DNA Replication and Repair

• DNA REPLICATION
• DNA REPAIR
DNA acts as a template for its own duplication. Because the nucleotide A will successfully pair only with
T, and G with C, each strand of a DNA double helix—labeled here as the S strand and its complementary
S′ strand—can serve as a template to specify the sequence of nucleotides in its complementary strand. In
this way, both strands of a DNA double helix can be copied precisely.

Note ANTI-PARALLEL nature of

replication

In each round of DNA replication, each of the two strands of DNA is used as a template for the
formation of a new, complementary strand. DNA replication is “semiconservative” because
each daughter DNA double helix is composed of one conserved strand and one newly
synthesized strand.
 origin of replication

A DNA double helix is opened at replication origins. DNA sequences at replication origins are recognized by initiator proteins (not shown), which locally pry
apart the two strands of the double helix. The exposed single strands can then serve as templates for copying the DNA.
 Three models for DNA replication make different predictions.

Three models for DNA replication make different predictions. (A) In the semiconservative model, each parent strand serves as a template for the synthesis of a
new daughter strand. The first round of replication would produce two hybrid molecules, each containing one strand from the original parent in addition to one
newly synthesized strand. A subsequent round of replication would yield two hybrid molecules and two molecules that contain none of the original parent DNA
(see Figure 6-3). (B) In the dispersive model, each generation of daughter DNA will contain a mixture of DNA from the parent strands and the newly synthesized
DNA. (C) In the conservative model, the parent molecule remains intact after being copied. In this case, the first round of replication would yield the original
parent double helix and an entirely new double helix. For each model, parent DNA molecules are shown in orange; newly replicated DNA is red. Note that only a
very small segment of DNA is shown for each model.
 Centrifugation in a cesium chloride gradient allows the separation
of heavy and light DNA.

Centrifugation in a cesium chloride gradient allows the separation of heavy and light DNA. Bacteria are grown for several generations in a medium containing either
15N (the heavy isotope) or 14N (the light isotope) to label their DNA. The cells are then broken open, and the DNA is loaded into an ultracentrifuge tube containing a
cesium chloride salt solution. These tubes are centrifuged at high speed for two days to allow the DNA to collect in a region where its density matches that of the salt
surrounding it. The heavy and light DNA molecules collect in different positions in the tube.
The first part of the Meselson-Stahl experiment ruled out the conservative model of DNA replication. (A) Bacteria grown in light medium (containing 14N) yield DNA
that forms a band high up in the centrifuge tube, whereas bacteria grown in 15N-containing heavy medium (B) produce DNA that migrates further down the tube. When
bacteria grown in a heavy medium are transferred to a light medium and allowed to continue dividing, they produce a band whose position falls somewhere between that
of the parent bands (C). These results rule out the conservative model of replication but do not distinguish between the semiconservative and dispersive models, both of
which predict the formation of hybrid daughter DNA molecules. The fact that the results came out looking so clean—with discrete bands forming at the expected
positions for newly replicated hybrid DNA molecules—was a happy accident of the experimental protocol. The researchers used a hypodermic syringe to load their DNA
samples into the ultracentrifuge tubes (see Figure 6-6). In the process, they unwittingly sheared the large bacterial chromosome into smaller fragments. Had the
chromosomes remained whole, the researchers might have isolated DNA molecules that were only partially replicated, because many cells would have been caught in the
middle of copying their DNA. Molecules in such an intermediate stage of replication would not have separated into such discrete bands. But because the researchers were
instead working with smaller pieces of DNA, the likelihood that any given fragment had been fully replicated—and contained a complete parent and daughter strand—
was high, thus yielding nice, clean results.
The two replication forks move away in opposite directions at each replication origin. (A) These drawings represent the same portion of a DNA molecule as it might
appear at different times during replication. The orange lines represent the two parental DNA strands; the red lines represent the newly synthesized DNA strands. (B) An
electron micrograph showing DNA replicating in an early fly embryo. The particles visible along the DNA are nucleosomes, structures made of DNA and the protein
complexes around which the DNA is wrapped (discussed in Chapter 5). The chromosome in this micrograph is the one that was redrawn in sketch (2) above. (Electron
micrograph courtesy of Victoria Foe.)
 DNA Helicase

Helicases separate nucleic acid duplexes into their component strands using energy from ATP hydrolysis. The crystal structure of this DNA helicase from
bacteriophage T7, reveals an hexagonal arrangement of six identical subunits. Surprisingly, the ring is not sixfold symmetric, but is slightly squished. A model for
the mechanism of how the enzyme might work explains this structural asymmetry. Of the six potential ATP binding sites, two opposing ones bind ATP tightly, two
are more likely to bind ADP and phosphate, and two are empty. These three states may interconvert in a coordinate fashion as ATP is hydrolyzed, creating a ripple
effect that continuously runs around the ring. Because of these conformational changes, the loops that extend into the center hole of the ring--that are proposed to
bind DNA--oscillate up and down, as seen in this cross section. The oscillating loops might pull a DNA strand through the central hole, thus unwinding the double
helix in the process. A frontal view shows the full dynamics of this fascinating protein machine.
DNA polymerase adds a deoxyribonucleotide to the 3′ end of a
growing DNA chain.

DNA polymerase adds a deoxyribonucleotide to the 3′ end of a growing DNA chain. (A) Nucleotides enter the reaction as deoxyribonucleoside triphosphates. This
incoming nucleotide forms a base pair with its partner in the template strand. It is then linked to the free 3′ hydroxyl on the growing DNA strand. The new DNA
strand is therefore synthesized in the 5′-to-3′ direction. Breakage of a high-energy phosphate bond in the incoming nucleoside triphosphate—accompanied by the
release of pyrophosphate—provides the energy for the polymerization reaction. (B) The reaction is catalyzed by the enzyme DNA polymerase (light green). The
polymerase guides the incoming nucleotide to the template strand and positions it such that its 5′ terminal phosphate will be able to react with the 3′-hydroxyl group
on the newly synthesized strand. The gray arrow indicates the direction of polymerase movement. (C) Structure of DNA polymerase, as determined by X-ray
crystallography, which shows the positioning of the DNA double helix. The template strand is the longer of the two DNA strands.
DNA polymerase adds a deoxyribonucleotide to the 3′ end of a growing DNA chain. (A) Nucleotides enter the reaction as deoxyribonucleoside triphosphates. This
incoming nucleotide forms a base pair with its partner in the template strand. It is then linked to the free 3′ hydroxyl on the growing DNA strand. The new DNA strand is
therefore synthesized in the 5′-to-3′ direction. Breakage of a high-energy phosphate bond in the incoming nucleoside triphosphate—accompanied by the release of
pyrophosphate—provides the energy for the polymerization reaction. (B) The reaction is catalyzed by the enzyme DNA polymerase (light green). The polymerase guides
the incoming nucleotide to the template strand and positions it such that its 5′ terminal phosphate will be able to react with the 3′-hydroxyl group on the newly
synthesized strand. The gray arrow indicates the direction of polymerase movement. (C) Structure of DNA polymerase, as determined by X-ray crystallography, which
shows the positioning of the DNA double helix. The template strand is the longer of the two DNA strands (Movie 6.1).
 During DNA synthesis, DNA
polymerase proofreads its own
work.

DNA polymerase contains

separate sites for DNA synthesis
and proofreading.
DNA polymerase faithfully replicates DNA by using the nucleotide sequence of the template strand, colored yellow, to select each new nucleotide to be added to the 3' end
of a growing strand, colored gray. In this animation, the different domains of DNA polymerase are colored differently. Before a nucleotide can be incorporated into DNA at
the 3' end of the growing strand, the blue finger domain of the polymerase moves inward to correctly position the nucleoside triphosphate. A pyrophosphate group is
released when each nucleotide is added. In this view, the details of nucleotide selection at the active site are shown with the incoming nucleoside triphosphate and the
template nucleotide in light blue. The growing strand is green, and the template strand is red. When the finger domain moves inward, the nucleoside triphosphate is tested
for its ability to form a proper base pair with the template nucleotide. When a base pair forms, the active site residues catalyze the covalent addition of the new nucleotide
to the 3' hydroxyl group on the growing strand, and the entire process repeats at speeds up to 500 nucleotides per second. On rare occasions, approximately once every
10,000 nucleotide additions, the polymerase makes an error and incorporates a nucleotide that does not form a proper base pair onto the end of the growing strand. When
this occurs, the polymerase changes conformation, and transfers the end of the growing strand to a second active site on the polymerase, where the erroneous, added
nucleotide is removed. The polymerase then flips back to its original conformation, allowing polymerization to continue. As a result, such a self-correcting DNA
polymerase will make a mistake only about once every 107 to 108 nucleotide pairs.
Multiple enzymes are required to
synthesize Okazaki fragments on the
lagging DNA strand.

Multiple enzymes are required to synthesize Okazaki fragments on the lagging

DNA strand. In eukaryotes, RNA primers are made at intervals of about 200
nucleotides on the lagging strand, and each RNA primer is approximately 10
nucleotides long. Primers are removed by nucleases that recognize an RNA
strand in an RNA/DNA helix and degrade it; this leaves gaps that are filled in by
a repair DNA polymerase that can proofread as it fills in the gaps. The
completed fragments are finally joined together by an enzyme called DNA
ligase, which catalyzes the formation of a phosphodiester bond between the 3′-
OH end of one fragment and the 5′-phosphate end of the next, thus linking up
the sugar-phosphate backbones. This nicksealing reaction requires an input of
energy in the form of ATP (not shown; see Figure 6-18).
DNA ligase joins together Okazaki fragments on the lagging
strand during DNA synthesis.

DNA ligase joins together Okazaki fragments on the lagging strand during DNA synthesis. The ligase enzyme uses a molecule of ATP to activate the 5′ end of
one fragment (step 1) before forming a new bond with the 3′ end of the other fragment (step 2).
DNA synthesis is carried out by a group of proteins that act together as
a replication machine. (A) DNA polymerases are held on the leading
and lagging strands by circular protein clamps that allow the
polymerases to slide. On the lagging-strand template, the clamp
detaches each time the polymerase completes an Okazaki fragment. A
clamp loader (not shown) is required to attach a sliding clamp each
time a new Okazaki fragment is begun. At the head of the fork, a DNA
helicase unwinds the strands of the parental DNA double helix. Single-
strand DNA-binding proteins keep the DNA strands apart to provide
access for the primase and polymerase. For simplicity, this diagram
shows the proteins working independently; in the cell, they are held
together in a large replication machine, as shown in (B). (B) This
diagram shows a current view of how the replication proteins are
arranged when a replication fork is moving. To generate this structure,
the lagging strand shown in (A) has been folded to bring its DNA
polymerase in contact with the leading-strand DNA polymerase. This
folding process also brings the 3′ end of each completed Okazaki
fragment close to the start site for the next Okazaki fragment. Because
the lagging-strand DNA polymerase is bound to the rest of the
replication proteins, it can be reused to synthesize successive Okazaki
fragments; in this diagram, the lagging strand DNA polymerase is
about to let go of its completed Okazaki fragment and move to the
RNA primer that is being synthesized by the nearby primase.
In a replication fork, two DNA polymerases collaborate to copy the leading strand template and the lagging-strand template DNA. In this picture, the DNA
polymerase that produces the lagging strand has just finished an Okazaki fragment. The clamp that keeps the lower DNA polymerase attached to the lagging
strand dissociates, and the DNA polymerase temporarily releases the lagging strand template DNA. As the DNA helicase continues to unwind the parental
DNA, the primase becomes activated and synthesizes a short RNA primer on the growing lagging strand. The DNA polymerase binds to the DNA again and
becomes locked in by the clamp. The polymerase uses the RNA primer to begin a short copy of the lagging strand-template DNA. The polymerase stalls when it
reaches the RNA primer of the preceding Okazaki fragment, and the entire cycle repeats.
Using computer animation based on molecular research, we are able to picture how DNA is replicated in living cells. You are looking at an assembly line of amazing
miniature biochemical machines that are pulling apart the DNA double helix and cranking out a copy of each strand. The DNA to be copied enters the production
line from bottom left. The whirling blue molecular machine is called a helicase. It spins the DNA as fast as a jet engine as it unwinds the double helix into two
strands. One strand is copied continuously and can be seen spooling off to the right. Things are not so simple for the other strand because it must be copied
backwards. It is drawn out repeatedly in loops, and copied one section at a time. The end result is two new DNA molecules.
Without a special mechanism to replicate the ends of linear
chromosomes, DNA would be lost during each round of cell division.
DNA synthesis begins at origins of replication and continues until the
replication machinery reaches the ends of the chromosome. The
leading strand is reproduced in its entirety. But the ends of the lagging
strand can′t be completed, because once the final RNA primer has
been removed there is no way to replace it with DNA. These gaps at
the ends of the lagging strand must be filled in by a special
mechanism to keep the chromosome ends from shrinking with each
cell division.
Telomeres and telomerase prevent linear eukaryotic chromosomes from shortening with each cell division. For clarity, only the template DNA (orange) and newly
synthesized DNA (red) of the lagging strand are shown (see bottom of Figure 6-21). To complete the replication of the lagging strand at the ends of a chromosome,
the template strand is first extended beyond the DNA that is to be copied. To achieve this, the enzyme telomerase adds more repeats to the telomere repeat sequences
at the 3′ end of the template strand, which then allows the lagging strand to be completed by DNA polymerase, as shown. The telomerase enzyme carries a short piece
of RNA (blue) with a sequence that is complementary to the DNA repeat sequence; this RNA acts as the template for telomere DNA synthesis. After the lagging-
strand replication is complete, a short stretch of single stranded DNA remains at the ends of the chromosome, as shown.
 DNA Primase produc
the name is “DNA”-P
it makes DNA.

DNA Primase is in fac

polymerase.

The ends of linear chromosomes pose unique problems during DNA replication. Because DNA polymerases can only elongate from a free 3' hydroxyl group, the
replication machinery builds the lagging strand by a backstitching mechanism. RNA primers provide 3'-hydroxyl groups at regular intervals along the lagging strand
template. Whereas the leading strand elongates continuously in the 5'-to-3' direction all the way to the end of the template, the lagging strand stops short of the end. Even
if a final RNA primer were built at the very end of the chromosome, the lagging strand would not be complete. The final primer would provide a 3'-OH group to
synthesize DNA, but the primers would later need to be removed. The 3'-hydroxyl groups on adjacent DNA fragments provide starting places for replacing the RNA
with DNA. However, at the end of the chromosome there is no 3'-OH group available to prime DNA synthesis. Because of this inability to replicate the ends,
chromosomes would progressively shorten during each replication cycle. This "end-replication" problem is solved by the enzyme telomerase. The ends of chromosomes
contain a G-rich series of repeats called a telomere. Telomerase recognizes the tip of an existing repeat sequence. Using an RNA template within the enzyme, telomerase
elongates the parental strand in the 5'-to-3' direction, and adds additional repeats as it moves down the parental strand. The lagging strand is then completed by DNA
polymerase alpha, which carries a DNA primase as one of its subunits. In this way, the original information at the ends of linear chromosomes is completely copied in
the new DNA.
Depurination and Deamination - and why there’s T instead of U in DNA

Depurination and deamination are the most frequent chemical reactions known to create serious DNA damage in cells. (A) Depurination can remove guanine (or
adenine) from DNA. (B) The major type of deamination reaction converts cytosine to an altered DNA base, uracil; however, deamination can also occur on other bases as
well. Both depurination and deamination take place on double-helical DNA, and neither break the phosphodiester backbone.
 UV radiation and thymidine dimers - (tanning
salons are bad!)

The ultraviolet radiation in sunlight can cause the formation of thymine dimers. Two adjacent thymine bases have become covalently attached to each other to
form a thymine dimer. Skin cells that are exposed to sunlight are especially susceptible to this type of DNA damage.
Chemical modifications of nucleotides, if left unrepaired, produce mutations

Chemical modifications of nucleotides, if left unrepaired, produce mutations. (A) Deamination of cytosine, if uncorrected, results in the substitution of one base
for another when the DNA is replicated. As shown in Figure 6-23B, deamination of cytosine produces uracil. Uracil differs from cytosine in its basepairing
properties and preferentially base-pairs with adenine. The DNA replication machinery therefore inserts an adenine when it encounters a uracil on the template
strand. (B) Depurination, if uncorrected, can lead to the loss of a nucleotide pair. When the replication machinery encounters a missing purine on the template
strand, it can skip to the next complete nucleotide, as shown, thus producing a daughter DNA molecule that is missing one nucleotide pair. In other cases (not
shown), the replication machinery places an incorrect nucleotide across from the missing base, again resulting in a mutation.
 The basic mechanism of DNA
repair involves three steps.

The basic mechanism of DNA repair involves three steps. In step 1 (excision), the
damage is cut out by one of a series of nucleases, each specialized for a type of DNA
damage. In step 2 (resynthesis), the original DNA sequence is restored by a repair
DNA polymerase, which fills in the gap created by the excision events. In step 3
(ligation), DNA ligase seals the nick left in the sugar-phosphate backbone of the
repaired strand. Nick sealing, which requires energy from ATP hydrolysis, remakes
the broken phosphodiester bond between the adjacent nucleotides (see Figure 6-18).
 Errors made during DNA replication must be corrected to
avoid mutations.

Mismatch repair eliminates replication errors and restores the

original DNA sequence.
Cells can repair double-strand breaks in one of two ways. (A) In nonhomologous end joining, the break is first “cleaned” by a nuclease that chews back the broken
ends to produce flush ends. The flush ends are then stitched together by a DNA ligase. Some nucleotides are lost in the repair process, as indicated by the black lines in
the repaired DNA. (B) If a double-strand break occurs in one of two daughter DNA double helices after DNA replication has occurred, but before the daughter
chromosomes have been separated, the undamaged double helix can be readily used as a template to repair the damaged double helix by homologous recombination.
This is a more involved process than non-homologous end joining, but it accurately restores the original DNA sequence at the site of the break.
 Homologous recombination
allows the flawless repair of
DNA double-strand breaks.
5’ ends are nucleased Homologous recombination allows the flawless repair of DNA
double-strand breaks. This is the preferred method for repairing
double-strand breaks that arise shortly after the DNA has been
replicated but before the cell has divided. See text for details.
(Adapted from M. McVey et al., Proc. Natl. Acad. Sci. USA
101:15694-15699, 2004. With permission from the National
Academy of Sciences.)