LECTURE 3

DNA REPLICATIoN

DNA replication is the process by which DNA makes a copy of itself during cell
division. DNA or deoxyribonucleic acid is a type of molecule known as
a nucleic acid. It consists of a 5-carbon de-oxyribose sugar, a phosphate, and
a nitrogenous base. Double-stranded DNA consists of two spiral nucleic acid
chains that are twisted into a double helix shape. This twisting allows DNA to
be more compact. In order to fit within the nucleus, DNA is packed into tightly
coiled structures called chromatin. Chromatin condenses to
form chromosomes during cell division. Prior to DNA replication, the
chromatin loosens giving cell replication machinery access to the DNA
strands. DNA is the genetic material that defines every cell. Before
a cell duplicates and is divided into new daughter cells through
either mitosis or meiosis, biomolecules and organelles must be copied to be
distributed among the cells. DNA found within the nucleus, must be replicated
in order to ensure that each new cell receives the correct number
of chromosomes. The process of DNA duplication is called DNA replication.
Replication follows several steps that involve multiple proteins called
replicons, enzymes and RNA. In eukaryotic cells, such as animal
cells and plant cells, DNA replication occurs in the S phase of
interphase during the cell cycle. The process of DNA replication is vital for cell
growth, repair, and reproduction in organisms.

Preparation for Replication

Step 1: Replication Fork Formation

Before DNA can be replicated, the double stranded molecule must be

“unzipped” into two single strands. DNA has four bases called adenine
(A), thymine (T), cytosine (C) and guanine (G) that form pairs between the
two strands. Adenine only pairs with thymine and cytosine only binds with
guanine. In order to unwind DNA, these interactions between base pairs must
be broken. This is performed by an enzyme known as DNA helicase. DNA
helicase disrupts the hydrogen bonding between base pairs to separate the
strands into a Y shape known as the replication fork. This area will be the
template for replication to begin.
DNA is directional in both strands, signified by a 5' and 3' end. This notation
signifies which side group is attached the DNA backbone. The 5' end has a
phosphate (P) group attached, while the 3' end has a hydroxyl (OH) group
attached. This directionality is important for replication as it only progresses
in the 5' to 3' direction. However, the replication fork is bi-directional; one
strand is oriented in the 5’ to 3’ direction (leading strand) while the other is
oriented 3' to 5’ (lagging strand). The two sides are therefore replicated with
two different processes to accommodate the directional difference.

Replication Begins

Step 2: Primer Binding

The leading strand is the simplest to replicate. Once the DNA strands have
been separated, a short piece of RNA called a primer binds to the 3' end of the
strand. The primer always binds as the starting point for replication. Primers
are generated by the enzyme DNA primase.

Step 3: Elongation

Enzymes known as DNA polymerases are responsible creating the new

strand by a process called elongation. There are five different known types of
DNA polymerases in bacteria and human cells. In bacteria such as E.
coli, polymerase III is the main replication enzyme, while polymerase I, II, IV
and V are responsible for error checking and repair. DNA polymerase III binds
to the strand at the site of the primer and begins adding new base pairs
complementary to the strand during replication. In eukaryotic cells,
polymerases alpha, delta, and epsilon are the primary polymerases involved
in DNA replication. Because replication proceeds in the 5' to 3' direction on
the leading strand, the newly formed strand is continuous.

The lagging strand begins replication by binding with multiple primers. Each
primer is only several bases apart. DNA polymerase then adds pieces of DNA,
called Okazaki fragments, to the strand between primers. This process of
replication is discontinuous as the newly created fragments are disjointed.
Step 4: Termination

Once both the continuous and discontinuous strands are formed, an enzyme
called exonuclease removes all RNA primers from the original strands. These
primers are then replaced with appropriate bases. Another exonuclease
“proofreads” the newly formed DNA to check, remove and replace any errors.
Another enzyme called DNA ligase joins Okazaki fragments together forming
a single unified strand. The ends of the linear DNA present a problem as DNA
polymerase can only add nucleotides in the 5′ to 3′ direction. The ends of the
parent strands consist of repeated DNA sequences called telomeres.
Telomeres act as protective caps at the end of chromosomes to prevent
nearby chromosomes from fusing. A special type of DNA polymerase enzyme
called telomerase catalyzes the synthesis of telomere sequences at the ends
of the DNA. Once completed, the parent strand and its complementary DNA
strand coils into the familiar double helix shape. In the end, replication
produces two DNA molecules, each with one strand from the parent molecule
and one new strand.

Replication Enzymes
DNA replication would not occur without enzymes that catalyze various steps
in the process. Enzymes that participate in the eukaryotic DNA replication
process include:

• DNA helicase - unwinds and separates double stranded DNA as it

moves along the DNA. It forms the replication fork by
breaking hydrogen bonds between nucleotide pairs in DNA.
• DNA primase - a type of RNA polymerase that generates RNA primers.
Primers are short RNA molecules that act as templates for the starting
point of DNA replication.
• DNA polymerases - synthesize new DNA molecules by
adding nucleotides to leading and lagging DNA strands.
• Topoisomerase or DNA Gyrase - unwinds and rewinds DNA strands to
prevent the DNA from becoming tangled or supercoiled.
• Exonucleases - group of enzymes that remove nucleotide bases from
the end of a DNA chain.
• DNA ligase - joins DNA fragments together by forming phosphodiester
bonds between nucleotides.

NB: DNA replication must take place before a cell divides

CELL REPRODUCTION
For any cell to reproduce successfully, three events must be met.
1. The genetic information must be copied (i.e. DNA replication)
2. The copies of the genetic information must be separated from one
another
3. The cell must divide.

CHROMOSOMES
A chromosome consists of a single molecule of DNA. This DNA in eukaryotes,
although linear, are highly folded and condensed and if stretched out, some
human chromosomes would be several centimeters long, to package this
tremendous length of DNA into small volume, each DNA molecule is coiled
again and again and tightly packed around histone proteins forming the rod
shaped chromosomes.
Most of the time, chromosomes are thin and difficult to observe but before cell
division, they condense further into readily observed structures. It is at this
stage that they are usually studied.
A functional chromosome has three essential elements- a Centromere, a pair
of Telomeres and Origin of replication.
Microtubules are filaments responsible for moving chromosomes during cell
division. Before cell division, a protein complex called the Kinetochore
assembles on the centromere and then the spindle microtubules later attach
to the centromere.
Chromosomes without a centromere cannot be drawn to newly formed nuclei;
these chromosomes are lost, often with catastrophic consequences to the cell.
The centromere of a chromosome can be classified into four locations. On the
basis of the location of the centromere, chromosomes are classified into
metacentric, submetacentric, acrocentric and telocentric.
The eukaryotic chromosomes exist in four major types on the position of the
centromere. The telomeres provide chromosomes stability, they are the
natural ends (tips) of the chromosome. If a chromosome breaks producing
new ends, these ends have a tendency to stick together and degrade and so,
the telomeres come in to provide stability. Origin of replication are sites
where DNA synthesis begins. Bacterial chromosomes have a single replication
origin but eukaryotes have many origins of replication.
In preparation for cell division, each chromosome (DNA) replicates, making a
copy of itself. These two initially identical copies (called sister chromatids) are
held together at the centromere. Each sister chromatid consists of a single
molecule of DNA.
THE CELL CYCLE
The cell cycle is a life history of a cell. It is the stages through which it passes
from one division to the next. This process is critical to genetics because
through the cell cycle, the genetic instructions are passed from parent to
daughter cells. A new cycle begins after a cell has divided and produced two
new cells. A new cell metabolizes, grows and develops and at the end of its
cycle, the cell divides to produce two cells which can then undergo additional
cell cycles. Progression through the cell cycle is regulated at key transition
points called checkpoint.
STAGES OF CELL CYCLE- It consists of two stages
✓ Interphase: The period in which the cell grows, develops and prepares
for cell division. Phases of interface include G1 phase, S phase and G2
phase.
• In G1 phase; the cell grows and synthesizes protein necessary for
cell division. At G1 phase each chromosome is composed of one
chromatid.
• S phase [DNA synthesis phase], in this phase each chromosome
duplicates [DNA replicates] i.e. [2 copies of each chromosome
emerges].
NB: Before the S phase, each chromosome is composed of one
chromatid but after the S phase each chromosome is composed of two
chromatids.
• G2 phase: After the S phase, the cell enters the G2 phase where
several biochemical events necessary for cell division takes place.
NB: Throughout interface, the chromosomes are relaxed and uncoiled
and are visible only as diffused chromatin with the microscope.

✓ The M phase [Mitotic phase]. The period of active cell division.

At the M phase copies of the cells chromosome [sister chromatids]
separate and the cell undergoes division. This stage of separation is critical
because it results in complete copying of genetic information for each of the
resulting cells. The M phase is divided into six stages i.e. five stages of mitosis
and then cytokinesis. The five stages of Mitosis [M phase] include; [prophase,
prometaphase, metaphase, anaphase, telophase] and then cytokinesis.

• Prophase: During prophase the diffuse chromatin condenses and

become visible under a light microscope.
• Prometaphase: Starts with the disintegration of the nuclear
membrane.
• Metaphase: In this phase, chromosomes arrange themselves in a
single plain [the metaphase plate] between the two centrosomes.
• Anaphase- Anaphase begins when sister chromatids separate and
move towards opposite spindle poles. When the sister chromatids
separate, each is then considered a separate chromosome.
• Telophase- Telophase is marked by the arrival of the
chromosomes at the spindle poles, nuclear membrane re-forms
around each set of chromosome producing two separate nuclei
within the cell.
The chromosome relaxes and then lengthens and once again
disappearing from view. In many cells, division of the cytoplasm
[cytokinesis] is simultaneous with telophase.
• In Cytokinesis – The cytoplasm divides and cell wall re-forms in
plant cells.
 SEXUAL REPRoDUCTIoN: GENETIC VARIATIoN.

If all reproduction were accomplished through cell cycle, life would be

quiet dull because mitosis produces only genetically identical progeny.
With only mitosis, you, your parents, your children, brothers, sisters and
cousins and many you do not know will be clones [i.e. copies of one
another]. Only occasional mutation would introduce any genetic
variability. This was how all organisms reproduced for the first 2 billion
years on earth’s existence and the way in which some organisms still
reproduce today. Then some 1.5-2 billion years ago, something
remarkable evolved cells that produce genetically variable offspring
through sexual reproduction. The evolution of sexual reproduction is
one of the most significant events of the history of life. The pace of
evolution depends on the amount of genetic variation present. By
shuffling the genetic information from two parents, sexual reproduction
greatly increases the amount of genetic variation and allows for
accelerated evolution. Sexual reproduction consists of two processes:

Meiosis- Leads to gamete formation in which chromosome number is

reduced by half.
Fertilization- This is the second process of sexual reproduction in
which two haploid gametes fuse and restore chromosome number to its
original diploid value.
MEIoSIS;
The words mitosis and meiosis are sometimes confusing and they
sound alike and both refer to chromosome division and
cytokinesis. But the outcomes of mitosis and meiosis are radically
different and several unique events that have important genetic
consequences take place only in meiosis. Like mitosis, meiosis is
preceded by an interface stage that includes G1, S and G2 phases.
Meiosis consists of two distinct processes, meiosis 1 and meiosis
11. Each of which includes a cell division. The cell division comes
at the end of meiosis 1 termed Reduction division because the
number of chromosomes per cell is reduced by half. The second
division which comes at the end of meiosis 11 is termed
Equational division. The events of meiosis 11 are similar to those
of mitosis, however, meiosis 11 differs from mitosis in that
chromosomes has already been halved in meiosis 1 and the cell
does not begin with the same number of chromosomes as it does
in mitosis.

STAGES oF MEIoSIS 1
Interface: GI, S and G2.
G1- Period of growth and development of the cell, protein necessary for
cell division are synthesized.
S Phase- [DNA synthesis] in which the chromosomes duplicate.
G2- Biochemical events necessary for the cell division takes place.
Throughout interface the chromosomes are relaxed as visible diffuse
chromatin.
Prophase 1: Is divided into; Leptotene, zygotene, pachytene, diplotene
and diakinesis
Leptotene- Chromosomes contract and become visible.
Zygotene- Condensation of chromosomes continues, homologous
chromosomes pair up, homologous pairs of chromosomes starts
synapsing [a very close association] each synapse chromosome consists
of four chromatids called bivalent/tetrad.
Pachytene- Chromosomes become shorter and thicker, a three-part
synaptomenal [chiasmata] develops between homologous
chromosomes, crossing over takes place in which homologous
chromosomes exchange genetic information.
Diplotene- Centromere of paired chromosomes move apart,
homologous chromosomes remain attached at each chiasma which is
the result of crossing over.
Diakinesis- Chromosome condensation still continues chiasmata moves
towards chromosome ends and the strand slip apart and the
homologous chromosomes remain paired only at the tips. Near the end
of prophase 1, the nuclear membrane breaks down and the spindle
forms.

Metaphase 1- Homologous pairs of chromosomes align along the

metaphase plate, a microtubule from each pole attaches to one
chromosome of a homologous pair.
Anaphase 1- Separation of homologous chromosomes in which the two
chromosomes of a homologous pair are pulled towards opposite poles.
Although the homologous chromosomes separate, their sister
chromatid remain attached and travel together.
Telophase 1- Chromosomes arrives at the spindle poles and the
cytoplasm divides.
The period between meiosis 1 and 11 are is called Interkinesis in
which nuclear membrane reforms around the chromosomes clustered
at each pole, the spindle [microtubule] breaks down and chromosomes
relax, the cell then passes into the next stage of meiosis.
MEIoSIS 11
Prophase 11- In which these events are reversed, the chromosomes
recondense, the spindle re-forms and the nuclear membrane once again
breaks down. In Interkinesis in some types of cells, the chromosomes
remain condensed and the spindle does not break down, these cells
move directly from cytokinesis to metaphase11.
Metaphase 11- This is similar to metaphase of mitosis. The individual
chromosomes line up on the metaphase plate with the sister chromatids
facing opposite poles.
Anaphase 11- The Kinetochore of the sister chromatids separate and
the chromatids are pulled to the opposite poles.
Telophase 11-Each chromatid is now a distinct chromosome and the
cytoplasm divides, the chromosomes relaxes and are no longer visible.
DIFFERENCE BETEWEEN MIToSIS AND MEIoSIS
• Mitosis consists of a single nuclear division and usually
accompanied by a single cell division while meiosis consists of
two successive cell divisions.
• After mitosis, chromosome number in newly formed cells is the
same as in the original cell whereas meiosis causes chromosome
number in the newly formed cell to be reduced by half.
• Mitosis produces two genetically identical cells [i.e. daughter cells
[somatic cells] that are genetically identical] whereas meiosis
produce four genetically variable daughter cells [germ/sex cells
that are genetically different.]
• Mitosis is the type of cell division that makes new body cells
[creates all body cells [somatic] whereas meiosis is a type of cell
division that creates egg and sperm cells. [I.e. creates sex cells
only.].
 • Mitosis ensures that a cell duplicates all its contents including its
chromosomes and splits to from identical daughter cells while
meiosis ensures that humans have the same number of
chromosomes in each generation. Meiosis also allows for genetic
variation through a process f gene shuffling while cells are
dividing.
• Mitosis results in diploid daughter cell [chromosome number
remains the same as the parent cell whereas meiosis results in
haploid daughter cells, chromosome number are halved from
parent cells.
• In mitosis, prophase is much shorter, no recombination/crossing
over occurs whereas in meiosis, prophase 1 takes much longer
and involves recombination/crossing over of chromosomes.
• In mitosis, during metaphase stage individual chromosomes
[pairs of chromatid] lines up along the equator whereas in meiosis
[metaphase 1] pairs of homologous chromosomes line up along
the equator.
• During anaphase stage in mitosis, the sister chromatids are
separated to opposite poles whereas in meiosis sister chromatids
move together to the same pole during anaphase 1 and are then
separated to opposite poles during anaphase 11.

SIMILARITIES oF MIToSIS AND MEIoSIS

• Both involves diploid parent cells

• Both involves interface, prophase, metaphase, anaphase and
telophase
• Both ends with cytokinesis