhibitor. This allows the transcription of G 1/S phase
declines, to bind to ORC on replication origins and load cyclins and other genes important for S phase.
MCM helicases during the following G 1 phase.
Extracellular signals such as nutritional state (in yeast)
and the presence of mitogens and anti-mitogens (in verte-
brates) regulate entry into the cell cycle.
Duplicated DNA Strands Become
Various polypeptide growth factors called mitogens stim-
Linked During Replication
ulate cultured mammalian cells to proliferate by inducing
During S phase, as chromosomes are duplicated to form sis- expression of early-response genes. Many of these encode
ter chromatids, they become tethered to each other by pro- transcription factors that stimulate expression of delayed re-
tein links. The linkages between sister chromatids established sponse genes encoding the GtfS phase cyclins and E2f tran-
during S phase will be essential for their accurate segregation scription factors.
during mitosis.
The G 1/S phase CDKs phosphorylate and inhibit Cdh 1,
The protein ci>mplexes that establish cohesion between sis-
the specificity factor that directs the anaphase-promoting
ter chromatids are called cohesins. The cohesin complex is
complex (APC/C) to ubiquitinylate S phase and M phase cy-
composed of four subunits: Smcl (sometimes also called
clins. This allows S phase cyclins to accumulate in late G 1
Rad21), Smc3, Scc1, and Sed. Smcl and Smd are members
of the SMC protein family, which is characterized by long In yeast, S phase CDKs initially are inhibited by Sicl.
coiled-coil domains that are flanked by a globular domain con- Phosphorylation marks Sic I for ubiquitinylation by the SCF
taining ATPase activity. The ATPase domains interact with ubiquitin-protein ligase and proteosomal degradation, re-
Sec I and Sed and, together, form a ring structure. The struc- leasing activated S phase CDKs that trigger onset of the S
tural mechanism by which cohesins link sister chromatids to- phase (see Figure 19-17).
gether is not understood, but it is likely that rings of cohesin DNA replication is initiated from helicase loading sites
embrace one or both coptes of the replicated DNA. However, known as origins of replication.
it is clear that cohesins are essential to hold the replicated DNA
Loading and activation of the MCM helicase occur in mu-
molecules together. When Xenotms egg extracts were depleted
tually exclusive cell cycle states: MCM helicasc loading can
of cohesin by treatment with antibodies specific for the cohesin
only occur when CDK activity is low (during early Gd;
SMC proteins, the depleted extracts were able to replicate the
MCM helicases are activated when CDK activity is high.
DNA in added sperm nuclei, but the resulting sister chromatids
did not associate properly with each other. S phase CDKs and DDK trigger the initiation of DNA rep-
Some details of how cohcsins arc loaded onto DNA to lication by the recruitment of MCM hclicase activators to
mediate sister chromatid cohesion are being unraveled. We origins (see Figure 1 9-19).
know that establishment of cohesion between sister chroma- Initiation of DNA replication occurs at each origin only
tids is tightly tied to DNA replication. Cohesins associate once during the cell cycle. This is achieved because S phase
with chromosomes during G 1 (Figure 19-20, step 0). During CDKs activate the helicases and at the same time prevent
DNA replication, they arc loaded onto chromosomes in such additional helicases from loading onto DNA.
a way that they can hold sister chromatids together. Most

896 CHAPTER 19 The Eukaryotic Cell Cycle

 Cohesins Sister chromatids
~

~ ~
~
~
~
m pliooHoo
D fJ fork
~ II
" Polo kinase

B
Aurora B kinase

Centromere
/

r G,
BJ

S phase Prophase
FIGURE 19-20 Model for establishing cohesin linkage of sister encircling the replicated sister chromatids). This conversion into
chromatids. There is strong evidence that the cohesin complex is cohesive cohesins requires cohesin loading factors. During G2, sister
circular, but it is not known whether a single cohesin ring links sister chromatids are replicated and linked along their entire length by
chromatids or whether two rings, one each around the separate sister cohesins. During this time, the Mei-5332/Sgo proteins recruit the
chromatids, are linked to each other like links in a chain. For simplicity, protein phosphatase 2A (PP2A) to centromeric regions. Step iJ: In
only one ring is shown here. Step 0 : Cohesins are loaded onto vertebrate cells, cohesins are released from chromosome arms during
chromosomes during G,, but they do not possess cohesive properties prophase and early metaphase by the action of Polo kinase and Aurora
(indicated as cohesins laterally associated with chromosomes). Step f): B kinase. By the end of metaphase, cohesins are retained only in the
Concomitant with DNA replication, and most likely closely behind the region of the centromere, where PP2A prevents cohesin phosphoryla-
replication fork, cohesins are converted into cohesive molecules, able tion and hence dissociation through recruiting PP2A.
to hold sister chromatids together (indicated as cohesin rings

rein kinases bring about the dramatic changes in the cell nec-
Cohesins establish linkages between the replicated DNA essary to facilitate sister chromatid segregation during
molecules. This linking mechanism is coupled to DNA anaphase, focusing on the events as they occur in metazoans.
replication.

Precipitous Activation of Mitotic

CDKs Initiates Mitosis
19.5 Entry into Mitosis Mitotic CDKs initiate mitosis. Whereas levels of the catalytic
Once S phase has been completed and the entire genome has CDK subunit are constant throughout the cell cycle, mitotic
been duplicated, the pairs of duplicated DNA chromosomes- cyclins gradually accumulate during S phase. Most eukaryotes
the sister chromatids-are segregated to the future daughter contain multiple mitotic cyclins, which for historical reasons
cells. This process requires not only the formation of the are called cyclin A and cyclin B. As they assemble, mitotic
apparatus that faci litates this segregation-the mitotic CDK complexes are maintained in an inactive state through
spindle-but essentially a complete remodeling of the cell. inhibitory phosphorylation on the CDK subunit. Recall from
Chromosomes condense and attach to the mitotic spindle, Section 19-3 that two highly conserved tyrosine and threonine
the nuclear envelope is disassembled, and almost all organ- residues in mammalian CDKs are subject to regulated phos-
elles are rebuilt or modified. All these events are triggered by phorylation. In CDKl, the mitotic CDK, phosphorylation of
mitotic CDKs. This section will first discuss how the mitotic tyrosine 15 and threonine 14 maintains mitotic cyclin-CDK
CDKs are precipitously activated after the completion of complexes in an inactivate state. The phosphorylation state of
DNA replication, during G2 We will then see how these pro- T L4 and Y15 is controlled by a dual-specificity protein kinase

19.5 Entry into Mitosis 897

 Mitotic cyclin Vertebrates contain multiple Weel protein kinases and
?: multiple Cdc25 phosphatases that collaborate to control not
:~
-
t/)
Q) ...
<.) only mitotic CDK activity but also the activity of G 1/S phase
> <tl
~~ CDKs. One member of the Cdc25 family of phosphatases,
cO
:.=u Cdc25A, is activated in late G 1 It rem oves the inhibitory
<.) '
>C
u:.= <.)
phosphorylation on tyrosine 15 of the G/S phase CDKs and S
>
<.) phase CDK catalytic subunit to activate the kinases. Another
family member, Cdc25C, is active during G 2 and removes
Mitosis the inhibitory phosphorylation on mitotic CDKs.
Activation of mitotic CDKs is the consequence of rapid
inactivation of Wee] and activation of Cdc25. Central to this
rapid transition are feedback loops, where mitotic CDKs acti-
vate Cdc25 and inactivate Wee 1 (see Figure 19-21 ). Phosphor-
ylation of Cdc25 by mitotic CDKs stimulates its phosphatase
activiry; phosphorylation ofWeel by mitotic CDKs inhibits its
Y15 T" kinase activity. Ongoing DNA replication inhibits Cdc25 ac-
tivity. A critical question that we know little about is how this

Inactive Active
positive feedback loop is started once DNA replication has
been completed. CDKs that function ea rlier in the cell cycle
FIGURE 19-21 Phosphorylation on the CDK subunit restrains have been suggested to start the positive feedback loop.
mitotic CDK activity during S phase and G2 Mitotic cyclins are Although it is not yet known how the precipitous activa-
synthesized during 5 phase and G2 and bind to CDK1. However, the tion of mitotic CDKs is initiated, it is clear that once active,
cyclin-CDK complex is not active because threonine 14 and tyrosine these protein kinases set in motion all the events necessary to
15 of the CDK1 subunit are phosphorylated by the protein kinase Wee1. ready the cell for chromosome segregation. The activation of
Once DNA replication has been completed, the protein phosphatase mitotic CDKs is associated with changes in the subcellular
Cdc25 is activated, and the phosphatase dephosphorylates CDK1. localization of these kinases. Mitotic CDKs initially associate
Active mitotic CDKs further stimulate Cdc25. At the same time, mitotic with centrosomes, where they are thought ro facilitate centro-
CDKs inhibit Wee 1, the protein kinase that places the inhibitory
some maturation. They then enter the nucleus, where they
phosphorylation on the CDK subunit. Ongoing DNA replication inhibits
bring about chromosome condensation and nuclear envelope
Cdc25 activity. How Cdc25 is initially activated upon completion of DNA
breakdown. In what follows we will discuss how the mitotic
replication to put in motion these feedback loops is not yet known.
CDKs accomplish the coordinated execution of mitosis.
During DNA replication initiation, S phase CDKs work to-
known as Weel and a dual-specificity phosphatase Cdc25 gether with DDK to promote MCM helicase activation. As in
(Figure 19-21). !he regulation of mitotic CDKs by these ac- the initiation of DNA replication, CDKs collaborate with other
tivities underlies the abrupt activation of their kinase activiry at protein kinases to bring about the mitotic events. The Polo ki-
the GrM phase transition and explains the observation that nase family is critical for formation of the mitotic spindle as
although mitotic cyclins gradually accumulate during S phase well as chromosome segregation. The family of Aurora kinases
and G 2, mitotic CDKs are not active until cells enter mitosis. plays key roles in mitotic spindle formation and ensuring that
Studies in the fission yeast Schizosaccharornyces pornbe chromosomes attach to the mitotic spindle in the correct way so
unraveled the mechanisms that lead to the precipitous acti- that they are segregated accurately during mitosis. Their contri-
vation of mitotic CDKs during G 2 . The dual-specificity pro- bution to the various mitotic events will also be discussed.
tein kinase Weel phosphorylates CDKs on the inhibitory
tyrosine 15. (Threonine 14 is not phosphorylated inS. pornbe
Mitotic CDKs Promote Nuclear
CDK 1.) Yeast cells with a defective weel gene activate mitotic
CDKs prematurely and hence experience premature entry Envelope Breakdown
into mitosis. Weel mutants not only enter mitosis prema- During interphase, chromosomes are surrounded by the nu-
turely but are also smaller. This is because unlike most other clear envelope. The centrosomes that form the mitotic spin-
cukaryotes, which coordinate cell size and cell division dur- dle are located in the cytoplasm. For chromosomes to
ing Gh this coordination occurs during G 2 in fission yeast. interact with the microtubules nucleated by the centrosomes,
Fission yeast cells carrying a mutant CDK 1 gene in which the nuclear envelope needs to be dismantled.
the tyrosine 15 residue is replaced hy phenylalanine (phenyl- The nuclear envelope is a double-membrane extension of
alanine is structurally similar to tyrosine but cannot be phos- the endoplasmic reticulum containing many nuclear pore
phorylated) show the same premature mitotic CDK complexes (see Figures 9-32 and 13-33). The lipid bilayer of
activation and entry into mitosis. The phosphatase that op- the inner nuclear membrane is associated with the nuclear
poses Wee 1 is Cdc25. Fission yeast cells carrying mutations lamina, a meshwork of lamin filaments adjacent to the inside
in the cdc2S gene arrest in G 2 , indicating that the phospha- face of the nuclear envelope (Figure 19-22a). The three
tase is essential for entry into mitosis. nuclear lamins (A, B, and C) present in vertebrate cells be-

898 CHAPTER 19 The Eukaryotic Cell Cycle

 long to a class of cytoskeletal proteins, the intermediate fila-
ments, that are critical in supporting cellular membranes.
Lamins A and C, which are encoded by the same transcnp-
tion unit and produced by alternative splicing of a single pre-
mRNA, are identical except for a 133-residue regwn at the
C-terminus of laminA, which is absent in lamin C. Lamin B,
encoded by a different transcription unit, is modified post-
transcriptionally by the addition of a hydrophobic isoprenyl
group near its carbo.>.yl terminus. This fatty acid becomes
embedded in the inner nuclear membrane, thereby ancho ring
the nuclear lamina to the membrane (see Figure 10-19). All
three nuclear lamins form dimers containing a rod-like a-
helical coiled-coil central section and globular head and tail
domains; polymerization of these dimers through head-to-
head and tail-to-tail associations generates the intermediate
filaments that compose the nuclear lamina (Figure 19-22b).
Once mitotic CDKs are activated at the end of G 2 , they
phosphorylate specific serine residues in all three nuclear lam ins.
This causes depolymerization of the lamin intermediate fila-
ments (see Figure 19-22b). The phosphorylated laminA and C
dimers are released into solution, whereas the phosphorylated
lamin B dimers remain associated with the nuclear membrane
via their isoprenyl anchor. Depolymerization of the nuclear lam-
ins leads to disintegration of the nuclear lamina meshwork and
contributes to disassembly of the nuclear env~lope.
Mitotic CDKs also affect other nuclear envelope compo-
nents (Figure 19-23). The CDKs phosphorylate specific
nu cleoporins, which causes nuclear pore complexes to dis-
sociate into subcomplexes during prophase. Phosphoryla-
tion of integral membrane proteins of the inner nuclear
Lamin tetramer membrane is thought to decrease their affinity for chromatin

1 MPF
and further contributes to the disassembl y of the nuclear
envelope. The weakening of the associations between the
inner nuclear membrane proteins and the nuclear lamina

... c~ cc and chromatin allows sheets of inner nuclear membrane to

retract into the endoplasmic reticulum, which is continuous
with the outer nuclear membrane.

Phosphorylated
lamin dimers Mitotic CDKs Promote Mitotic Spindle Formation
A key function of mitotic CDKs is to induce the formation of
FIGURE 19-22 The nuclear lamina and its regulation by the mitotic spindle, also known as the mitotic apparatus. As
phosphorylation. (a) Electro11 micrograph of the nuclear lamina we saw in Chapter 18, the mitotic spindle is made of micro-
from a Xenopus oocyte. Note the regular mesh-like network of Ia min tubules that attach to chromosomes via specialized protein
intermediate filaments. This structure lies adjacent to the inner structures associated with chromosomes known as kincto-
nuclear membrane (see Figure 18-46). (b) Schematic diagrams of
chores. In most organisms, the mitotic spindle is organized
the structure of the nuclear lamina. Two perpendicular sets of
by centrosomes, sometimes called spindle pole bodies. They
10-nm-diameter filaments built of lamins A, B. and C form the nuclear
contain a specialized tubulin, -y tubulin, which together with
lamina (top). lndividuallamin filaments are formed by end-to-end
polymerization of Ia min tetramers, which consist of two Iamin
associated proteins nucleates microtubules. Notable excep-
coiled-coi l dimers (middle). The red and blue circles represent the tions to centrosome-based spindle assembly mechamsms are
' globular N-tPrminal and (-terminal domains, respectively. Phosphor- higher plants and metazoan oocytes. In these cells, (- ) end~
ylation of specific serine residues near the ends of the rod-like central of microtubules are cross-linked and the microtubules self-
section of Ia min dimers causes the tetramers to depolymerize assemble into a spindle.
(botrom). As a result, the nuclear lamina disintegrates. [Part (a) from U. The function of the mitotic spindle is to segregate chromo
Aebi et al., 1986, Nature 323:560; courtesy of U. Aebi; part (b) adapted from A. somes so that the sister chromatids separate from each other
Murray and T. Hunt, 1993, The Cell Cycle: An Introduction, W. H. Freeman and and are moved to opposite poles of the mitotic spi ndle (see Fig-
Company.] ure 18-37). To achieve this, chromosomes have to attach to the

19.5 Entry into Mitosis 899

 Mitotic spindle formation begins at the G 1-S phase transi-
tion with the duplication of ccn trosomes. The mechanism
whereby this duplication occurs is poorly understood, but at
the heart of this process is the duplication of the pair of cen-
trioles, sho rt microtubules arranged orthogonally to each
other. As discussed in Chapter 18, G 1 cells contain a single
pair of centrioles. Concomitant wi th entry into S phase and
triggered by the G 1/S phase CDKs, the two centrioles split
Cytoplasm
apart, and each centnole begins to grow a daughter centriole
/ fJINM . (sec Figure 18-35). The new centrioles grow and mature dur-
protems
ing S phase, each centriole pair begins to assemble centro-
somal material, and by G 2 the two centrosomes have formed.
Several additional protein kinases have been identified that
control centrosome dup lication . Chief among them is a
member of the conserved Polo kinase family, Plk4 . How
G 1/S phase CDKs and Plk4 promote centrosome duplication
is not yet understood but is thought to involve the phos-
phorylation of multiple centrosome components, which fa-
cilitates their duplication and growth. As we will see, the
Polo kinases not only play a key role in centrosome duplica-
FIGURE 19-23 Nuclear envelope proteins phosphorylated by
tion but also participate in essentially all aspects of mitosis.
mitotic CDKs. Step 0: Components of the nuclear pore complex (NPC)
The key initiating step of mitotic spindle formation is the
are phosphorylated by mitotic CDKs in prophase, causing NPCs to
dissociate into soluble and membrane-associated NPC subcomplexes.
severing of the ties that link the duplicated c~ntrosomcs. This
Step fJ: Mitotic CDK phosphorylation of inner nuclear membrane (INM) centrosome disjunction occurs in G 2 and is triggered by
proteins inhibits their interactions with the nuclear lamina and mitotic CDKs (see Figure 18-35). As soon as this separation
chromatin. Step 0 :Mitotic CDK phosphorylation of nuclear lam ins occurs, microtubules arc nucleated from both centrosomes
causes their depolymerization and dissolution of the nuclear lamina. and they move away from each other, pulled by the motor
Step B : Mitotic CDK phosphorylation of chromatin proteins induces protein dynein . The specifics of microtubule array formation
chromatin condensation and inhibits interactions between chromatin and mitotic spindle assembly were discussed in Chapter 18.
and the nuclear envelope. [Adapted from B. Burke and J. Ellenberg, 2002, Here we will briefly consider how chromosomes attach to the
Nat. Rev. Mol. Cell Bioi. 3:487.] mitotic spindle and how mistakes in the process are corrected.
For chromosomes to be accurately segregated during mi-
mitotic spindle so that one kinetochore of each sister chromatid tosis, the sister chromatid pair has to be stably hi-oriented on
pair attaches to ll)icrotubules emanating from opposite poles. the mitotic spindle (Figure 19-24). How is this accomplished?
Sister chromatids are then said to be hi-oriented. In what fol- Once centrosomes have moved apart from each other, micro-
lows, we will discuss how the mitotic spindle forms, how chro- tubules, in a search-and-capture mechanism, begin to interact
mosomes attach to it, and how cells correct faulty attachments. wi th kinetochores of sister chromatid pairs. Initially, chro-
During Gh cells contain a single centrosome that func- mosomes glide along the length of microtuhules propelled by
tions as the major microtubule nucleating center of the cell. motor proteins. When the chromosome reaches the end of a

0 VIDEO: Normal Cell Division in C. elegans Embryogenesis

FIGURE 19-24 Chromosome attachment to the mitotic spindle.

Chromosomes attach to the mitotic spindle and congress to the
spindle center. They then attach via their kinetochores to the ends of
microtubules (called end-on attachments), and these attachments are
stabilized by additional microtubules. The final chromosome attach-
ment where the chromosome has stably bi-oriented on the mitotic
spindle is shown.

900 CHAPTER 19 The Eukaryotic Cell Cycle .

~ VIDEO: Abnormal Chromosome Segregation in C. elegans Embryo in the Absence of Kinetochore Protein KNL-3

FIGURE 19-25 Stable and unstable chromosome attachments.

(a) Amphitelic attachment (b) Merotelic attachment
When sister kinetochores attach to microtubules emanating from
opposite spindle poles, they are stably attached. This configuration
is called amphitelic attachment (a). Microtubules (green) pull
~Cohesins
kinetochores (tan); cohesins resist this pulling force. This leads to J Aurora B

~ti ~~\~
kinetochores being pulled away from the protein kinase Aurora B (red
zone), which localizes to the inter sister kinetochore space. As a result,
Aurora ~can no longer phosphorylate the microtubule binding factors Microtubules
at the kinetochore and kinetochore-microtubule attachments are
stable. When one kinetochore attaches to microtubules emanating
from two opposite spind le poles (merotelic attachment, b), or both
v
sister kinetochores attach to microtubules emanating from the same Sister chromatids
spindle pole (syntelic attachment, c), or only one of the two sister (d) Monotelic attachment
(c) Syntelic attachment
kinetochores attaches to microtubules (monotelic attachment, d),
kinetochores are not pulled out of the Aurora B zone. As a result,
Aurora B phosphorylates the microtubule binding subunits of the
kinetochore and microtubule attachments are destabilized.

microtubule, the kinetochores attach to microtubules in an

end-on attachment, the final configuration in which chromo-
somes are linked to the mitotic spindle. Kinetochores of sister
chromatids then bind microrubules emanating from the op-
posite spindle pole. In the end, the sister chromatid pair is
said to be stably bi-oriented on the mitotic spindle.
The ultimate goal of chromosome attachment to the mi- second chance to get the attachment right. The molecular basis
totic spindle is that each and every chromosome is attached to for this sensing mechanism is partly understood. Aurora B
the mitotic spindle in a bi-orientcd manner (also known as phosphorylates a number of kinetochore components involved
amphitelic attachment; Figure 19-25a). How does the cell in microtubule binding. When phosphorylated, these proteins
"know" that this has occurred? Microscopic analysis of lose their microtubule-binding activity. Aurora B localizes to
chromosome attachment has shown that initially many chro- the region of sister chromatids between the two kinetochores.
mosomes attach to microtubules in faulty ways. A kinetochore When kinetochores are not under tension, they are in close
can attach to microtubules emanating from both poles, a situ- proximity to Aurora Band the protein kinase can phosphory-
ation called merotelic attachment (Figure 19-25b). Kineto- late the microtubule-binding subunits of the kinetochores, de-
chores of a sister chromatid pair can attach to microtubules stabilizing any kinetochore-microtubule attachments (see
from the same pole (syntelic attachment; Figure 19-25c) or Figure 19-25h-d). When microrubules are attached correctly
only one kinetochore can attach to microtubules (monotelic to kinetochores, microtubule forces pull kinctochores away
attachment; Figure 19-25d). Clearly, none of these attach- from Aurora Band the kinase can no longer reach its targets at
ments are productive in the sense that they would not result in kinetochores (see Figure 19-25a).
accurate chromosome segregation. Thus mechanisms must be Microtubules continuously pull on chromosomes. Once all
in place that detect and correct such faulty attachments. chromosomes have attached to microtubules in an amphitelic
The sensing mechanism used by cells to detect incorrect manner, the only things that prevent chromosomes from segre-
attachments is based on tension. When sister chromatids arc gating to the poles and that hold them back in the middle of
correctly attached to microrubulcs, their kinetochores are the spindle are the cohesin complexes (see Figure 19-25a). As
under tension (see Figure 19-25a). Microtubules attached to we will see in Section 19.6, it is the severing of these cohesin
the kinetochores pull at them and the cohesin molecules that molecules that initiates anaphase chromosome segregation.
hold the sister chromatids together withstand these forces,
creating tension at kinctochores. Merotelic, syntelic, or
Chromosome Condensation Facilitates
monotelic attachment leads to insufficient tension at kineto-
chores, allowing the cell to distinguish these faulty forms of Chromosome Segregation
attachment from the correct amphitelic one. Chromosome segregation not only requires the building of
How does the cell sense w hether or not kinctochores are the apparatus that segregates chromosomes, it also requires
under tension? The protein kinase Aurora B and its associated that the DNA is compacted into travel-friendly structures.
regulatory factors, together known as the chromosomal pas- An attempt to segregate the long and intertwined DNA-
senger complex (CPC), sense kinetochores that are not under protein complexes present in interphase cells would lead to
tension and sever these microtubule attachments, giving cells a breakage of the DNA and hence loss of genetic material. To

19.5 Entry mto Mitosis 901

 known, but it appears that mitotic CDKs induce the process at
least in part through activating condensins. Two of the non-SMC
condensin su bunits are targets of mitotic CDKs. Their phosphor-
ylation stimulates condensins to supercoil DNA in vitro.
Finally, processes acting in parallel to condensins also
contribute to chromosome compaction. The dissociation of
cohesins from chromosomes is such a parallel process. A
large fraction of cohesins is removed from chromosomes
during prophase (see Figure 19-20, step lJ). This process is
mediated by phosphorylation of cohesins by Polo kinase and
Aurora B kinase. In most organisms, cohesins are only main-
tained around centromeres. Cohesins around centromeres
are protected from phosphorylation-dependent removal by
protein phosphatase 2A (PP2A). This phosphatase is re-
cruited to centromeric regions by a member of a family of
PP2A targeting factors known as the Mei-S332/Shugoshin
family of proteins. The protected 'pool of cohesins provides
the resistance to the pulling force exerted by microtubulcs
necessary to establish tension at bi-oriented kinetochores. As
we will see in Section 19.8, this protection mechanism also
plays an essential role in establishing the meiotic chromo-
some segregation pattern.

FIGURE 19-26 Scanning electron micrograph of a metaphase

chromosome. During metaphase, the chromosomes are fully KfY CONCFOTS of Section 19.5
condensed and the two individual sister chromatids are visible.
[Biophoto Associates/Photo Researchers.) Entry into Mitosis
Mitotic CDKs induce entry into mitosis in all eukaryotes.
avoid this fate, cells compact their chromosomes during pro- Mitotic CDKs are held inactive until the completion of
phase into the dense structures we have become acquainted DNA replication by inhibitory phosphorylation on the CDK
with through light or electron microscopy (Figure 19-26). subunit.
Chromosome condensation results in a dramatic reduc- Mitotic CDKs promote their own activation through pos-
tion in chromos9me length, up to 10,000-fold in vertebrates. itive feedback loops leading to the rapid inactivation of
The second key aspect of the compaction process is the un- Wee! kinase and activation of Cdc25 phosphatase.
tangling of the intertwined sister chromatids. This process is
Mitotic CDKs induce nuclear envelope breakdown 111
called sister chromatid resolution and is mediated in part by
most eukaryotes by phosphorylating lamins.
the decatenation activity of topoisomerase II and goes hand
in hand with the condensation process. Centrosome duplication occurs during S phase. Mitotic
Condensation of chromosomes is not very well under- CDKs induce the separation of the duplicated centrosomes,
stood, but central to the process is a protem complex known which initiates mitotic spindle formation.
as the condensin complex. This protein complex is related to Sister chromatids attach to the mitotic spindle via their ki-
the cohesin complex that links sister chromatids together after netochores in a hi-oriented manner, with one sister kineto-
DNA replication and was first identified based on its ability to chore attaching to microtubules emanating from one spindle
promote chromosome condensation in frog extracts. Like the pole and the other one to microtubules nucleated by the other
cohcsin complex, condensins are composed of two large spindle pole.
coiled-coil SMC protein subunits that associate through thetr
Cells sense hi-orientation of sister chromatids through a
ATPase domains with non-SMC subunits. When condensin
tension-based mechanism. When kinetochores are not under
function is lost in cells, chromosomes do not condense and
tension, the protein kinase Aurora B phosphorylates the mi-
sister chromatid tangles are not resolved. How condensins
crotubule binding subunits of the kinetochore, which de-
compact chromosomes is not understood, but in analogy ro
creases their microtubule binding affinity.
cohesin rings, condensins may create intrachromosomallink-
ages that package chromosomes into the characteristic loops Chromosomes must be compacted for segregation.
seen in electron micrographs of compacted chromosomes. Condensins, protein complexes that are related to cohes-
Like all early mitotic events, chromosome condensation is ins, facilitate chromosome condensation and are activated
ultimately triggered by mitotic CDKs. The exact mechanisms by mitotic CDKs.
whereby these kinases induce chromosome condensation is not

902 CHAPTER 19 The Eukaryotic Cell Cycle

 19.6 Completion of M itosis: Chromosome Cohesin cleavage was discovered in budding yeast. Anal-
ysis of the Sec I subunit by Western blot analysis showed
Segregation and Exit from Mitosis that the protein migrated in SDS PAGE gels according to its
Once all chromosomes have condensed and correctly at- predicted molecular weight from G 1 until metaphase, but
tached to the mitotic spindle, chromosome segregation com- during anaphase the protein ran considerably faster 111 SDS
mences. In this section, we will discuss how cleavage of PAGE, indicating the protein became somehow smaller. Sub-
cohesins by a protease known as separase triggers anaphase sequent studies showed that the faster-migrating form of
chromosome movement and how this cleavage is regulated. Sccl was indeed a cleavage product. Insight into the identity
We will then explore how thf' machinery that initiates cohc- of the protein that was rf'~ponsihle for the cleavage of cohc-
sin cleavage at the metaphase-anaphase transition, the sin came from the analysis of previously identified yeast mu-
APC/C, also initiates mitotic CDK inactivation. Next, we tants that failed to segregate chromosomes during anaphase.
will see how phosphatases activated at the end of mitosis A mutant in the gene encoding SP7-what we now know
participate in mitotic CDK inactivation, bringing about the to be scparase-failed to produce the cleavage fragment.
disassembly of mitotic structures and the resetting of the cell Subsequent analyses revealed not only that separase is a pro-
tO the G 1 state. We will end this section with a discussion of tease but that cleavage of cohesin is essential for chromo-
cytokinesis, the process that produces two daughter cells. some segregation. Cells expressing a form of Sccl with its
cleavage sites mutated fail to segregate their chromosomes.
Given the irreversible nature of Sccl cleavage, it is absolutely
Separase-Mediated Cleavage of Cohesins essential that scparase activity is tightly controlled. In what
Initiates Chromosome Segregation follows we will discuss this regulation.
As mentioned in the previous section, each sister chromatid
of a metaphase chromosome is attached to microtubules via
The APC/C Activates Separase Through
its kinetochore (see Figure 19-24). At metaphase, the spindle
is in a state of tension, with forces pulling the two kineto- Securin Ubiquitinylation
chores toward the opposite spindle poles. Sister chromatids Prior to anaphase, a protein known as securin binds to and
do not separate because the} arc held together at thetr cen- inhibits separase (see Figure 19-2 7). Once. all kinetochores
tromeres by cohesin complexes. In all organisms analy1.ed to have attached to spindle microtubules in the correct hi-
date, loss of cohesins from chromosomes triggers anaphase oriented manner, the APC/C ubiquitin ligase directed by
chromosome movement. The mechanism that brings about specificity factor Cdc20 ubiquitinylates securin (note that
this loss of cohesins from chromosomes is conserved too. A this specificity factor is distinct from Cdh 1, which targets
protease known as separase cleaves a subunit of cohesin APC/C substrates for degradation later during mitosis).
called Sccl or Rad21, breaking the protein circles linking Polyubiquitinylated securin is rapidly degraded by protea-
sister chromatids (Figure 19-27). Once this link is broken, somes, thereby releasing separase.
anaphase begins as poleward force exerted on kinetochores APC/CcdclO is activated in prophase by mitotic CDK
moves the split sister chromatids toward spindle poles. phosphorylation of several APC/C subunits. However, this

Ub
/
Ub
/
Ub

Smc3
Scc3
Separase d

. FIGURE 1927 Regulation of cohesin cleavage. Separase, a the APC/C directs it to ubiquitinylate securin and mitotic cyclins.
protease that can cleave the Sccl subunit of cohesin complexes, is Following securin degradation and a decrease in mitotic CDK activity,
inhibited before anaphase by the binding of securin. Mitotic CDKs also the released and dephosphorylated separase cleaves the Sccl subunit,
inhibit separase by phosphorylating it. When all the kinetochores have breaking the cohesin circles and allowing sister chromatids to be
attached to spindle microtubules and the spindle apparatus is properly pulled apart by the spindle apparatus that is pulling them toward
assembled and oriented, the Cdc20 specificity factor associated with opposite spindle poles.

19.6 Completion of Mitosis: Chromosome Segregation and Exit from Mitosis 903
phosphorylated APC/CcdclO is not active until all chromo- Mitosis G,
somes have hi-oriented on the mitotic spindle. As we will sec ----11 Sic1
Mitotic CDKs f - - - - APC/C-Cdhl
in Section 19.7, APC/CCJc20 is inhibited by a checkpoint path-
way that ensures that mitosis does not continue until all chro-
mosomes have achieved proper attachment to the mitotic t
Cdc14
apparatus. Cdc20 is inhibited until every kinetochore has at-
tached to microrubules and tension is applied to the kineto- t
Mitotic exit network
chores of all sister chromatids, pulling them toward opposite
spindle poles. In vertebrate cells, separase is also regulated by FIGURE 19 28 The protein phosphatase Cdc:14 triggers exit
phosphorylation. Mitotic CDK activity inhibits scparasc dur- from mitosis i n budding yeast. During mitosis, mitotic CDK activity
ing prophase and metaphase. Only when mitotic CDK activity inhibits its inhibitors, APC/CCdht and Sicl. During G,, APC/Ccdht and Sicl
begins to decline ar the metaphase-anaphase transition inhibit mitotic CDKs. During exit from mitosis, the protein phosphatase
through APC/CCdclo_mcdiated protein degradation can sepa- Cdc14 throws the switch between these two antagonistic states. The
rase become active and trigger chromosome segregation. mitotic exit network activates the phosphatase during anaphase,
Once cohesins are cleaved, anaphase chromosome move- allowing it to dephosphorylate APC/Ccdht, thereby activating it. The
ment ensues. As discussed in Chapter J 8, chromosome segrega- phosphatase also promotes the accumulation of Sicl. In addition,
Cdc14 dephosphorylates the many mito~ic CDK substrates, which
tion is mediated by microtubule depolymerization and motor
leads to rapid exit from mitosis.
proteins as spindle poles move away from each other. Decline
in mitotic CDK activity is important for these anaphase chro-
mosome movements. When mitotic CDK inactivation is inhib- CDKs. Conversely, APC/Ccdhi and Sicl inhibit mitotic CDKs
ited, anaphase does occur, but it is abnormal. Dephosphorylation (Figure 19-28). The protein phosphatase Cdc14 throws the
of a number of microtubule-associated proteins that affect mi- switch between these two mutually antagonistic states dur-
crotubule dynamics appears important for this process. In bud- ing anaphase. Cdcl4 is kept inactive during most of the cell
ding yeast, this dephosphorylation is brought about by the cycle, but the phosphatase is activated during anaphase by a
protein phosphatase Cdcl4, which we will see, plays an essen- GTPase signaling pathway known as the mitotic exit net-
tial role in the final cell cycle stage, exit from mitosis. work (MEN). This signaling cascade is, as we will sec in
Section 19.7, responsive to spindle position and only be-
comes active in anaphase, when the anaphase spindle is
Mitotic CDK Inactivation Triggers
properly positioned within the cell. Once activa ted during
Exit from Mitosis anaphase, Cdcl4 dephosphorylates APC/Ccdhl and Sicl to
Anaphase spindle elongation and the events associated with promote mitotic cyclin degradation and mitotic CDK inacti-
exit from mitosis-mitotic spindle disassembly, chromo- vation, respectively. This leads to exit from mitosis.
some decondensation, and nuclear envelope re-formation- Phosphatase activity is also essential for exit from mitosis
are brought about by the dephosphorylation of CDK in vertebrates. Simple inactivation of mitotic CDKs is nor suf-
substrates. In other words, exit from mitosis can be viewed ficient to trigger the timely exit from mitosis. It is not yet clear
as a reversal of entry into mitosis. The phosphorylation which phosphatase dephosphorylates CDK substrates to reset
events that triggered the different mitotic events need to be the cell to the G 1 stage. Both protein phosphatase 1 and pro-
undone for the cell to reverse to the G 1 state. tein phosphatase 2A have been implicated in the process.
Dephosphorylation of mitotic CDK substrates is caused Ultimately, reversal of mitotic CDK phosphorylation
by the inactivation of mitotic CDKs. In most organisms, mi- changes the activities of many proteins back to their inter-
tottc CDK inactivation is triggered by APC/CCd.:lO_mcdiated phase state. Dephosphorylation of condensins, histone Hl,
degradation of mitotic cyclins. As mitotic CDKs activate and other chromatin-associated proteins leads to the decon-
APCtc<ddO, they initiate their own demise. In budding yeast densation of mitotic chromosomes in telophase. The targets of
only about 50 percent of mitotic cyclins are degraded by CDKs whose dephosphorylation is important for mitotic spin-
APC/CCJdo. As we will sec in Section 19.7, a pool of mitotic dle disassembly arc not known, but likely multiple proteins are
cyclins is protected from APC/Ccd,lO ro allow for enough targets. More is known about how the nuclear envelope re-
time to position the mitotic spindle accurately within the forms. Dephosphorylated inner nuclear membrane proteins
cell. How a fraction of mitotic cyclins is protected from are thought to bind to chromatin once again. As a result, mul-
APC!CcJ,lO is not known, but it is clear that a second mitotic tiple projections of regions of the ER membrane containing
CDK-inactivating step i~ needed for exit from mitosis to these protems are thought to associate with the surface of the
occur. The conserved protein phosphatase Cdc14 brings dPcondensing chromosomes and then fuse with one another
about this second step in mitotic CDK inhibition. directed by an unknown mechanism to form a continuous
In budding yeast, complete inactivation of mitotic CDKs double membrane around each chromosome (Figure 19-29).
requires the destruction of mitotic cyclins by APC/CCJhl and Dephosphorylation of nuclear pore subcomplexes allows them
the accumulation of the CDK inhibitor Sicl, which-re- to reassemble into complete NPCs traversing the inner and
call-holds S phase CDKs in check until cells enter the cell outer membranes soon after fusion of the ER projections.
cycle. Both APCtc<Jhl and Sicl are inhibited by mitotic Ran-GTP, required for driving most nuclear import and

904 CHAPTER 19 The Eukaryotic Cell Cycle

.
 ~ VIDEO: Nuclear Envelope Dynamics During Mitosis

... FIGURE 19-29 Model for reassembly of the nuclear envelope

Cytosol
during telophase. Extensions of the endoplasmic reticulum (ER)
associate with each decondensing chromosome and then fuse with
one another, forming a double membrane around the chromosome.
Dephosphorylated nuclear pore subcomplexes reassemble into nuclear
pores, forming individual mini-nuclei called karyomeres. The enclosed
chromosome further decondenses, and subsequent fusion of the
nuclear envelopes of all the karyomeres at each spindle pole forms Chromatin
a single nucleus containing a full set of chromosomes. NPC, nuclear
pore complex. [Adapted from B. Burke and J. Ellenberg, 2002, Nature Rev. Mol. Membrane!
Cell Bioi. 3:487.] recruitment

export (sec Chapter 13), stimulates both fusion of the ER pro-

jections to form daughter nuclear envelopes and assembly of
. NPCs (see Figure 19-29). The Ran-GTP concentration is high-
est in the microvicinity of the decondensing chromosomes be-
cause the Ran-guanine nucleotide-exchange factor (Ran-GEF)
is bound to chromatin. Consequently, membrane fusion is
stimulated at the surfaces of decondensing chromosomes,
forming sheets of nuclear membrane with inserted NPCs.
Membrane fusion,
NPC assembly
l
:"--- ....-_:) -~
l( Outernuclear~ ~
Cytokinesis Creates Two Daughter Cells
When chromosome segregation is completed, the cytoplasm
.......-'-n.-"' .-J ~ - m~r-a~~ l
and organelles arc distributed between the two future daugh- Inner nuclear
membrane '
ter cells. This process is called cytokinesis. With the excep-
tion of higher plants, the division of the cell is brought about
by a contractile ring made of actin and the actin motor myo-
sin (see Figure 17-36). During cytokinesis, the ring contracts
in a manner similar to muscle contraction, pulling the mem- In budding yeast and fission yeast, the site of cytokmesis is
brane inward and eventually dosing the connection between determined prior to mitosis. In budding yeast, this occurs during
the two daughter cells. G~. when the bud site is determined. In fission yeast, proteins
Cytokinesis must be coordinated with other cell cycle important for contractile ring formation accumulate in the mid-
events in spaceand time. For cell division to produce two dle of the cell during G2 Irrespective of the sequence of events-
daughter cells each containing the components necessary for site of mitosis first, then site of cytokinesis or vice versa-it is
survival, the division plane must be placed so that each cell clear that these events must be tightly coordinated. As we will
receives approximately half of the mother cell's cytoplasmic see in Section 19. 7, cells have developed surveillance mecha-
content and exactly half of the genetic content. Cytokinesis nisms that ensure that the site of cytokinesis is coordinated with
must also be coordinated with the temporal sequence of cell spindle position. This is especially important during asymmetric
cycle events, the completjon of mitosis. In what follows, we cell divisions, which give rise to cells of different size and/or fate.
will explore both these aspects of cytokinesis regu lation. Such cell divisions are essential during development and in stem
In animal cells, the contractile ring forms during ana- cell divisions (see Chapter 21). Cytokinesis must also be coordi-
phase and is placed in the middle of the anaphase spindle. nated with other cell cycle events. The major signal for cytokine-
This ensures that each daughter cell receives half of the ge- sis is the inactivation of mitotic CDKs. Cells expressing a
netic mate rial. Despite the importance of this coordination, stabilized version of mitotic cyclins progress through anaphase
surprisingly little is known about it. Some experiments but do not undergo cytokinesis. The CDK targets in the cytoki-
support the idea that signals sent from the spindle midzone nesis machinery have not yet been discovered.
to the cell cortex are important to coordinate the site of This concludes our discussion of the molecular events of
cywkinesis with spindle position. Other research suggests cell division. As we saw, cyclin-dependent kinases and ubiq-
that microtubules of t he spindle interact with the cell cor- uitin-mediated protein degradation are at the center of this
tex, positioning the cleavage furrow with respect to spindle control (Figure 19-30). In what follows, we will discuss the
pole position. Most likely, a combination of these path- mechanisms that ensure a subsequent cell cycle stage is not
ways governs the formation of the cleavage furrow during initiated until the previous has been completed and that each
cyrok inesis. cell cycle step occurs accurately.

19.6 Completion of Mitosis: Chromosome Segregation and Exit from Mitosis 905
 Phosphatases activate

8
Cdh1 and APC/C-Cdh1/
proteasome degrades ~
mitotic cyclins

~8
APC/C-Ccic?O/ Telophase and cytokinesis

Anaphase DNA prereplication

securin
complexes assemble
Early at origins
G, G 1/S phase-CDKs inactivate Cdh1
M id-late G1/S phase-C DKs activate
G, expression of S phase cyclin
CDKs components
'
START G 1/S phase-CDKs phosphorylate
S phase inhibitor
Cdc25 phosphatase s
Metaphase activates mitotic CDKs,
which activate early
mitotic events

S phase CDKs activate

prereplication complexes

DNA replication
FIGURE 19-30 Fundamental processes in the eukaryotic cell cycle. See text for discussion.

KEY CONCEPTS of Section 1 9.6 19.7 Surveillance Mechanisms

Completion of Mitosis: Chromosome Segregation in Cell Cycle Regulation
and Exit from Mitosis
Su rveillance mechanisms known as checkpo int path ways op-
Cleavage of cohesin by separase induces chromosome seg- erate to ensure that t he next cell cycle event is not initiated
regation during anaphase. until the previous one has been completed. Checkpoint path-
At the onset of anaphase, the APC/C is directed by Cdc20 ways consist of sensors that monitor a pa rticular cell ular
to ubiquitinylate securin, which is subsequently degraded by event, a signaling cascade that ini tiates the response, and an
proteasomes. T h is activates separase (see figure 19-27). effector that ha lts cell cycle p rogression and acti,ates repair
Exit from mitosis is triggered by mitotic CDK inactivation pathways when necessary. Cell cycle even ts moni tored by
mainly brought about by mitotic cyclin degrad ation. checkpoint pathways include grow th , DNA replication,
Ex1t from mitosis requires the activity of protein phospha- DNA damage, kinetochore attachment to t he mitotic spin-
tases such as Cdcl4 to remove mitotic phosphorylations d le, and position of the spind le within t he cell. They are re-
from many different proreins, permitting mitotic spindle dis- sponsible for the extraordinarily high fidelity of cell division,
assembly, the decondensation of c h romosome~, and the re- ensuring that each daughter cell receives t he correct number
assembly of the nuclear envelope. of accurately replicated chromo~omes. They function by
Cytokinesis finalizes cell fission and must be coordinated controlling t he protei n kinase activities of the cyclin-CDKs
with the site of nuclear division. This coordination is espe- through a variety of mechanisms: regulation of the synthesis
cially important in cells undergoing an asymmetric division. and degradation of cyclins, phosphorylation of CDKs at
inhibitory ~ites, regulation of the synthesis and stability of

906 CH APTER 19 The Eukaryotic Cell Cycle

 CDK inhibitors (CKis) that inactivate cyclin-CDK com- the permissive temperature, the temperature-sensitive pro-
plexes, and regulation of the APC/C ubiquitin-protein ligase. rein is functional again, and cells continue to proliferate.
Thus, although the cdc13 mutant cells failed to di\ide at the
restrictive temperature, they retained their viability and so
Checkpoint Pathways Establish Dependencies
were able to resume proliferation once they were returned to
and Prevent Errors in the Cell Cycle the permissive temperature where the temperature-sensitive
The experiments that led to the idea of surveillance mecha- cdc13 protein was functional again.
nisms or checkpoint pathways establishing dependencies in To characterize the cdc13 mutant in more detail, Hartwell
the cell cycle were simple and beautiful in their interpretation. and colleagues examined the effecr~ of introducing a second
Recall that Lee Hartwell and colleagues isolated temperature- mutation in another gene, a deletion in the RAD9 gene. The
sensitive cdc mutants in S. cerevisiae. Their characterization RAD9 gene is not essential for viability, but when it is deleted,
and the study of the genes affected in these mutants pro- cells are extremely sensitive to DNA damaging agents such as
foundly shaped our understand ing of the eukaryotic cell x-rays. This mutation on its own does not affect growth of
cycle. It was the characterization of one of these cdc mu- cells at any temperature but had a dramatic effect on cdc13
tants, cdc 13, that led Hartwell and colleagues to formulate mutants. When the researcher~ examined the cdc13 rad9 dou-
the checkpoint pathway concept. ble mutant at the restrictive temperature, they observed that
For the purpose of our discussion here, the only thing the mutant no longer arrested tn G 2 bur instead the cells con-
that is important to know about CDC7J function is that the tinued to divide for a few divisions (Figure 19-3lb). When
gene is required for telomere replication and, in its absence, they returned these cells to the permissive temperature, the
large stretches of incompletely replicated telomeric DNA double mutant failed to resume proliferation. This indicates
persist in cells. Cells carrying a temperature-sensitive allele that while the cells continued to divide a few times at the re-
of the cdc13 gene as the sole source of CDCJJ arrest with a strictive temperature, they lost viability.
G 2 DNA content when shifted to the restrictive temperature Hartwell and colleagues proposed the following explana-
(Figure 19-3 1a). This arrest is indicative of a defect in late S tion for this observation: cdcl 3 mutants arrest at the restnc-
phase or entry into mitosis. When the cells are returned to tive temperature because they harbor incompletely replicated
DNA. This damaged DNA signals the cell tO arrest cell cycle
progression and induce repair of the damage because mitosis
(a) cdc13 mutant Cell cycle arrest
of cells with damaged DNA would almost certainly lead to
Resume cell death. The RAD9 gene is part of the machinery that con-
proliferation; veys this cell cycle arrest. In cells lacking RAD9, the "halt cell
f ---+ colony cycle progression" signal does not work and cells undergo
will form
mitosis despite incompletely replicated DNA. This kills the
cells. Hartwell and his colleagues called this surveillance
Permissive Restrictive Permissive mechanism a checkpoint pathway.
temperature temperature temperature
(25 cJ (37 cJ (25 cJ Today we know that cells harbor multiple checkpoint
pathways to ensure that a cell cycle phase does not commence
before the pre\ ious one has been completed. In addition to
(b) cdc13 rad9.l No cell cycle arrest;
mutant few divisions establishing dependencies, the checkpoint pathways ensure
that each aspect of chromosome replication and division oc-
Q
---+
G
W
V 7

---+
Cells are dead
because of cell
division with
curs with accuracy. For example, a single kinetochore that fails
to attach to the mitotic spindle can halt cell cycle progression
~
\_u
0 (i'r(\
~
incompletely
replicated DNA
in metaphase by activating the spindle assembly checkpoint.
Each checkpoint is built in the same manner. A sensor
Permissive Restrictive Permissive detects a defect in a particu lar cellular process and in re-
temperature temperature temperature sponse to this defect activates a signal transduction pathway.
(25 cJ (37 cJ (25 cJ
Effectors activated by the signaling pathway initiate repair
EXPERIMENTAL FIGURE 19-31 An experiment that led to of the defect and halt cell cycle progression until the defect is
the checkpoint pathway concept. (a) When shihed to the restrictive
corrected. In what follows we will discuss the major check-
temperature, cdc 13 mutants arrest cell cycle progression because
point pathways that govern cell cycle progression.
of incomplete DNA replication. When the cells are returned to the
permissive temperature, they resume proliferation because they
maintained viability during the cell cycle arrest. (b) cdc13 rad9.l double The Growth Checkpoint Pathway Ensures
mutants do not arrest when shifted to the restrictive temperature That Cells Only Enter the Cell Cycle After
because these cells cannot sense that their DNA is incompletely
Sufficient Macromolecule Biosynthesis
replicated. Cells undergo mitosis, and this leads to cell death because
genetic information is lost. Therefore, cells quickly lose viability at the Cell proliferation requires that cells multiply through the pro-
restrictive temperature and can no longer resume proliferation when cess of cell division and that the indi,idual cells grow through
they are returned to the permissive temperature. macromolecule biosynthesis. Cell growth and division are

19.7 Surveillance Mechan isms in Cell Cycle Regulation 907

separate processes, but for cells to maintain a constant size as (a) Small cells (b) Large cells
they multiply, cell growth and division must be tightly coor- Cdr2
dinated. For example, when nutrients are limited, cells reduce
their growth rate, and cell division must be down-regulated
accordingly. This type of coordination between cell growth
and division is especially important in unicellular organisms
that experience changes in nutrient availability as part of
their natural life cycle. It is therefore not surprising that sur-
veillance mechanisms exist th:~t adjmt cell division rate ac- Entry into mitosis Entry into
cording to growth rate. inhibited by Pom1 mitosis promoted
ln budding yeast, cell growth and division are coordi- FIGURE 19-32 A protein gradient measures cell length in
nated in G 1 In this stage of the cell cycle, the growth and S. pombe. The protein kinase Pom1 forms a gradient from each pole
division cycles are linked by making the activity of G 1 CDKs toward the middle of the cell. Wee1 and its inhibitor Cdr2 are localized
dependent on cell growth. Which aspect of growth is linked to patches of cell cortex in the middle of the cell. Pom 1 inhibits Cdr2,
to the cell cycle? Classical experiments using protein synthe- and hence the pathway that inhibits Wee1. (a) In small cells, the
sis inhibitors indicate that growth rate and hence cell cycle concentration of Pom 1 in the middle of the cell is higher. Cdr2 is
control by growth is determined by protein synthesis. How inhibited, allowing Wee1 to remain active and prohibit entry into
mitosis. (b) As cells grow in length, the c~ncentration of Pom1 in the
protein synthesis controls G 1 CDK activity is an area of ac-
middle of the cell declines. Pom1 inhibition of Cdr2 decreases, and the
tive investigation. The G 1 cyclin Cln3 is subject to transla-
Cdr2 signaling pathway is now able to inhibit Wee1, promoting entry
tional control, making levels of this cyclin especially sensitive
into mitosis. [Adapted from Moseley et al.. 2009; Nature 459:857-860.)
to protein synthesis rate. However, it is clear that this is not
the only mechanism. Although the molecular pathways that
coordinate cell division with cell growth are not yet under- indirectly, G 1 CDKs. Thus this transcription factor appears
stood, it appears that this control is highly plastic. The length to integrate cell growth and division. However, the details of
of G 1 and the crittcal cell size, that is, the size at which cells this coordination remain to be elucidated.
enter the cell cycle, change with nutrient availability.
S. pombe grows as a rod -shaped cell that increases in
The DNA Damage Response Halts Cell Cycle
length as it grows and then divides in the middle during mi-
tosis to produce two daughter cells of equal size (see Figure Progression When DNA Is Compromised
19-4 ). Unlike budding yeast and most metazoan cells that The complete and accurate duplication of the genetic ma-
grow primarily during G" this yeast does most of its grow- terial is essential for cell division. If cells enter mitosis when
ing during the G1 phase of the cell cycle and it is entry into DNA is not completely replicated or otherwise damaged, ge-
mitosis that is carefully regulated in response to cell size. netic changes occur. In many instances, those changes will
Recall entry into mitosis is regulated by the protein kinase lead to cell death, but as we will see in Chapter 24, they can
Wee I, which inliibits CDK 1 by phosphorylating tyrosine 15. also lead to genetic alterations that result in loss of growth
When nutrients are limited, Weel phosphorylates CDKl; and division control and eventually cancer. This is under-
hence cells remain in G 2 until they reach the critical size for scored by the finding that many proteins involved in sensing
mitotic entry. This size control is brought about by regulated DNA damage and its repair are frequently found mutated in
protein localization. The protein kinase Porn 1 forms a gradi- human cancers.
ent from each pole toward the middle of the cell. The CDK The enzymes that replicate DNA are highly accurate, but
inhibitor Weel and its inhibitor Cdr2 localize in patches to this exactness is not enough to ensure complete accuracy
the cell cortex in the middle of the cell (Figure 19-32 ). Poml during DNA synthesis. Furthermore, environmental insults
prevents Cdr2 from inhibiting Weel. When cells are small, such as x-rays and UV light can cause DNA damage, and
Porn] inhibits Cdr2. Weel is active and prevents entry into this damage must be repaired before a cell's entry into mito-
mitosis. As cells grow, the local concentration of Pom 1 in sis. Cells have a DNA damage response system in place that
the middle of the cell declines and Cdr2 becomes active and senses many different types of DNA damage and responds
inhibits Weel. Cells can now enter mitosis. Hence in this by activating repair pathways and halting cell cycle progres-
organism, cell length is measured by a protein gradient. sion until the damage has been repaired. Cell cycle arrest can
Nutrients are usually not limiting in multicellular organ- occur either in G" S phase, or G 2 , depending on whether
isms. Instead, cell growth is controlled by growth factor sig- DNA damage occurred before cell cycle entry or during
naling pathways such as the Ras, AMPK, and TOR pathways DNA replication. [n multicellular organisms, the strategy to
(see Chapters 8 and 16). These pathways also appear to be deal with particularly severe DNA damage is different.
important for coordinating cell growth and division. Muta- Rather than attempting to repair the damage, cells undergo
tions in key components of growth factor signaling path- programmed cell death or apoptosis, a mechanism that we
ways, such as Myc, cause dramatic changes in cell size in will discuss in detail in Chapter 21.
Drosophzla. Myc regulates the transcription of many genes DNA damage exists in many different forms and ranges in
important for macromolecule biosynthesis and also, more severity. A break of the DNA helix, known as a double-strand

908 CHAPTER 19 The Eukaryotic Cell Cycle

break, is perhaps the most severe form of damage since such a thought to activate its protein kinase activity, leading to the
lesion would almost certainly lead to DNA loss if mitosis en- recruitment of adapter proteins whose function is to recruit
sued in its presence. More subtle defects include single-strand and help activate the Chk 1 kinase. Active Chk 1 then induces
breaks, structural changes in nucleotides, or DNA mis- repair pathways and inhibits cell cycle progression.
matches. For our discussion here, it is important to note that Chkl and Chk2 halt the cell cycle. The protem kinascs
cells have sensors for all these different types of damage. These phosphorylate Cdc25, thereby inactivating it (sec Figure 19-33).
sensors scan the genome and, when they detect a lesion, as- When the DNA damage occurs during G" Cdc25A inhibi-
semble signaling and repair factors onto the site of the lesion. tion results in inhibition of G 1/S phase CDKs and S phase
Central ro the detection of the different lesions is a pair of l.DKs (Figure 19-34). As a result, these kinases cannot initi-
homologous protein kinases called ATM and ATR. These ate DNA replication. When the DNA damage occurs during
protein kinases get recruited to sites of DNA damage. They S phase or in G 2, Cdc25C inhibition by Chkl/2 results in the
then initiate the sequential recruitment of adapter proteins mhibition of mitotic CDKs and hence arrest in G 2 Acnve
and another set of protein kinases called Chk I and Chk2. DNA replication also inhibits entry into mitosis. ATR con-
These kinases then activate repair mechanisms and cause cell tinues to inhibit Cdc25C via Chk 1 until all replication forks
cycle arrest or apoptosis in animals (Figure 19-33). ATR and complete DNA replication and disassemble. This mechanism
ATM recognize different types of DNA damage. ATM is very makes the initiation of mitosis dependent on the completion
specialized in that it only responds to double-strand breaks. of chromosome replication. Finally, cells also sense DNA
ATR is able to recognize more diverse types of DNA damage, replication stress that results in stalled or slowing of replica-
such as stalled replication forks, damaged nucleotides, and tion forks. lt triggers activation of the ATR-Chk l check-
double-strand breaks. ATR recognizes these diverse types of point pathway and results in down-regulation of S phase
damage because all of them contain some amount of single- CDK activity and prevents firing of late-replicating origins.
stranded DNA, either as part of the damage itself or because Chkl-mediated inhibition of the Cdc25 famil) of phos-
repair enzymes create single-stranded DNA as part of there- phatases is not the only mechanism whereby DNA damage
pair process. Stalled replication forks, for example, are recog- or incomplete replication inhibits cell cycle progression. As
nized by ATR. The association of ATR with stalled forks is we will see below, DNA damage leads to the activation of
the transcription factor p53, which transcribes the CDK in-
hibitor p21. p21 binds to and inhibits all metazoan cyclin-
CDK complexes. As a result, cells arc arrested in G 1 and G 2
Stalled . (see Figure 19-34 ).
Double-strand re lication DNA Nucleotide ATM recognizes double-strand breaks (sec Figure 19-33 ).

bc:'.:...............':~~'?'9'
The protein kinase gets directly recruited to the DNA ends b;
a complex known as the MRN complex, which binds to bro-
ken ends and holds them together. Activated ATM then phos-
phorylates and activates Chk2 and recruits repair protems.
These repair proteins initiate homologous recombination, as

~
discussed in Chapter 4. This process involves the creation of
single-stranded overhangs, which in turn recruit and activate
[hk2, Chk}) ATR and its effectors, further enhancing the DNA damage
response. ATM can also recruit an alternative repair pathwa}

Repair

Cdc25
where two double-strand breaks are directly fused with each
other in a repair process known as nonhomologous end join-
ing. Like ATR, ATM activation also halts cell cycle progres-

l
CDKs Apoptosis p21
sion by a Chk2-mediatcd inhibition of Cdc25, thus preventing
activation of CDKs. This inhibition can occur in G 1 or G 2
A key effector of the DNA damage response in metazoan
FIGURE 19-33 The DNA damage response system. The protein cells is the transcription factor p53 (sec Figure 19-33 ). It is
kinases ATM and ATR are activated by damaged DNA. ATR responds to known as a tumor suppressor because its normal function is
a variety of DNA damage-most likely to the single-stranded DNA that
to limit cell proliferation in the face of DNA damage. The
exists either as a result of the damage itself or as a result of repair. ATM
protein is extremely unstable and generally does not accu-
is specifically activated by double-strand breaks. Because double-
mulate to high enough levels to stimulate transcription under
strand breaks are converted into single-stranded DNA as a result of
repair, they also, albeit indirectly and hence depicted as a dashed line,
normal conditions. The instability of p53 results from its
activate ATR. ATM and ATR, once activated by DNA damage, activate ubiquitinylation by a ubiquitin-protein ligase called Mdm2
another pair of related protein kinases, Chkl and Chk2. These kinases and subsequent proteasomal degradation. The rapid degra-
then induce the DNA repair machinery and cause cell cycle arrest by dation of p53 is inhibited by ATM and ATR, which phos-
inhibiting Cdc25. In metazoan cells, when the DNA damage is severe, phorylate p53 at a sire that interferes with Mdm2 binding.
Chkl and Chk2 also activate the transcription factor p53. p53 induces This and other modifications of p53 in re~ponsc to DNA
cell cycle arrest by inducing transcription of the CKI p21 and apoptosis. damage greatly increase its ability to activate transcription of

19.7 Surveillance Mechanisms in Cell Cycle Regulation 909

 Ongoing DNA damage DNA damage
DNA
replication ! !
ATM/R ATM/R

!
ATR
+
Chkl/2
I
~
p53 p53
!
! 1 ! ! DNA damage

Chk1 ~ Cdc25C p21 CIP p21 CIP

!
! 1 1 ATM/R--+ Chk1/2
Mitotic CDKs

!
M phase
entry
+ 1
p21 CIP Cdc25A

1 !
S phase +-- G 1/S phase and S phase CDKs
Late entry
replication S phase

i
S phase CDKs

i
Cdc25A

.__--Chkl
T
i
ATR

i
Replication stress
FIGURE 19-34 Overview of DNA damage checkpoint controls in ATR protein kinases (ATM/R) inhibit Cdc25 via the Chkl / 2 protein
the cell cycle. During G1, the p53-p21 CIP pathway inhibits G1 CDKs. kinases. They also activate p53, which induces production of the CKI
During ongoing DNA replication and in response to replication stress p2 1. During G,, the DNA damage checkpoint pathway inhibits Cdc2SA
(slow DNA replication fork movement or DNA replication fork collapse), inhibiting G1/S phase CDKs and S phase CDKs, thereby blocking entry
the ATR-Chkl protein kinase cascade phosphorylates and inactivates into or passage through S phase. During G2, ATM/R-Chkl /2 inhibit
Cdc25C, thereby preventing the activation of mitotic CDKs and Cdc25C. The p53-p21 P pathway is also activated. Red symbols indicate
inhibiting entry into mitosis. In response to DNA damage, the ATM or pathways that inhibit progression through the cell cycle.

specific genes that help the cell cope with DNA damage. One The Spindle Assembly Checkpoint Pathway
of th ese genes encodes the CKI p21 (see Figure 19-34 ). Prevents Chromosome Segregation Until
Under some circumstances, such as when DNA damage
Chromosomes Are Accurately Attached
ts extensive, p53 also activates expression of genes that lead
to apoptosis, the process of programmed cell death that nor- to the Mitotic Spindle
mally occurs in specific cells during the development of mul- The spindle assembly checkpoint pathway prevents entry into
ticellular animals. In metazoans, the p53 response evolved to anaphase until every kinetochore of every chromatid is prop-
induce apoptosis in the face of extensive DNA damage, pre- erly attached to spindle microtubules. If even a single kineto-
sumably to prevent the accumulation of multiple mutations chore is unattached or not under tension, anaphase is
that might convert a normal cell into a cancer cell. The dual inhibited. Clues about how this checkpoint operates initially
role of p5 3 in both cell cycle arrest and the induction of came from the isolation of yeast mutants with a defective re-
apoptosis may account for the observation that nearly all sponse to benomyl, a microtubule-depolymerizing drug. Low
cancer cells have mutations in both alleles of the p53 gene or concentrations of benomyl increase the time required for
in the pathways that stabilize p53 in response to DNA damage yeast cells to assemble the mitotic spindle and attach kineto-
(see Chapter 24). The consequences of mutations in p53, chores to microtubules. Wild-type cells exposed to benomyl
A TM, and Chk2 provide dramatic examples of the signifi- do not begin anaphase until these processes are completed
cance of cell cycle checkpoint pathways to the health of a and then proceed on through mitosis, producing normal
multicellular organism. daughter cells. In contrast, mutants defective in the spindle

910 CHAPTER 19 The Eukaryotic Cell Cycle

assembly checkpoint pathway proceed through anaphase be- Figure 19-27). Mad2 is recruited to kinetochores that arc not
fore assembly of the spindle and attachment of kinetochores attached to microrubules. Madl appears critical for this pro-
is complete; consequently, they mis-segregate their chromo- cess. Kinerochore-bound Mad2 rapidly exchanges with a solu-
somes, producing abnormal daughter cells that die. ble form of Mad2 that inhibits all the Cdc20 in the cell (Figure
We now know that cells harbor a surveillance mecha- 19-35a). When microtubules attach to kinetochores, the kinet-
nism that prevents anaphase entry in the presence of unat- ochores release the bound Mad2 and cease the process by
tached kinetochores. Components of the spindle assembly which the inhibitory, soluble form of Mad2 is produced (figure
checkpoint recognize and bind to unoccupied microtubule 19-35b). However, when even a single kinetochore is unat-
binding sites at kinetochores and create an anaphase inhibi- tached to microtubules from the opposite spindle pole of its
tory signal (Figure 19-35a). A protein known as Mad2 (mitotic sister, sufficient soluble inhibitory Mad2 is produced at the un-
arrest defective 2) is central to the creation of this inhibitory attached kinetochore to inhibit all the Cdc20 in the cell. This
signal. Mad2 regulates Cdc20, the specificity factor required elegant model for the spindle assembly checkpoint pathway
to target the APGC to securin. Recall that APGCCJdo_mediated can account for the ability of a single unattached kinetochore
ubiquitinylation of sccurin and its subsequent degradation is to inhibit all the cellular Cdc20 until the kinetochore becomes
required for activation of separase and entry into anaphase (see properly associated with spindle microtubules.

(a) Checkpoint activation (b) Checkpoint inactivation

a
~~~~:,";~ I!J: a ~~~ 8&8_/'9
~ ~riiSO-.:
IJ

~
11 eso~ .,
Mad1 \ 1:.1
Release of 9,1
Mad1-Mad2 lO
~cse
tetra mer from

ottoohm~
kinetochore
Attachment
L.ck of

--~--------::t~ --~
completed

'-----
Mad1
li3 Mad2 in open conformation
Microtubules
@ Mad2 in closed conformation
FIGURE 19-35 The spindle assembly checkpoint pathway. The as this cycle repeats (step 1';1). The source of closed Mad2 that initiates this
spindle assembly checkpoint pathway is active until every single chain reaction is the closed Mad2 bound to Mad 1 associated with a
kinetochore has attached properly to spindle microtubules. (a) The Mad2 kinetochore, explaining how a single unattached kinetochore can cause
protein exists in two conformations, one "open" (red squares) and the inactivation of all the Cdc20 in the cell through the formation of closed
other "closed" (orange circles). According to the current model, Mad1 Mad2-Cdc20 complexes. How are unattached kinetochores that recruit
and the closed Mad2 form a tetramer that binds to unattached kineto- Mad 1-Mad2 complexes generated? Either microtubules fail to attach or
chores via the Mad1 subunit (step 0 ). Open Mad2 can bind transiently to Aurora B severs kinetochore-microtubule attachments that are not under
closed Mad2 bound to Mad 1 at the kinetochore (step 6 ). This interac- tension (see Figure 19-25). (b) Silencing of the spindle assembly
tion with closed Mad2 stimulates open Mad2 to bind a Cdc20. Open checkpoint pathway: Attachment of microtubules (green) to kinetochores
Mad2 can bind Cdc20 only while it is interacting with a closed Mad2. This causes the displacement of the Mad 1-Mad2 tetra mer. Mad2 in the
converts the open Mad2 protein to the closed conformation, causing it displaced tetra mer cannot interact with open Mad2 but rather binds to
to dissociate from the closed Mad2 at the kinetochore (step 0 ). The p31 comet. p31 comet is always active and disassembles Mad2-Cdc20
stable interaction of closed Mad2 with Cdc20 prevents Cdc20 from complexes, releasing active Cdc20 (step fJ). However, a small number of
binding to the APC/C. Further, the closed Mad2 bound to (dc20 can Mad1 Mad2 tetramers bound to kinetochores can generate enough
interact transiently with another Mad2 in the open conformation (step!)), Mad2-Cdc20 complexes by the mechanism shown in (a) to overcome the
causing it to bind another Cdc20 molecule. This converts the open Mad2 activity of p31. Once all kinetochores have attached to microtubules,
to the closed conformation bound to Cdc20. This newly formed closed causing the release of all Mad 1-Mad2 tetramers, p31 activity predomi-
Mad2-Cdc20 complex dissociates from the first Mad2-Cdc20 pair, nates, releasing active Cdc20, which binds to the APC/C, resulting in
generating two Mad2-Cdc20 complexes (step 1,'1). Thus free Mad2 in the ubiquitinylation and proteasomal degradation of securin and the onset
open conformation is quickly converted to closed Mad2 bound to Cdc20 of anaphase. [Modified from A. De Antoni et al., 2005, Curr. Bioi. 15:214.]

19.7 Surveillance Mechanisms in Cell Cycle Regulation 911

 Entry into anaphase is also inhibited when the attach-
ments of microtubules to kinetochores are faulty. As we saw
in Section 19.5, kinetochores of sister chromatids often at-
Kin 4
tach to microtubules emanating from the same pole (syntelic Tem1-GDP ---,.,..__,
attachment) or a single k inetochore attaches to microtubules (mactive)
that originate from two different poles (merotelic attach- M etaphase -'----\-- Nucleus
ment). These faulty attachments result in insufficient or no ten- Nucleolus Spindle
sion at sister kinetochores, and such microtubule-kinetochore Cdc14 lf - -'--f - microtubule
(inactive)
interactions are quick ly dest::~hilind hy Aurora B phosphory-
lation of the microtubule-binding proteins of the kineto-
chore. This leads to the generation of unattached kinetochores,
which are recognized by the spindle assembly checkpoint. In Proper nucl ear D ~ fJ Improper nuclear
this manner Aurora B and the spindle assembly checkpoint positio n / ~ position

collaborate during every cell cycle to accurately attach every

single pair of sister chromatids on the mitotic spindle in the
correct, hi-oriented manner.
The spindle assembly checkpoint pathway is essential for NI.Jcleolus
viability in mice, highlighting the importance of this quality- Cdc14
control pathway during every cell division. If anaphase is Anaphase
(inactive)
initiated before both kinetochorcs of a rep licated chromo-
some become attached to microtubules from opposite spin- Cdc14
(ACTIVE)
dle poles, daughter cells are produced that have missing or
extra chromosomes, an outcome called twndisjunction.

~
When nondisjunction occurs in mitotic cells, it can lead to
the misregulation of genes and contribute to the develop-
Exit from Late mitotic
ment of cancer. When nondisjunction occurs during the mei- mitosis arrest
otic division that generates a human egg or sperm, trisomy
of any chromosome can occur. Down syndrome is caused by Checkpoint IJ
failure
trisomy of chromosome 21, resulting in developmental ab-
normalities and mental retardation. Every other trisomy re-

An"'~
sults in embryonic lethality or death shortly after birth.
.
The Spindle Position Checkpoint Pathway
Ensures That the Nucleus Is Accurately multinucleate cells

Partitioned Between Two Daughter Cells

The coordination of the site of nuclear division with that of
cytokinesis is essential for the production of two identical
daughter cells. If cytokinesis occurred in such a way that
each daughter cell failed to receive a complete genetic com-
plement, chromosome loss or gain would ensue. In many FIGURE 19-36 The spindle position checkpoint in budding
systems, surveillance mechanisms have been described that yeast. Cdcl4 phosphatase activity is required for exit from mitosis.
ensure cytokinesis does not occur when the mitotic spindle is Top: In 5. cerevisiae, during interphase and early mitosis, Cdcl4 (red dots)
not correctly positioned in the cell. This surveillance mecha- is sequestered and inactivated in the nucleolus. Inactive Tem1 -GDP
nism, known as the spindle position checkpoint pathway, is (purple) associates with the spindle pole body (SPB) nearest to the bud
best understood in budding yeast. l n budding yeast, the site as soon as the mitotic spindle forms. If chromosome segregation
occurs properly (step O J, extension of the spindle microtubules inserts
of bud formation and therefore the site of cytokinesis is de-
the daughter SPB into the bud, causing Teml to be activated by an
termined during G 1 Thus the axis of division is defined prior
unknown mechanism. Teml-GTP activates a protein kinase cascade,
to mitosis and the mitotic spindle must be aligned along this
which then promotes the release of active Cdcl4 from the nucleolus
mother-bud axis during every cell division (Figure 19-36, and exit from mitosis. If the spindle apparatus fails to place the
step 0 ). When this process fails, the spindle position check- dilughter SPB in the bud (step f) ), Kin4 (c.yan), an inhibitor ofTeml, IS
point prevents mitotic CDK inactivation, giving the cell an recruited from the mother cell cortex to t he mother-cell-located SPB
opportunity to reposition the spindle prior to spindle disas- and maintains Teml in the GOP bound form and mitotic exit does not
sembly and cytokinesis (Figure 19-36, step H ). If the spindle occur. Ltel (orange) is an inhibitor of Kin4 and localized to the bud.
position checkpoint fails, cells that misposition their spindles Ltel prevents Kin4 that leaks into the bud from inhibiting Teml.lfthe
give rise to mitotic products with too many or too few nuclei checkpoint fails (step !D), cells with mispositioned spindles inappropri-
(Figure 19-36, step !)). ately exit from mitosis and produce anucleate and multi-nucleate cells.

912 CHAPTER 19 The Eukaryotic Cel l Cycle

 Recall that in budding yeast, a pool of mitotic cyclins are
spared from degradation by APCICCdclO to facilitate the In response to DNA damage two related protein kinases
sometimes difficult process of aligning the mitotic spindle in AT~l and ATR are recruited to the site of damage, where
such a way that half the nucleus squeeze~ through the tiny they activate signaling pathways that lead to cell cycle arrest,
bud neck during anaphase spindle elongation. Recall further repa ir, and, under some circumstances, apoptosis.
that inactivation of this protected pool of mitotic cyclin-CDK The spindle assembly checkpoint pathway, which prevents
complexes is triggered by the protein phosphatase Cdc l 4, premature initiation of anaphase, utilizes Mad2 and other
which in turn is activated h} a signal transduction pathway proteins to regulate the APC/C specificity factor Cdc20, which
known as the mitotic exit 11etwork (see rigure 19-28). The t;Irget~ <;ecurin and mitotic cyclins for ubiquitinylation.
mitotic exit network is controlled by a small (monomeric) The spindle position checkpoint pathway prevents mitotic
GTPase called Tern 1. This member of the GTPase superfam- CDK inactivation when the spindle is mispositioned. ln thts
ily of switch proteins controls the activity of a protein kinase pathway localized activators and inhibitors and a sensor that
cascade similarly to the way Ras controls MAP kinase path- shuttles between them allows the cells to sense spindle position.
ways (see Chapter 16). Tern 1 associates with spindle pole
bodies (SPBs) as soon as they form. An inhibitor of the GTPase
called Kin4 localizes to the mother cell but is absent from the
bud (sec Figure 19-36). An inhibitor ofKin4 called Ltel local- 19.8 Meiosis: A Special Type
izes to the bud but is absent from the mother cell and inhibits
any residual Kin4 that leaks into the bud. When spindle micro- of Cell Division
tubule elongation at the end of anaphase has correctly posi- In nearly all diploid eukaryotes, meiosis generates haploid
tioned segregating daughter chromosomes into the bud, germ cells (eggs and sperm), which can then fuse wtth a germ
Teml inhibition by Kin4 is relieved. As a consequence, Teml cell from another individual to generate a diploid zygote that
is converted into its active GTP-bound state, activating the develops into a new individual. Meiosis is a fundamental as-
protein kinase signaling cascade. The terminal kinase in the pect of the biology and evolution of all eukaryotes because it
cascade then phosphorylates the nucleolar anchor that binds resu lts in the reassortment of the chromosome sets received
and inhibits Cdcl4, releasing the Cdc14 phosphatase into the from an individual's two parents. Both chromosome reas-
cytoplasm and nucleoplasm in both the bud and mother cell sortmcnt and homologous recombination between parental
(see rigure 19-36, step 0 ). Once active Cdc14 is available, DNA molecules during meiosis guarantees that each haploid
mitotic CDKs are inactivated and cells exit from mitosis. germ cell generated will receive a unique combination of
When the spindle fails to position correctly, the Teml-bear- gene alleles that is distinct from each parent as well as from
ing spindle pole body fai ls to enter the bud and the mitotic every other haploid germ cell formed.
exit network cannot be activated. Cells arrest in anaphase. The mechanisms of meiosis are analogous to those of mi-
Thus spatial restriction of inhibitors and activators of a signal tosis. However, several key differences in meiosis allow this
transduction pathway allows the cell to sense a spatial cue, process to generate haploid cells with generic diversity (see
spindle position, and translate it into regulation of a signal Figure 5-3). In mitosis, each S phase is followed by chromo-
transduction pathway. some segregation and cell division. In contrast, during meio-
sis, one round of DNA replication is followed by two
consecutive chromosome segregation phases. This leads to
'-'~V CO C"EPTc; of c;('>rtio--- 19 7 the formation of haploid rather than diploid daughter cells.
During the two divisions, maternal and paternal chromo-
Surveillance Mechanisms in Cell Cycle Regulation somes are shuffled and divided so that the daughter cells are
Surveillance mechanisms known as checkpoint pathways different in genetic makeup from the parent cell. In this sec-
establish dependencies among cell cycle events and ensure tion, we will discuss the similarities between mitosis and
that cell cycle progression does not occur prior to the com- meiosis as well as the meiosis-specific mechanisms that
pletion of a preceding event. transform the canonical mitotic cell cycle machinery so that
Checkpoint pathways consist of sensors that monitor a it brings about the unusual cell division that leads to the
particular cellular event or defect therein, a signaling path- formation of haploid daughter cells.
way, and an effector that halts cell cycle progression and
activates repa ir pathways when necessary. Extracellular and Intracellular Cues
Growth and cell division are integrated during G 1 in most Regulate Entry into Meiosis
systems. Reduced macromolecule biosynthesis delays cell cycle The signals triggering entry into the meiotic divisions in
entry. metazoans are a very active area of research and much is snll
Cells arc able to detect and respond to a wide variety of unknown. However, the same basic principles govern the de-
DNA damage, and the response differs depending on the cell cision to enter the meiotic program in all organisms where
cycle stage that cells are in. this has been studied. Extracellular signals induce a tran-
scriptional program that produces meiosis-specific cell cycle

19.8 Me1osis: A Special Type of Cell Division 913

factors that bring about the unusual meiotic cell divisions. G 1 Depletion of nitrogen and carbon sources induces diploid
This modification of the cell cycle goes hand in hand with a cells to undergo meiosis instead of mitosis, yielding haploid
developmental program that induces the features character- spores (see Figure 1-17). During the meiotic divisions, bud-
istic of gametes, such as the development of a flagellum in ding is repressed and pre-meiotic S phase and the two mei-
sperm or the production of a stress-resistant cell wall during otic divisions occur within the confines of the mother cell.
spore formation in fungi. At least one of the extracellular Spore walls are then produced around the four meiotic prod-
signals inducing entry into the meiotic divisions in mammals ucts. Recall that budding and the initiation of DNA repl ica-
is retinoic acid, a steroid hormone that functions in many tion are induced by G/S phase CDKs. Their expression
differc>nr developmental processes. The cellular targets of needs to he inhibited tu prevem budding. Nutritional starva-
this hormone and how it functions to specify the meiotic fate tion represses expression of G 1/S phase cyclins, inhibiting
remain to be dtscovered. budding. However, DNA replication also relies on GtfS
The molecular mechanisms underlying the decision to phase CDKs. How can pre-meiotic DNA replication occur in
enter the meiotic divisions are well understood in S. cerez,r- the absence of G 1/S phase CDKs? The sporulation-specific
srae. The decision to enter the meiotic divtsions is made in protein kinase lme2 takes over the role of G tfS phase CDKs

Row Mitosis Meiosis

In somatic cells In cells in the sexual cycle

One cell division,

Parental Q ---+ 0 Daughter
Two cell divisions,
0 0 ___. 0 0 Products

0 00
resulting in
cell
resulting in four Meiocyte ---+
two daughter cells o cells products of meiosis of meiosis

Chromosome number @ Chromosome number

0 00
2 per nucleus maintained
{e.g., for a diploid cell}
(---+
@)
halved in the products
of meiosis e ---.. 0 ---.. 00

i~ ~ i~ ~
One premitotic .,a..,"
~II)

One premeiotic .,
~

a..,"
(/)

<(<3 <(<3
3 S phase per cell Z=> S phase for both z"c:
division oc: cell divisions 0

G1 S G2 M Gt s M, Mu

4
Normally, no pairing
of homologous
Chrom7 / Full synapsis of
of homologous Bivalent
{:!
~ ...:-- Chromatid
chromosomes in prophase chromosomes in prophase
1-;>- ...} Chromosome
Chromosome/

At least one recombination

Normally, no recombination
5
in prophase
between nonsister Chiasma- 8::
::;;._._ ,.:_
chromatids

Bi-oriented sister Co-orientation of

++
6 sister kinetochores in meiosis I
kinetochores

Loss of cohesion Maintenance of cohesion

7 between sister chromatid between sister chromatid arms
arms during prophase during prophase of meiosis I

Centromeres Centromeres do not

......--.....
~
8 divide at
anaphase
divide at anaphase I
but do at anaphase II ......--.....
~
Anaphase I Anaphase II

Conservative process: daughter cells' genotypes

Promotes variation among the products of meiosis
identical with parental genotype

Cell undergoing mitosis can be diploid or haploid Cell undergoing meiosis is diploid or multiple thereof

FIGURE 19-37 Comparison of the main features of mitosis and meiosis. [Adapted from A. J. F. Griffiths et al., 1999, Modern Genetic Analysis,
W. H. Freeman and Company.]

914 CHAPTER 19 The Eukaryotic Cell Cycle

 in promoting DNA replication. lme2 promotes (1) phos- (see Figure 19-38). The chiasmata now provide the resis-
. phorylation of the APC/C specificity factor Cdh 1, inactivat- tance to the pulling force exerted by microtubules on the
ing it so that S phase and M phase cyclins can accumulate; metaphase I spindle (Figu rc 19-39).
(2) phosphorylation of transcription factors to induce genes The recombination between nonsister chromatids that
required for S phase, including DNA polymerases and occurs in prophase of meiosis I has at least two functional
S phase cyclins; and (3) phosphorylation of the S phase CDK consequences: First, it holds homologous chromosomes to-
inhibitor Sic I, leading to release of activeS phase CDKs and gether during meiosis I metaphase. Second, it contributes to
the onset of pre-meiotic DNA replication. genetic diversity among individuals of a species b} ensuring
new combinations of gene alleles in different individuals.
(Note: genetic diversity primarily arises from the indepen-
Several Key Features Distinguish Meiosis
dent reassortment of maternal and paternal homologs dur-
from Mitosis ing the meiotic divisions). The homologs connected through
The meiotic divisions differ in several fundamental aspects from at least one chiasma, which formed during prophase of mei-
the mitotic divisions. These are summarized in Figure ] 9-37. osis I, must now align on the meiosis I spindle so that mater-
During meiosis, a single round of DNA replication is followed nal and paternal chromosomes are segregated away from
by two cycles of cell division, termed meiosrs I and meiosis 11 each other during anaphase of meiosis I. This requires that
(Figure 19-38). Meiosis II resembles mitosis in that sister the kinetochorcs of sister chromatids attach to spindle fibers
chromatids are segregated. However, meiosis I is very differ- emanating from the same spindle pole rather than from op-
ent. During this division, homologous chromosomes-the posite spindle poles as in mitosis (see Figure 19-39). Sister
chromosome inherited from your mother and the same chro- chromatids arc said to be co-oriented. However, the kineto-
mosome inherited from your father-arc segregated. This chores of the maternal and paternal chromosomes of each
unusual chromosome segregation requires three meiosis- bivalent attach ro spindle microtubules from opposite spin-
specific modifications to the chromosome segregation ma- dle poles; they are hi-oriented.
chinery. In what follows we will discuss these and explain Finally, to facilitate two consecutive chromosome segre-
why they are needed. gation phases, cohesins have to be lost from chromosomes in
The tension-based sensing mechanism responsible for ac- a stepwise manner. Recall that during mit(>sis, all cohesins
curately attaching chromosomes to the spindle during mitosis is are lost by the onset of anaphase (Figure 19-40a). In con-
also responsible for segregating chromosomes during meiosis I. trast, during meiosis, cohesins are lost from chromosome
Thus homologous chromosomes must be linked so that the arms by the end of meiosis I, but a pool of cohesins around
tension-based mechanism of accurate attachment to the spindle kinetochores is protected from removal (Figure 19-40b).
can function. Homologous recombination between homolo- This pool of cohesins persists throughout meiosis I but is
gous chromosomes creates these linkages (see Figure 19-38). removed at the onset of anaphase II. As we will see next, loss
The molecular mechanisms of homologous recombination of cohesins from chromosome arms is required for homolo-
are discussed in detail in Chapter 4. Here, we will restrict our gous chromosomes to segregate away from each other dur-
discussion to the importance of homologous recombination ing meiosis I.
for the meiotic divisions to occur successfully. The mechanisms that remove cohcsins during meiosis arc
In G 2 and pr'o phase of meiosis I, the two replicated chro- the same as during mitosis. Securin degradation releases sep-
matids of each chromosome are associated with each other arase, which then cleaves the cohesins holding the chromo-
by cohesin complexes along the full length of the chromo- some arms together. This allows the recombined maternal
some arms, just as they arc following DNA replication in a and paternal chromosomes to separate, bur each pair of
mitotic cell cycle (see Figure 19-38). In prophase of meiosis chromatids remains associated at the centromere. During
I, homologous chromosomes (i.e., the maternal and paternal metaphase II, sister chromatids align on the metaphase II
chromosome I, the maternal and paternal chromosome 2, spindle and separase is activated yet again, cleaving the re-
etc.) pair with each other and undergo homologous recom- sidual cohesin around centromeres, facilitating anaphase II
bination. Significantly, at least one recombination event oc- (see Figure 19-40b).
curs between a maternal and a paternal chromosome. The
crossing over of chromatids produced by recombination can Recombination and a Meiosis-Specific Cohesin
be observed microscopically in the first meiotic prophase
Subunit Are Necessary for the Specialized
and metaphase as structures called chiasmata (singular, chi-
asma). In contrast, no pairing between homologous chromo- Chromosome Segregation in Meiosis I
somes occurs during mitosis, and recombination between As discussed earlier, in metaphase of meiosis I, both sister
nonsister chromatids is rare. Concomitant with homologous chromatids in one (replicated) chromosome associate with mi-
recombination, homologous chromosomes associate with crotubules emanating from the same spindle pole rather than
. each other in a process known as synapsis. In most organ- from opposite poles as they do in mitosis (see Figure 19-39).
isms this synapsis is mediated by a proteinaceous complex Two physical links between homologous chromosomes resist
known as the synaptonemal complex (SC). Homologous the pulling force of the spindle until anaphase: (a) crossing
chromosomes linked through chiasmata are called bivalents over between chromatids, one from each pair of homologous

19.8 Meiosis: A Special Type of Cell DivisiOn 91 s

0 FOCUS ANIMATION: Meiosis

FIGURE 19-38 Meiosis. Pre-meiotic cells have Paternal

two copies of each chromosome (2n), one derived homolog
Premeiotic
from the paternal parent and one from the maternal
cell (2n)
parent. For simplicity, the paternal and maternal Maternal
homologs of only one chromosome are dia- homolog
grammed. Step 0 : All chromosomes are replicated
during S phase before the first meiotic division,
giving a 4n chromosomal complement. Cohesin
complexes (not shown) link the sister chromatids
composing each replicated chromosome along
Repl icated
their full lengths. Step f) : As chromosomes chromosomes (4n)
condense during the first meiotic prophase,
replicated homologs pair and undergo homologous
recombination, leading to at least one crossover
event. At metaphase, shown here, both chromatids
of one chromosome associate with microtubules
emanating from one spindle pole, but each
D
1
Recombination and synapsis
betw~en homologs in prophase

member of a homologous chromosome pair

associates with microtubules emanating from Metaphase I
opposite poles. Step iJ : During anaphase of meiosis II)

I, the homologous chromosomes, each consisting of 'iii

0
two chromatids, are pulled to opposite spindle 'iii
::iE
poles. Step [1 : Cytokinesis yields the two daughter
cells (now 2n). which enter meiosis II without
undergoing DNA replication. At metaphase of meio-
sis II, shown here, the sister chromatids associate
with spindle microtubules from opposite spindle
poles, as they do in mitosis. Steps lit and r!l: Anaphase I
Segregation of sister chromatids to opposite
spindle poles during the second meiotic anaphase
followed by cytokinesis generates haploid gametes
(1 n) containing one copy of each ch romosome.
Micrographs on the left show meiotic metaphase I
and metaphase II in developing gametes from
Li/ium (lily) ovules. Chromosomes are aligned at the
metaphase plate. [Photos courtesy of Ed Reschke/Peter
Arnold, Inc.] Daughter cells
in metaphase II
(2n)

=
II)
'iii
0 Anaphase II
'Gi
::iE

ln ln 1n 1n
 Centriole (a) Mitosis
___.-(spindle pole)
<:J{' Metaphase Anaphase

' - . Spindle microtubules

..!,
Separase
\

".!.
/.--~.....
, .... -
-~~
.
()'- :-.~
'
.' ......
. '\
~

;- -
~ ;

(b) Meiosis
Metaphase I Anaphase I

,;;;~- _..... ~\; ..!,

:_
-.~. \
Separase
~~- ~'
Sgoi-PP2A Rec8
' FIGURE 19-39 Chiasmata and cohesins distal to them link
Metaphase II Anaphase II
homologous chromosomes in meiosis I metaphase. Connections
between chromosomes during meiosis I are most easily visualized in
organisms with acrocentric centromeres, such as the grasshopper. The
kinetochores at the cent rome res of sister chromatids attach to spindle
,~lid
_.!. :-:..
~~~!.
\ ..! ,
, \\::....... - - . (4,\ Separase
microtubules emanating from the same spindle pole, with the
kinetochores ofthe maternal (red) and paternal (blue) chromosomes '""'-
"
attaching to spindle microtubules from opposite spindle poles. The Rec8
maternal and paternal chromosomes are attached at the chiasmata FIGURE 19-40 Cohesin function during mitosis and meiosis.
which are formed by recombination between them and the cohesion (a) During mitosis, sister chromatids generated by DNA replication inS
between sister chromatid arms that persists throughout meiosis I phase are initially linked by cohesin complexes along the length of the
metaphase. Note that elimination of cohesion between sister chroma- chromatids. During chromosome condensation, cohesin complexes
tid arms is all that is required for the homologous chromosomes to (yellow) become restricted to the region of the centromere at meta-
separate at anaphase. [Adapted from L. V. Paliulis and R. B. Nicklas, 2000, phase. Mei-5332/Sgo 1 (purple) recruits PP2A to centromeres, where they
J. Cell Bioi. 150:1223.] antagonize Polo kinase and Aurora B, preventing the dissociation of
cohesins from centromeric regions. Dissociation of Mei-5332/Sgo 1 from
centro meres and activation of separase leads to removal of cohesins at
chromosomes, and (b) cohesins distal to the crossover point the centromere. Sister chromatids now separate, marking the onset of
(see figure 19-40b, top) . Evidence for the function of rccombi anaphase. (b) In prophase of meiosis I, maternal and paternal chroma-
nation during meiosis comes from the observation that when tids establish linkages between each other by homologous recombina-
recombination is blocked by mutations in proteins essential tion. By metaphase I, the chromatids of each replicated chromosome
for the process, chromosomes segregate randomly during are cross-linked by cohesin complexes along their full length. Rec8, a
meiosis I; that is, homologous chromosomes do not neces- meiosis-specific homolog of Sec 1, is cleaved along chromosome arms
but not around the centromere, allowing homologous chromosome
sari ly segregate to opposite spindle poles.
pairs to segregate to daughter cells. Centromeric Rec8 is protected from
At the onset of meiotic anaphase 1, cohesins between chro-
cleavage by PP2A, recruited to centromeric regions by the PP2A
mosome arms are cleaved by separase. This cleavage is re-
regulator Mei-5332/Sgol (shown in purple). By metaphase II, the
quired for homologous chromosomes to segregate. If cohesins Mei-5332/ Sgo 1-PP2A complex dissociates from chromosomes. Cohesins
were not lost from chromosome arms, the recombined sister can now be cleaved during meiosis II, allowing sister chromatids to
chromatid would rip during anaphase I. The maintenance of segregate. [Modified from F. Uhlmann, 2001 , Curr. Opin. Cell Bioi. 1 3:754.]
centromeric cohesion during meiosis I is necessary for the
proper segregation of sister chromatids during meiosis II.
Studies in many organisms have shown that a specialized cleaved by separase, so the sister chromatids can segregate,
cohesin subunit, Rec8, is necessary for the stepwise loss of as they do in mitosis (see figure 19-40b, bottom). Conse-
cohesins from chromosomes during meiosis. Expressed only quently, understanding the regulation of Rec8-cohesin com-
during meiosis, Rec8 is homologous to Sccl, the cohesin plex cleavage is central to understanding chromosome
subunit that closes the cohesin ring in the cohesin complex segregation in meiosis I.
of mitotic cel ls. Immunolocalization experiments revealed The mechanism that protects Rec8 from clea\age at cen-
that during early anaphase of meiosis I, Rec8 is lost from tromeres during meiosis I is similar to the mechanism that
chromosome arms but is retained at centromeres. However, protects Scc1 at centromeres during mitosis. Recall that dur-
during early anaphase of meiosis II, centromeric Rec8 is ing mitotic prophase, protein kinascs, chief among them Polo

19.8 Meiosis: A Special Type of Cell Division 917

kinase, phosphorylate cohesins in the chromatid arms, caus- nism. During meiotic metaphase I, kinetochore-associated mi-
ing them to dissociate and eliminating cohesion on chromatid crotubules are also under tension (even though the co-oriented
arms by metaphase. However, cohesion at the centromeres is kinetochores of sister chromatids attach to microtubules com-
maintained because a specific isoform of protein phosphatase ing from the same spindle pole) because chiasmata generated
2A (PP2A) is localized to centromeric chromatin by members by recombination between homologous chromosomes and the
of a family of proteins known as the Mei-S332/Shugoshin. cohesins distal to the chiasmata prevent them from being
PP2A keeps cohesin in a hypophosphorylated state that does pulled to the poles (see Figure 19-39). Since kinetochore-
not dissociate from chromatin (see Figure 19-40a). During microtubule attachments are unstable in the absence of tension
mt:taphase II, Mei-S332/Sgol dissociates from chromo- (due to Aurora B-mediated phosphorylatiOn), kinetochores
somes. In addition, when the last kinetochore is properly as- that attach to the wrong spindle fibers release the incorrect
sociated with spindle microtubules, Cdc20 is derepressed microtubules, enabling them to bind microtubules again
and associates with the APC/C, causing ubiquitinylation of until attachments are made that generate tension. As in mi-
securin. This releases separase activity, which cleaves Sccl tosis, once tension is generated, microtubule attachment to
whether it is phosphorylated or not, eliminating cohesion at the kinetochores is stabilized.
the centromere and allowing chromatid separation in ana-
phase (see Figure 19-40a).
DNA Replication Is Inhibited, Between
Cohesin removal differs for meiosis I because when
Rec8 replaces Sccl in the cohesin complex, the complex the Two Meiotic Divisions
does not dissociate in prophase when it is phosphorylated. The mechanism by which DNA replication is suppressed be-
The meiotic cohesin complex can only be removed from tween meiosis I and II is currently an active area of investiga-
chromatin via the action of separase. Rec8 also differs from tion, but it is thought that a change in the regulation of CDK
Sec I in that it must be phosphorylated by several protein activity is at least in part responsible for this suppression. The
kinases to be cleaved by separase. During meiosis I, the same S phase CDKs that promote DNA replication prior to
centromere-specific isoform of PP2A targeted to centromeric mitosis are needed for pre-meiotic DNA repli~ation. The same
chromatin by Mei-5332/Shugoshin prevents this phosphor- mitotic CDKs that promote mitosis also promote the meiotic
ylation. The PP2A targeting factor and PP2A then dissociate divisions, except we now call mitotic CDKs meiotic CDKs
from chromosomes by metaphase II, allowing separase since they promote the meiotic divisions and not mitosis.
cleavage of Rec8. So how is DNA replication prevented between the two
meiotic divisions? Following anaphase of meiosis l, meiotic
CDK activity does not fall as low as it does following mitotic
Co-orienting Sister Kinetochores Is Critical
anaphase. This partial drop in CDK activity is thought to be
for Meiosis I Chromosome Segregation sufficient to promote the disassembly of the meiosis I spindle
As discussed earlier, in mitosis and meiosis 11, sister kineto- but insufficient to promote MCM helicase loading (recall a
chores attach to. spindle microtu buies emanating from op- state of very low or no CDK activity is needed to load MCM
posite spindle poles; the kinetochores are said to be helicases). During prophase of meiosis II, meiotic CDK activ-
br-orzented. This is essential for segregation of sister chroma- ity rises again and the meiosis II spindle forms. After all
tids to different daughter cells. In contrast, at meiosis I meta- sister kinetochores have attached to microtubules from
phase, sister kinetochores attach to spindle microtubules opposite spindle poles, separase is activated and cells pro-
emanating from the same spindle pole; the sister kineto- ceed through meiosis II anaphase, telophase, and cytokinesis
chores are said to be co-oriented (see Figure 19-39). Obvi- to generate haploid germ cells.
ously, attachment of sister kinetochores to the proper
microtubules in meiosis I and II is critical for correct meiotic
segregation of chromosomes.
Proteins required for meiosis I sister kinetochore co- KEY CONCEPTS of Section 19.8
orientation were first identified in S. cerevisiae. We now Meiosis: A Special Type of Cell Division
know that a protein complex known as the monopolin com-
Meiosis is a specialized division in which meiosis-specific
plex associates with kinetochores during meiosis I and links
gene products modulate the mitotic cell division program
sister kinetochores to favor attachment to microtubules em-
(see Figure 19-38).
anating from the same spindle pole. In organisms where ki-
netochores attach to multiple microtubules, Rec8-containing The meiotic division comprises one cycle of chromosome
cohesins are essential for sister kinetochore co-orientation. replication followed by two cycles of cell division to produce
These meiosis-specific cohesins impose a rigid kinetochore haploid germ cells from a diploid pre-meiotic cell. During
structure, restricting the movement of sister kinetochores meiosis I, homologous chromosomes are segregated; during
and thereby favoring attachment to microtubules from the meiosis li sister chromatids separate.
same spindle pole. Specialized environmental conditions induce a develop-
Like during mitosis and meiosis II, correct attachment of mental program that leads to the meiotic divisions.
meiosis I chromosomes is mediated by a tension-based mecha-

918 CHAPTER 19 The Eukaryotic Cell Cycle

 the spindle assembly checkpoint. Many questions remain
During prophase of meiosis I, homologous chromosomes about how the plane of cytokinesis and the localization of
undergo recombination. At least one recombination event daughter chromosomes are determined in cells that divide
occurs between chromatids of homologous chromosomes, symmetrically and asymmetrically as is often seen as part of
linking the homologous chromosomes. development of complex tissues and organ structures. How
Cohesins distal to the chiasma are responsible for holding the cell cycle machinery is modulated by developmental cues
the homologous chromosomes together during prophase to bring about specialized divisions is also an intense area of
and metaphase of meiosis I. investigation.
Understanding the::.e detailed aspects of cell cycle control
At the omt!t of anaphase of meiosis I, cohesms on chromo-
will have significant consequences, particularly for the treat-
some arms are phosphorylated and as a result cleaved by
ment of cancers. Cancer cell!. often have defects in cell cycle
separase, but cohesins in the region of the centromere are
checkpoints that led to the accumulation of multiple muta-
protected from phosphorylation and cleavage. This protec-
tions and DNA rearrangements that result in the cancer
tion is brought about by a meiosis-specific cohesin subunit
phenotype. However, the absence of these checkpoints can
and a protein phosphatase that associates with centromeres.
make specific types of cancers particularly vulnerable to ex-
As a result, the chromatids of homologous chromosomes re-
tensive DNA damage induced with radiation therapy or
main associated during segregation in meiosis I.
chemotherapy. Normal cells activate cell cycle checkpoints
Cleavage of centromeric cohesins during anaphase of that arrest the cell cycle until the DNA damage is repaired.
meiosis IT allows individual chromatids to segregate into Bur these cancer cells do nor and as a consequence suffer
germ cells. sufficient genetic damage to induce apoptosis. If more were
A complex of meiosis-specific kinetochore proteins, known understood about cell cycle controls and checkpoint path-
as the monopolin complex, promote the co-orientation of sis- ways, it might be possible to design ever more effective ther-
ter chromatids during meiosis I. Both sister chromatids attach apeutic strategies, especially against types of cancer that are
to microtubules emanating from the same spindle pole. largely resistant to today's conventional therapies. It seems
very likely that better understanding of tht: molecular pro-
Incomplete CDK inactivation between the two meiotic di-
cesses involved will allow the design of more effective treat-
visions inhibits DNA replication.
ments in the future.

Perspectives for the Future

The remarkable pace of cell cycle research over the last 25 Key Terms
years has led to a detailed model of eukaryotic cell cycle con- anaphase-promoting maturation-promoting
trol. A beautiful logic underlies these molecular controls. complex/cyclosome factor (MPF) 880
Each regulatory event has two important functions: to acti- (APC/C) 888 meiosis 913
vate a step of the cell cycle and to prepare the cell for the
APC/C specificity factor 888 mitogen 892
next event of th~ cycle. This strategy ensures that the phases
of the cycle occur in the proper order. ATM/ATR 909 mitosis 875
Although the general logic of cell cycle regulation now Aurora B kinase 901 mitotic CDKs 883
seems well established, many critical details remain to be dis- Cdc14 phosphatase 913 monopolin complex 918
covered. For instance, how cell growth and division are co- Cdc25 phosphatase 898 p53 protein 909
ordinated and how the metabolic stare of a cell feeds into its CDK-activating kinases Polo kinases 898
cell cycle machinery remain to be discovered. A number of (CAK) 888
key nutrient and growth-factor-sensing pathways such as the Rh protein 891
CDK inhibitor (CKls) 888 SCf (Skp 1, Cullin, F-box
AMPK, Ras, and TOR pathways have been identified and
their inner workings recently revealed. Understanding how checkpoint pathway 874 proteins) 888
they impact the cell cycle machinery will be a critical ques- cohesin 896 S phase CDKs 883
tion to be addressed in the years to come. Substantial prog- condensin 902 START 875
ress has been made recently in identifying substrates critical cell size 892 securin 903
phosphorylated by different CDKs, but much work remains crossing over (or cross sensor 906
to be done ro understand how the modification of these pro- ovcr)915
teins leads to the multiple events that these CDKs trigger. separase 903
cyclin 874 sister chromatids 875
Much has been discovered recently about the operation
of cell cycle checkpoint pathways, but the mechanisms that cyclin-dependent kinase synaptonemal complex 915
activate ATM and ATR in the DNA damage checkpoint are (CDK) 874
Weel protein-tyrosine
poorly understood. Likewise, much remains to be learned E2F transcription factor 887 kinase 898
about the control and mechanism of regulation of Mad2 in G 1 CDKs 883

Key Terms 919

 gene. What did the characterization of the wee 1 gene tell us
Review the Concepts
about cell cycle control?
1. What cellular mechanism(s) ensure that passage through 14. Describe how cells know whether sister kinetochores are
rhe cell cycle is unidirectional and irreversible? What mo- properly attached to the mitotic spindle.
lecular machinery underlies these mechanism(~)? 15. Describe the series of events by which the APC/C pro-
2. What types of experimental strategies do researchers em- motes the separation of sister chromatids at anaphase.
ploy to study cell cycle progression? How can these strate- 16. Meiosis and mitosis are overall analogous processes in-
gies differ based on generic versus biochemical approaches? volving many of the same proteim. However, some proteins
3. Tim Hunt shared the 2001 Nobel Prize in Physiology or function uniquely in each of these cell-division events. Ex-
Medicine for his work in the discovery and characterization plain the meio'>is-specific function of the following: (a) lme2,
of cyclin proteins in eggs and embryos. Describe the experi- (b) Rcc8, (c) monopolin.
mental steps that led him to his discovery of cyclins. 17. Leland Hartwell, the third recipient of the 2001 Nobel
4. What experimental evidence indicates that cyclin B is re- Prize in Physiology or Medicine, was acknowledged for his
quired for a cell to enter mitosis? What evidence indicates characterization of cell cycle checkpoint pathways in the
that cyclin B must be destroyed for a cell to exit mitosis? budding yeastS. cerevisiae. What is a cell cycle checkpoint
5. What physiological differences between S. pombe and S. pathway? When during the cell cycle do checkpoint path-
cerevisiae make them useful yet complementary tools for ways function? How do cell cycle checkpoint pathways help
studying the molecular mechanisms involved in cell cycle to preserve the genome?
regulation and control? 18. What role do tumor suppressors, including p53, play in
6. ln Xenofms, one of the substrates of mitotic CDKs is the mediating cell cycle arrest for cells with DNA damage?
Cdc25 phosphatase. When phosphorylated by mitotic 19. Individuals with the hereditary disorder ataxia telangi-
CDKs, Cdc25 is activated. What is the substrate of Cdc25? ectasia suffer from neurodegeneration, immunodeficiency,
How does this information help to explain the rapid rise in and increased incidence of cancer. The g"enetic basis for
mitotic CDK activity as cells enter mitosis? ataxia telangiectasia is a loss-of-function mutation in the
7. Explain how CDK activity is modulated by the following ATM gene (ATM 5; ataxia telangiectasia mutated ). Besides
proteins: (a ) cyclin, (b) CAK, (c) Weel, (d) p21. p53, what other substrate is phosphorylated by ATM? How
does the phosphorylation of this substrate lead to inactiva-
8. Explain the role of CDK inhibitors. If cyclin-CDK com-
tion of CDKs to enforce cell cycle arrest?
plexes are required to allow regulated progression through
the eukaryonc cell cycle, what would be the physiological
rationale for CDK inhibitors?
Analyze the Data
9. What is the functional definition of START? Cancer cells
typically lose START control. Explain how the following 1. Many of the proteins that regulate transit through the cell
mutations, which arc found in some cancer cells, lead to a cycle have been characterized. Xnf7, identified in extracts of
bypass of START controls: (a) overexpression of cyclin D, Xenopus eggs, binds to the anaphase-promoting complex/
(b) loss of Rb function, (c) loss of p 16 function, (d) hyperac- cyclosome (A PC/C). To elucidate the function of this pro-
tive E2F. tein, studies have been undertaken in which Xnf7 either has
10. The Rb protein has been called the "master brake" of been depleted from extracts using an antibody raised against
the cell cycle. Describe how the Rb protein acts as a cell cycle it or has been augmented in the extracts through addition of
brake. How is the brake released in mid- to late G 1 to allow extra Xnf7. The consequences on transit through mitosis
the cell to proceed to the S phase? were then assessed (see J. B. Casaletto eta!., 2005, f. Cell Bioi.
11. A common feature of cell cycle regulation is that the 169:61-71 ).
events of one phase ensure progression into a subsequent a. Xenopus egg extracts, arrested in metaphase, were
phase. InS. cerevisiae, G 1 and G 1/S phase CDKs promote either depleted of Xnf7 or were mock depleted (subjected to
S-phase entry. Name two ways in which they promote the the same treatment as the first sample but without Xnf7
activation of S phase. antibody), then released from metaphase arrest by addition
of Ca 2 . Aliquots of the extract were then sampled at vari-
12. For S phase to be completed in a timely manner, DNA
ous times after Ca 2 addition and the amounts of mitotic
replication initiates from multiple origins in eukaryotes. InS.
cyclin determined, as shown on the Western blot below.
cereuisiae, what role do S-phase CDKs and DDK complexes
What information do these data provide about a possible
play to ensure th;:~t the entire genome is replicated once and
function for Xnf7?
only once per cell cycle?
13. In 2001, the Nobel Prize in Physiology or Medicine was Time (mon) 0 5 10 15 20 30 40 60
awarded to three cell-cycle scientists. Paul Nurse was recog- Mock depleted
nii'ed for his studies with the fission yeastS. pombe, in par-
Xnf7 depleted -
ticular for the discovery and characterization of the weer+-

920 CHAPTER 19 The Eukaryotic Cell Cycle

 b. In additional studies, exogenous Xnf7 was added to a. What type of assay(s) could be employed to determme
Xenopus egg extracts, arrested at metaphase, so that the whether the expression of cyclin B is regulated at the tran-
total amount of this protein in the extracts was higher than scriptional or translational level, if either?
normal. The extracts, released from arrest by Cal+ addition, b. Based on what you've learned in Chapter 19, is it pos-
were then assessed at various times after release for mitotic sible that the activity of cyclin B could be regulated at the
cyclin ubiquitinylation (cyclin-Ub conjugates). What is the post-translational level? Describe a cellular mechanism
rationale for examining ubiquitinylation? Using the follow- through which this might happen.
ing figure, determine what information these studies add be- c. How might it be possible for cyclin B expression and/
. yond that obtained from p:nr (a). or activity to be at least partially regulated by events in the
cell's external environment?

No addition Xnf7 addition

T1me (mon) 0 4 6 8 10 12 16 20 30 0 4 6 8 10 12 16 20 30

References
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Meiosis: A Special Type of Cell Division
Surveillance Mechanisms in Cell Cycle Reg ulation lshiguro, K., and Y. Watanabe. 2007. Chromosome cohe,ion in
Jorgensen, P., and M. Tyers. 2004. How cells coordinJte growth mitosis and me10sas . .f. Cell Sc1. 120(Pt. 3):367-369.
and division. Curr. Bzol. 14(23):R 1014-1027. .'v1arston, A. L., and A. Amon. 2004. Meiosis: cell-cycle controls
Bartek, J., and J. Lukas. 2007. DNA damage checkpoints: shuffle and deal. Nature Rev. Mol. Cell Bioi. 5:983-997.
from imnation to recovery or adaptation. Curr. Opin. Cell Bwl. Zickler, D., and N. Kleckner. 1999. Meiotic chromosomes:
19(2):238-245. mtegrarmg structure and function. Am!. Rev. Genet. 33:603-754.

922 CHAPTER 19 The Eukaryotic Cell Cycle

 CLASSIC EXPERIMENT 19.1

Cell Biology Emerging from the Sea:

The Discovery of Cyclins
T. Evans et al., 1983, Ce//33:391

rom the first cell divisions after fer-

F tilization to aberrant divisions that
occur in cancers, biologists have long
stages of development. Ruderman and
Hunt, while teaching a physiology
Soon afterward, in a third study,
Hunt exammed the changes in protein
course at the Marine Biological Labo- expression during the maturation and
been interested in how cells control ratory in Woods Hole, Massachusetts, fertilization of sea urchin oocytes. This
when they divide. The processes of cell began a set of experiments designed to time he performed the experiment in a
division have been separated into uncover the genes that were expressed slightly different manner. Rather than
stages known collectively as the cell at this point as well as the mechanism treating the oocytes and embryo with
cycle. While studying early develop- by which this burst of protein synthesis radioactively labeled amino aCids for a
ment in marine invertebrates in the was controlled. set time period, he labeled the cells
early 1980s, Joan Ruderman and Tim continuously for more than 2 hours,
Hunt discovered the cyclins, key regu- removing samples for analysis at
lators of the cell cycle. The Experiment 10-minute intervals. Now he could
In a collaborative project, Ruderman monitor the changes in protein expres-
and Hunt looked at regulation of gene sion throughout the early stages of de-
Background
expression in the fertilized egg of the velopment. As had been shown 111
The question of how an organism de- surf clam Spisula solidissima. Whereas other organisms, the pattern of protein
velops from a fertilized egg continues it was known that overall protein syn- synthesis was altered when the sea ur-
to drive a large body of scientific re- thesis rapidly increased on fertiliza- chin oocyte was fertilized. Three pro-
search. Whereas such research was tion, they wanted to find out whether teins-represented by three prominent
classically the concern of embryolo- the proteins expressed in the earliest bands on an autoradiograph-were
gists, the developing understanding of stage of development, the two-cell em- expressed in the embryos, but not in
gene expression in the 1980s brought bryo, were different from those ex- the oocytes. Interestingly, the intensity
new approaches to answer this ques- pressed in the unfertilized egg. When of one of these bands changed over
tion. One such approach was to exam- either eggs or two-cell clam embryos time: the band was intense at the early
ine the pattern of gene expression in are treated with radioactively labeled time points, then barely visible after 85
both the oocyte and the newly fertilized amino acids, the cell takes up the minutes. It increased in intensity again
egg. Ruderman and Hunt were among amino acids, which are subsequently between 95 and 105 minutes. The in-
the biologists who took this approach incorporated into newly synthesized tensity of the hand, representing the
to the study of e11rly development. proteins. Using this technique, Ruder- amount of the protein in the cell, ap-
Biologists had well characterized man and Hunt monitored the pattern peared to be oscillating over time (Fig-
the early development of a number of of protein synthesis by breaking open ure la). This suggested that the protein
marine invertebrate systems. Their the cells, separating the proteins using had been quickly degraded and then
eggs arc fertilized externally, allowing SDS-polyacrylamide gel electrophore- synthesized again.
researchers to study their development sis (SDS-PAGE), and then visualizing Because the time frame of the ex-
in a plastic dish. During the early the radioactively labeled proteins by periment coincided w1th early embry-
stages of development, tl1c embryonic autoradiography. When they com- onic cell divisions, Hunt next asked
cells divide synchronously, which al- pared the pattern of protein synthesis whether the synthesis and destruction
lows an entire population of cells to be in the egg with that in the two-cell em- of the protein was correlated with pro-
studied at the same stage of the cell bryo, they saw that three different pro- gression of the cell cycle. He examined
cycle. Researchers had established that teins that were either not expressed or a portion of cells from each time point
a large part of the mRNA in the unfer- expressed at an extremely low levels in under a microscope, counting the
tilized oocyte is not translated. On fer- the egg were highly expressed in the number of cells dividing at each time
tilization, these maternal mRNA are embryo. ln a subsequent study, Ruder- point where samples had been taken
rapidly translated. Previous studit><; man examined the pattern of protein for protein analy~is. Hunt then corre-
had shown that when fertilized eggs expression in the oocytes of the star- lated the amount of the protein present
are treated with drugs that inhibit pro- fish Asterias forbesi as they mature. in the cell with the proportion of cells
tein synthesis, cell division could not She again observed the increased ex- dividing at each time point. He noticed
take place. This suggested that the initial pression of three proteins of similar that the level of expression of one of
burst of protein synthesis from the ma- size to those that she and Hunt had the proteins was highest before the cell
ternal mRNA is required at the earliest seen in surf clam embryos. divided and lowest on cell division

Cell Biology Emerging from the Sea: The Discovery of Cyclins 923
(a) Min (postfertil ization) FIGURE 1 Autoradiography permits the detection of cyclical
(() (() (() (() synthesis and destruction of mitotic cyclin in sea urchin embryos.
(() (() (() (() (O(O(O(OO N C"l
N C"l '<t LO (() r-- 00 0) A suspension of sea urchin eggs was synchronously fertilized by the ad-
dition of sea urchin sperm, and 35 5-methionine was added. At
10-minute intervals beginning 26 minutes after fertilization, samples
Cyclin-+
were taken for protein analysis on an 50S-polyacrylamide gel and for
detection of cell cleavage by microscopy. (a) Autoradiogram of the SDS
B-+ gel showing samples removed at each time point. Most proteins, such
as Band C. continuously increased in intensity. In contrast, cyclin
C-+ suddenly decreased in intensity at 76 minutes after fertilization and
then began increasing again at 86 minutes. The cyclin band peaked
again at 106 min and decreased again at 126 min. (b) Plot of the
intensity of the cyclin band (red line) and the fraction of cells that had
(b) undergone cleavage during the previous 10-minute interval (cyan line).
Note that the amount of cyclin fell precipitously just before cell
"'C
50
c cleavage. [From T. Evans et al., 1983, Ce// 33:389; courtesy of R. Timothy Hunt,
It)
.0 Cl- Imperial Cancer Research Fund.)
.s :Ec .g Q)

~ >
- -a
u
0
~
'iii
.!!!.
ai~
u
25

c ~.
Q)

c ~
"'C

60 120
Min (postfertilization)

(Figure lb), suggesting a correlation proteins regulate the cell cycle by as- the first cyclins, scientists have identi-
with the stage of the cell cycle. When sociating with cyclin-depcndent ki- fied at least 15 other cyclins that regu-
the same experiment was performed in nases, which in turn regulate the late all phases of the cell cycle.
the surf clam, Hunt saw that two of activities of a variety of transcription In addition to the basic resea rch in-
the proteins that he and Ruderman and replication factors, as well as other terest in these proteins, the cyclins' cen-
had described previously displayed the proteins involved in the complex al- tral role in cell division has made them
same pattern of synthesis and destruc- terations in cell architecture and chro- a focal point in cancer research. Cyclins
tion. Hunt called these proteins eye/ins mosome structure that occur during arc involved in the regu lation of several
to reflect their changing expression mitosis. In brief, cyclin-CDK com- genes that are known to play prominent
through the cell cycle. plexes direct and regulate progression roles in tumor development. Scientists
through the cell cycle. As with so many have shown that at least one cyclin, cy- .
key regulators of cellular functions, it clin 01, is overexprcssed in a number of
Discussion was soon shown that the cyclins dis- tumors. The role of these proteins in
The discovery of the cyclins heralded covered in sea urchins and surf dams both normal and aberrant cell division
an explosion of investigation into the are conserved in eukaryotes from yeast continues to be an active and exciting
cell cycle. It is now known that these to humans. Since the identification of area of research today.

924 CHAPTER 19 The Eukaryotic Cell Cycle