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DNA Replication and Repair
13.1
13.2
13.3
DNA Replication
DNA Repair
Between Replication and Repair
The Human Perspective:
The Consequences of DNA Repair Deficiencies
R
eproduction is a fundamental property of all living systems. The process
of reproduction can be observed at several levels: organisms duplicate by
asexual or sexual reproduction; cells duplicate by cellular division; and
the genetic material duplicates by DNA replication. The machinery that replicates
DNA is also called into action in another capacity: to repair the genetic material after
it has sustained damage. These two processes—DNA replication and DNA repair—
are the subjects addressed in this chapter.
The capacity for self-duplication is presumed to have been one of the first critical properties to have appeared in the evolution of the earliest primitive life forms.
Without the ability to propagate, any primitive assemblage of biological molecules
would be destined for oblivion. The early carriers of genetic information were probably RNA molecules that were able to self-replicate. As evolution progressed and
RNA molecules were replaced by DNA molecules as the genetic material, the
process of replication became more complex, requiring a large number of auxiliary
components. Thus, although a DNA molecule contains the information for its own
duplication, it lacks the ability to perform the activity itself. As Richard Lewontin expressed it, “the common image of DNA as a self-replicating molecule is about as true
as describing a letter as a self-replicating document. The letter needs a photocopier;
the DNA needs a cell.” Let us see then how the cell carries out this activity. ■
Three-dimensional model of a DNA helicase encoded by the bacteriophage T7. The protein
consists of a ring of six subunits. Each subunit contains two domains. In this model, the
central hole encircles only one of the two DNA strands. Driven by ATP hydrolysis, the protein moves in a 5⬘ → 3⬘ direction along the strand to which it is bound, displacing the complementary strand and unwinding the duplex. DNA helicase activity is required for DNA
replication. (COURTESY OF EDWARD H. EGELMAN, UNIVERSITY OF VIRGINIA.)
533
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Chapter 13 DNA REPLICATION AND REPAIR
13.1DNA REPLICATION
The proposal for the structure of DNA by Watson and
Crick in 1953 was accompanied by a suggested mechanism for its “self-duplication.” The two strands of the double
helix are held together by hydrogen bonds between the bases.
Individually, these hydrogen bonds are weak and readily broken. Watson and Crick envisioned that replication occurred
by gradual separation of the strands of the double helix (Figure 13.1), much like the separation of two halves of a zipper.
Because the two strands are complementary to each other,
each strand contains the information required for construction
of the other strand. Thus once the strands are separated, each
can act as a template to direct the synthesis of the complementary strand and restore the double-stranded state.
1
Parental DNA
molecules
DNA molecules from
1st generation progeny
DNA molecules from
2nd generation progeny
Semiconservative Replication
The Watson-Crick proposal shown in Figure 13.1 made certain predictions concerning the behavior of DNA during
replication. According to the proposal, each of the daughter
duplexes should consist of one complete strand inherited from
the parental duplex and one complete strand that has been
newly synthesized. Replication of this type (Figure 13.2,
scheme 1) is said to be semiconservative because each daughter duplex contains one strand from the parent structure. In
the absence of information on the mechanism responsible for
replication, two other types of replication had to be considOld
SEMICONSERVATIVE REPLICATION
2
Parental DNA
molecules
DNA molecules from
1st generation progeny
Old
T
A
T
A
A
T
DNA molecules from
2nd generation progeny
G
C
G
C
G
A
T
C G
CONSERVATIVE REPLICATION
T
A T
G C
C G
A T
3
Parental DNA
molecules
T
T A
G
C
A
T
C
G
C
C
G
New
T A
A
G
A
Old New
DNA molecules from
1st generation progeny
T A
A
T
T
T
New
G
A
C
T
T
G
G
A T
C
T
G
A
A
C
G
G
A T
C
DNA molecules from
2nd generation progeny
New Old
FIGURE 13.1 The original Watson-Crick proposal for the replication
of a double-helical molecule of DNA. During replication, the double
helix unwinds, and each of the parental strands serves as a template for
the synthesis of a new complementary strand. As discussed in this chapter, these basic tenets have been borne out.
DISPERSIVE REPLICATION
FIGURE 13.2 Three alternate schemes of replication. Semiconservative
replication is depicted in scheme 1, conservative replication in scheme 2,
and dispersive replication in scheme 3. A description of the three alternate modes of replication is given in the text.
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13.1 DNA REPLICATION
Transfer to 14N
Generations
Light
Parental
Hybrid
Heavy
ered. In conservative replication (Figure 13.2, scheme 2), the
two original strands would remain together (after serving as
templates), as would the two newly synthesized strands. As a
result, one of the daughter duplexes would contain only
parental DNA, while the other daughter duplex would contain only newly synthesized DNA. In dispersive replication
(Figure 13.2, scheme 3), the parental strands would be broken
into fragments, and the new strands would be synthesized in
short segments. Then the old fragments and new segments
would be joined together to form a complete strand. As a result, the daughter duplexes would contain strands that were
composites of old and new DNA. At first glance, dispersive
replication might seem like an unlikely solution, but it appeared
to Max Delbrück at the time as the only way to avoid the seemingly impossible task of unwinding two intertwined strands of a
DNA duplex as it replicated (discussed on page 538).
535
To decide among these three possibilities, it was necessary
to distinguish newly synthesized DNA strands from the original
DNA strands that served as templates. This was accomplished
in studies on bacteria in 1957 by Matthew Meselson and
Franklin Stahl of the California Institute of Technology who
used heavy (15N) and light (14N) isotopes of nitrogen to distinguish between parental and newly synthesized DNA strands
(Figure 13.3). These researchers grew bacteria in medium containing 15N-ammonium chloride as the sole nitrogen source.
Consequently, the nitrogen-containing bases of the DNA of
these cells contained only the heavy nitrogen isotope. Cultures
of “heavy” bacteria were washed free of the old medium and
incubated in fresh medium with light, 14N-containing compounds, and samples were removed at increasing intervals over a
period of several generations. DNA was extracted from the samples of bacteria and subjected to equilibrium density-gradient
centrifugation (see Figure 18.35). In this procedure, the DNA is
mixed with a concentrated solution of cesium chloride and centrifuged until the double-stranded DNA molecules reach equilibrium according to their density.
I
Generations
Light 14N DNA
(a)
Hybrid 14N15N DNA
II
0 (parental)
Heavy 15N DNA
III
0.3
Semiconservative
0.7
I
(b)
1.0
II
1.1
III
1.5
Conservative
1.9
I
(c)
2.5
II
3.0
III
Dispersive
4.1
(a)
(b)
FIGURE 13.3 Experiment demonstrating that DNA replication in bac-
on the ratio of 15N/14N that is present in their nucleotides. The greater
the 14N content, the higher in the tube the DNA fragment is found at
equilibrium. (a) The results expected in this type of experiment for each
of the three possible schemes of replication. The single tube on the left
indicates the position of the parental DNA and the positions at which
totally light or hybrid DNA fragments would band. (b) Experimental results obtained by Meselson and Stahl. The appearance of a hybrid band
and the disappearance of the heavy band after one generation eliminates
conservative replication. The subsequent appearance of two bands,
one light and one hybrid, eliminates the dispersive scheme. (B: FROM
teria is semiconservative. DNA was extracted from bacteria at different
stages in the experiment, mixed with a concentrated solution of the salt
cesium chloride (CsCl), placed into a centrifuge tube, and centrifuged to
equilibrium at high speed in an ultracentrifuge. Cesium ions have sufficient atomic mass to be affected by the centrifugal force, and they form
a density gradient during the centrifugation period with the lowest concentration (lowest density) of Cs at the top of the tube and the greatest
concentration (highest density) at the bottom of the tube. During centrifugation, DNA fragments within the tube become localized at a position having a density equal to their own density, which in turn depends
M. MESELSON AND F. STAHL, PROC. NAT ’L. ACAD. SCI. U.S.A. 44:671, 1958.)
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Chapter 13 DNA REPLICATION AND REPAIR
In the Meselson-Stahl experiment, the density of a DNA
molecule is directly proportional to the percentage of 15N or
14
N atoms it contains. If replication is semiconservative, one
would expect that the density of DNA molecules would decrease
during culture in the 14N-containing medium in the manner
shown in the upper set of centrifuge tubes of Figure 13.3a.
After one generation, all DNA molecules would be 15N-14N
hybrids, and their buoyant density would be halfway between
that expected for totally heavy and totally light DNA (Figure 13.3a). As replication continued beyond the first generation, the newly synthesized strands would continue to contain
only light isotopes, and two types of duplexes would appear in
the gradients: those containing 15N–14N hybrids and those
containing only 14N. As the time of growth in the light
medium continued, a greater and greater percentage of the
DNA molecules present would be light. However, as long as
replication continued semiconservatively, the original heavy
Chromosome
DNA strand
parental strands would remain intact and present in hybrid
DNA molecules that occupied a smaller and smaller percentage of the total DNA (Figure 13.3a). The results of the
density-gradient experiments obtained by Meselson and Stahl
are shown in Figure 13.13b, and they demonstrate unequivocally that replication occurs semiconservatively. The results
that would have been obtained if replication occurred by conservative or dispersive mechanisms are indicated in the two
lower sets of centrifuge tubes of Figure 13.3a.1
By 1960, replication had been demonstrated to occur semiconservatively in eukaryotes as well. The original experiments
were carried out by J. Herbert Taylor of Columbia University.
The drawing and photograph of Figure 13.4 show the results of
a more recent experiment in which cultured mammalian cells
were allowed to undergo replication in bromodeoxyuridine
(BrdU), a compound that is incorporated into DNA in place of
thymidine. Following replication, a chromosome is made up of
two chromatids. After one round of replication in BrdU, both
chromatids of each chromosome contained BrdU (Figure
13.4a). After two rounds of replication in BrdU, one chromatid
1
Anyone looking to explore the circumstances leading up to this heralded research effort and examine the experimental twists and turns as they unfolded
might want to read the book Meselson, Stahl, and the Replication of DNA by
Frederick Lawrence Holmes, 2001. A discussion of the experiment can also
be found in PNAS 101:17889, 2004, which is on the Web.
Chromosome contains
only thymidine
Replicates
in BrdU
Chromatid
Both chromatids contain one strand with BrdU and
one strand with thymidine
Continued replication
in BrdU-containing
medium
One chromatid of each chromosome
contains thymidine
(a)
(b)
FIGURE 13.4 Experimental demonstration that DNA replication occurs
semiconservatively in eukaryotic cells. (a) Schematic diagram of the
results of an experiment in which cells were transferred from a medium
containing thymidine to one containing bromodeoxyuridine (BrdU) and
allowed to complete two successive rounds of replication. DNA strands
containing BrdU are shown in red. (b) The results of an experiment similar to that shown in a. In this experiment, cultured mammalian cells were
grown in BrdU for two rounds of replication before mitotic chromosomes
were prepared and stained by a procedure using fluorescent dyes and
Giemsa stain. Using this procedure, chromatids containing thymidine
within one or both strands stain darkly, whereas chromatids containing
only BrdU stain lightly. The photograph indicates that, after two rounds
of replication in BrdU, one chromatid of each duplicated chromosome
contains only BrdU, while the other chromatid contains a strand of
thymidine-labeled DNA. (Some of the chromosomes are seen to have exchanged homologous portions between sister chromatids. This process of
sister chromatid exchange is common during mitosis but is not discussed
in the text.) (B: COURTESY OF SHELDON WOLFF.)
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13.1 DNA REPLICATION
of each chromosome was composed of two BrdU-containing
strands, whereas the other chromatid was a hybrid consisting of
a BrdU-containing strand and a thymidine-containing strand
(Figure 13.4a,b). The thymidine-containing strand had been
part of the original parental DNA molecule prior to addition of
BrdU to the culture.
Replication in Bacterial Cells
We will focus in this section of the chapter on replication in
bacterial cells, which is better understood than the corresponding process in eukaryotes. The early progress in bacterial research was driven by genetic and biochemical approaches
including:
■
■
The availability of mutants that cannot synthesize one or
another protein required for the replication process. The
isolation of mutants unable to replicate their chromosome
may seem paradoxical: how can cells with a defect in this
vital process be cultured? This paradox was solved by the
isolation of temperature-sensitive (ts) mutants, in which
the deficiency only reveals itself at an elevated temperature, termed the nonpermissive (or restrictive) temperature.
When grown at the lower (permissive) temperature, the
mutant protein can function sufficiently well to carry out
its required activity, and the cells can continue to grow and
divide. Temperature-sensitive mutants have been isolated
that affect virtually every type of physiologic activity (see
also page 269), and they have been particularly important
in the study of DNA synthesis as it occurs in replication,
DNA repair, and genetic recombination.
The development of in vitro systems in which replication
can be studied using purified cellular components. In some
studies, the DNA molecule to be replicated is incubated
with cellular extracts from which specific proteins suspected of being essential have been removed. In other
studies, the DNA is incubated with a variety of purified
proteins whose activity is to be tested.
Taken together, these approaches have revealed the activity of more than 30 different proteins that are required to
replicate the chromosome of E. coli. In the following pages, we
will discuss the activities of several of these proteins whose
functions have been clearly defined. Replication in bacteria
and eukaryotes occurs by very similar mechanisms, and thus
most of the information presented in the discussion of bacterial replication applies to eukaryotic cells as well.
Replication begins at a specific site on the bacterial chromosome
called the origin. The origin of replication on the E. coli chromosome is a specific sequence called oriC where a number of
proteins bind to initiate the process of replication.2 Once initiated, replication proceeds outward from the origin in both
directions, that is, bidirectionally (Figure 13.5). The sites in
Figure 13.5 where the pair of replicated segments come toReplication Forks and Bidirectional Replication
2
The subject of initiation of replication is discussed in detail on page 547 as it
occurs in eukaryotes.
537
Replication fork
Origin
Daughter strand
Parental strand
Replication
fork
FIGURE 13.5 Model of a circular bacterial chromosome undergoing bidirectional, semiconservative replication. Two replication forks move in
opposite directions from a single origin. When the replication forks
meet at the opposite point on the circle, replication is terminated, and
the two replicated duplexes detach from one another. New DNA strands
are shown in red.
gether and join the nonreplicated DNA are termed replication forks. Each replication fork corresponds to a site where
(1) the parental double helix is undergoing strand separation,
and (2) nucleotides are being incorporated into the newly synthesized complementary strands. The two replication forks
move in opposite directions until they meet at a point across
the circle from the origin, where replication is terminated.
The two newly replicated duplexes detach from one another
and are ultimately directed into two different cells.
Separation of the strands of a circular, helical DNA duplex poses
major topological problems. To visualize the difficulties, we
can briefly consider an analogy between a DNA duplex and a
two-stranded helical rope. Consider what would happen if
you placed a linear piece of this rope on the ground, took hold
Unwinding the Duplex and Separating the Strands
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Chapter 13 DNA REPLICATION AND REPAIR
of the two strands at one end, and began to pull the strands
apart just as DNA is pulled apart during replication. It is apparent that separation of the strands of a double helix is also a
process of unwinding the structure. In the case of a rope,
which is free to rotate around its axis, separation of the strands
at one end would be accompanied by rotation of the entire
fiber as it resisted the development of tension. Now, consider
what would happen if the other end of the rope were attached
to a hook on a wall (Figure 13.6a). Under these circumstances,
separation of the two strands at the free end would generate
increasing torsional stress in the rope and cause the unseparated portion to become more tightly wound. Separation of
the two strands of a circular DNA molecule (or a linear molecule that is not free to rotate, as is the case in a large eukaryotic chromosome) is analogous to having one end of a linear
molecule attached to a wall; in all of these cases, tension that
develops in the molecule cannot be relieved by rotation of the
entire molecule. Unlike a rope, which can become tightly
overwound (as in Figure 13.6a), an overwound DNA molecule becomes positively supercoiled (page 391). Consequently,
movement of the replication fork generates positive supercoils
in the unreplicated portion of the DNA ahead of the fork
(Figure 13.6b). When one considers that a complete circular
chromosome of E. coli contains approximately 400,000 turns
and is replicated by two forks within 40 minutes, the magnitude of the problem becomes apparent.
(a)
Point of attachment
of DNA
Replication machinery
(b)
FIGURE 13.6 The unwinding problem. (a) The effect of unwinding a
two-stranded rope that has one end attached to a hook. The unseparated
portion becomes more tightly wound. (b) When a circular or attached
DNA molecule is replicated, the DNA ahead of the replication machinery becomes overwound and accumulates positive supercoils. Cells possess topoisomerases, such as the E. coli DNA gyrase, that remove positive
supercoils. (B: REPRINTED WITH PERMISSION FROM J. C. WANG, NATURE
REVIEWS MOL. CELL BIOL. 3:434, 2002; ©
LAN MAGAZINES LIMITED.)
COPYRIGHT
2002, BY MACMIL-
It was noted on page 391 that cells contain enzymes,
called topoisomerases, that can change the state of supercoiling in a DNA molecule. One enzyme of this type, called
DNA gyrase, a type II topoisomerase, relieves the mechanical
strain that builds up during replication in E. coli. DNA gyrase
molecules travel along the DNA ahead of the replication fork,
removing positive supercoils. DNA gyrase accomplishes this
feat by cleaving both strands of the DNA duplex, passing a
segment of DNA through the double-stranded break to the
other side, and then sealing the cuts, a process that is driven by
the energy released during ATP hydrolysis (shown in detail in
Figure 10.14b). Eukaryotic cells possess similar enzymes that
carry out this required function.
We begin our discussion of the mechanism of DNA replication by describing some
of the properties of DNA polymerases, the enzymes that synthesize new DNA strands. Study of these enzymes was begun
in the 1950s by Arthur Kornberg at Washington University. In
their initial experiments, Kornberg and his colleagues purified
an enzyme from bacterial extracts that incorporated radioactively labeled DNA precursors into an acid-insoluble polymer
identified as DNA. The enzyme was named DNA polymerase
(and later, after the discovery of additional DNA-polymerizing
enzymes, it was named DNA polymerase I). For the reaction to
proceed, the enzyme required the presence of DNA and all
four deoxyribonucleoside triphosphates (dTTP, dATP, dCTP,
and dGTP). The newly synthesized, radioactively labeled
DNA had the same base composition as the original unlabeled
DNA, which strongly suggested that the original DNA strands
had served as templates for the polymerization reaction.
As additional properties of the DNA polymerase were uncovered, it became apparent that replication was more complex
than previously thought. When various types of template
DNAs were tested, it was found that the template DNA had to
meet certain structural requirements if it was to promote the
incorporation of labeled precursors (Figure 13.7). An intact,
double-stranded DNA molecule, for example, did not stimulate incorporation. This was not surprising considering the requirement that the strands of the helix must be separated for
replication to occur. It was less obvious why a single-stranded,
circular molecule was also devoid of activity; one might expect
this structure to be an ideal template to direct the manufacture
of a complementary strand. In contrast, addition of a partially
double-stranded DNA molecule to the reaction mixture produced an immediate incorporation of nucleotides.
It was soon discovered that a single-stranded DNA circle
cannot serve as a template for DNA polymerase because
the enzyme cannot initiate the formation of a DNA strand.
Rather, it can only add nucleotides to the 3⬘ hydroxyl terminus
of an existing strand. The strand that provides the necessary 3⬘
OH terminus is called a primer. All DNA polymerases—both
prokaryotic and eukaryotic—have these same two basic requirements (Figure 13.8a): a template DNA strand to copy
and a primer strand to which nucleotides can be added. These
requirements explain why certain DNA structures fail to promote DNA synthesis (Figure 13.7a). An intact, linear double
helix provides the 3⬘ hydroxyl terminus but lacks a template. A
The Properties of DNA Polymerases
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13.1 DNA REPLICATION
circular single strand, on the other hand, provides a template
but lacks a primer. The partially double-stranded molecule
(Figure 13.7b) satisfies both requirements and thus promotes
nucleotide incorporation. The finding that DNA polymerase
cannot initiate the synthesis of a DNA strand raises a critical
question: how is the synthesis of a new strand initiated in the
cell? We will return to this question shortly.
The DNA polymerase purified by Kornberg had another
property that was difficult to understand in terms of its presumed role as a replicating enzyme: it only synthesized DNA
in a 5⬘-to-3⬘ (written 5⬘ → 3⬘) direction. The diagram first
presented by Watson and Crick (see Figure 13.1) depicted
events as they would be expected to occur at the replication fork.
The diagram suggested that one of the newly synthesized
strands is polymerized in a 5⬘ → 3⬘ direction, while the other
strand is polymerized in a 3⬘ → 5⬘ direction. Is there some
other enzyme responsible for the construction of the 3⬘ → 5⬘
strand? Does the enzyme work differently in the cell than under in vitro conditions? We will return to this question as well.
During the 1960s, there were hints that the “Kornberg enzyme” was not the only DNA polymerase in a bacterial cell.
Then in 1969, a mutant strain of E. coli was isolated that had
less than 1 percent of the normal activity of the enzyme, yet was
able to multiply at the normal rate. Further studies revealed that
the Kornberg enzyme, or DNA polymerase I, was only one of
several distinct DNA polymerases present in bacterial cells. The
major enzyme responsible for DNA replication (i.e., the
replicative polymerase) is DNA polymerase III. A typical bacterial cell contains 300 to 400 molecules of DNA polymerase I but
5'
3'
3'
5' 3'
5'
3' 5'
5'
3'
5'
3'
3'
5'
3'
5'
3'
(b)
FIGURE 13.7 Templates and nontemplates for DNA polymerase activity.
(a) Examples of DNA structures that do not stimulate the synthesis of
DNA in vitro by DNA polymerase isolated from E. coli. (b) Examples of
DNA structures that stimulate the synthesis of DNA in vitro. In all
cases, the molecules in b contain a template strand to copy and a primer
strand with a 3⬘ OH on which to add nucleotides.
5'
3'
Primer P
P
Primer
5'
O
Base
Base
Base
Base
Base
Base
Template
3'
O
(a)
539
O
5'
O
O
G
C
S
OHO
P
γP
O
A
OH
T
OO P O
β
O
NH2
O
OP O
O
α
OH
N
N
N
N
O
Base
Base
P
Base
Base
5'
(a)
A
T
HN
N
S
3'
Growing
DNA strand
O
CH3 O P
O
Mg2+
Mg2+
Base
DNA
template
Base
(b)
A FIGURE 13.8 The activity of a DNA polymerase. (a) The poly-
merization of a nucleotide onto the 3⬘ end of the primer strand.
The enzyme selects nucleotides for incorporation based on their ability
to pair with the nucleotide of the template strand. (b) A simplified
model of the two-metal ion mechanism for the reaction in which nucleotides are incorporated into a growing DNA strand by a DNA polymerase. In this model, one of the magnesium ions draws the proton away
_
O
3'
5'
O
O
P
5'
DNA polymerase
New DNA strands
under construction
5'
3'
O
O-
O
S
OH
..
P P P
S
3'
O
P
S
O
T
O
A
O
S
5'
(c)
from the 3⬘ hydroxyl group of the terminal nucleotide of the primer, facilitating the nucleophilic attack of the negatively charged 3⬘ oxygen
atom on the ␣ phosphate of the incoming nucleoside triphosphate. The
second magnesium ion promotes the release of the pyrophosphate. The
two metal ions are bound to the enzyme by highly conserved aspartic
acid residues of the active site. (c) Schematic diagram showing the direction of movement of each polymerase along the two template strands.
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Chapter 13 DNA REPLICATION AND REPAIR
only about 10 copies of DNA polymerase III. The presence of
DNA polymerase III had been masked by the much greater
amounts of DNA polymerase I in the cell. But the discovery of
other DNA polymerases did not answer the two basic questions
posed above; none of the enzymes can initiate DNA chains, nor
can any of them construct strands in a 3⬘ → 5⬘ direction.
The lack of polymerization activity in the 3⬘ → 5⬘ direction has a straightforward
explanation: DNA strands cannot be synthesized in that
direction. Rather, both newly synthesized strands are assembled in a 5⬘ → 3⬘ direction. During the polymerization reaction, the —OH group at the 3⬘ end of the primer carries out a
nucleophilic attack on the 5⬘ ␣-phosphate of the incoming
nucleoside triphosphate, as shown in Figure 13.8b. The polymerase molecules responsible for construction of the two new
strands of DNA both move in a 3⬘-to-5⬘ direction along the
template, and both construct a chain that grows from its 5⬘-P
terminus (Figure 13.8c). Consequently, one of the newly synthesized strands grows toward the replication fork where the
parental DNA strands are being separated, while the other
strand grows away from the fork.
Although this solves the problem concerning an enzyme
that synthesizes a strand in only one direction, it creates an even
more complicated dilemma. It is apparent that the strand that
Semidiscontinuous Replication
grows toward the fork in Figure 13.8c can be constructed by the
continuous addition of nucleotides to its 3⬘ end. But how is the
other strand synthesized? Evidence was soon gathered to indicate that the strand that grows away from the replication fork is
synthesized discontinuously, that is, as fragments (Figure 13.9).
Before the synthesis of a fragment can be initiated, a suitable
stretch of template must be exposed by movement of the replication fork. Once initiated, each fragment grows away from the
replication fork toward the 5⬘ end of a previously synthesized
fragment to which it is subsequently linked. Thus, the two
newly synthesized strands of the daughter duplexes are synthesized by very different processes. The strand that is synthesized
continuously is called the leading strand because its synthesis
continues as the replication fork advances. The strand that is
synthesized discontinuously is called the lagging strand because initiation of each fragment must wait for the parental
strands to separate and expose additional template (Figure
13.9). As discussed on page 542, both strands are probably synthesized simultaneously, so that the terms leading and lagging
may not be as appropriate as thought when they were first
coined. Because one strand is synthesized continuously and the
other discontinuously, replication is said to be semidiscontinuous.
The discovery that one strand was synthesized as small fragments was made by Reiji Okazaki of Nagoya University, Japan,
following various types of labeling experiments. Okazaki found
3'
5'
Leading strand template
Leading strand
Replication fork
Lagging strand template
3'
5'
Lagging
strand
3'
5'
3'
5'
(a)
(b)
A FIGURE 13.9 The two strands of a double helix are synthesized
by a different sequence of events. DNA polymerase molecules
move along a template only in a 3⬘ → 5⬘ direction. As a result, the two
newly assembled strands grow in opposite directions, one growing toward the replication fork and the other growing away from it. One
strand is assembled in continuous fashion, the other as fragments that
are joined together enzymatically. (a) Schematic diagram depicting the
differences in synthesis of the two strands. (b) Electron micrograph of a
replicating bacteriophage DNA molecule. The left two limbs are the
replicated duplexes, and the right end is the unreplicated duplex. The
lagging strand of the newly replicated DNA is seen to contain an
exposed, single-stranded (thinner) portion, which runs from the replication fork to the arrow. (B: FROM J. WOLFSON AND DAVID DRESSLER,
ANNUAL REVIEW OF MICROBIOL29; © 1975, BY ANNUAL REVIEWS, INC.)
REPRINTED WITH PERMISSION FROM THE
OGY, VOLUME
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13.1 DNA REPLICATION
that if bacteria were incubated in [3H]thymidine for a few seconds and immediately killed, most of the radioactivity could be
found as part of small DNA fragments 1000 to 2000 nucleotides
in length. In contrast, if cells were incubated in the labeled DNA
precursor for a minute or two, most of the incorporated radioactivity became part of much larger DNA molecules (Figure
13.10). These results indicated that a portion of the DNA was
constructed in small segments (later called Okazaki fragments)
that were rapidly linked to longer pieces that had been synthesized previously. The enzyme that joins the Okazaki fragments
into a continuous strand is called DNA ligase.
The discovery that the lagging strand is synthesized in
pieces raised a new set of perplexing questions about the initiation of DNA synthesis. How does the synthesis of each of
these fragments begin when none of the DNA polymerases are
capable of strand initiation? Further studies revealed that initiation is not accomplished by a DNA polymerase but, rather, by
a distinct type of RNA polymerase, called primase, that conSedimentation velocity
20
40
60 S
120 sec
structs a short primer composed of RNA, not DNA. The leading strand, whose synthesis begins at the origin of replication,
is also initiated by a primase molecule. The short RNAs synthesized by the primase at the 5⬘ end of the leading strand and
the 5⬘ end of each Okazaki fragment serve as the required
primer for the synthesis of DNA by a DNA polymerase. The
RNA primers are subsequently removed, and the resulting
gaps in the strand are filled with DNA and then sealed by
DNA ligase. These events are illustrated schematically in Figure 13.11. The formation of transient RNA primers during the
process of DNA replication is a curious activity. It is thought
that the likelihood of mistakes is greater during initiation than
during elongation, and the use of a short removable segment of
RNA avoids the inclusion of mismatched bases.
Replication involves more than incorporating nucleotides. Unwinding the duplex and separating the strands require the
aid of two types of proteins that bind to the DNA, a helicase
(or DNA unwinding enzyme) and single-stranded DNAbinding (SSB) proteins. DNA helicases unwind a DNA duplex in a reaction that uses energy released by ATP hydrolysis
to move along one of the DNA strands, breaking the hydrogen bonds that hold the two strands together and exposing
the single-stranded DNA templates. E. coli has at least 12 different helicases for use in various aspects of DNA (and RNA)
The Machinery Operating at the Replication Fork
Radioactivity (103 cts/min per 0.1 ml)
3'
5'
Leading strand
60 sec
5'
3'
Lagging strand
1 Primer synthesis
3'
5'
by primase
2
30 sec
Elongation by
DNA polymerase III
15 sec
7 sec
2 sec
1
P OH
2
3
Distance from top
3 Primer removal and
gap filling by DNA
polymerase I
FIGURE 13.10 Results of an experiment showing that part of the DNA
is synthesized as small fragments. Sucrose density gradient profiles of
DNA from a culture of phage-infected E. coli cells. The cells were labeled for increasing amounts of time, and the sedimentation velocity of
the labeled DNA was determined. When DNA was prepared after very
short pulses, a significant percentage of the radioactivity appeared in
very short pieces of DNA (represented by the peak near the top of the
tube on the left). After periods of 60–120 seconds, the relative height of
this peak falls as labeled DNA fragments become joined to the ends of
high-molecular-weight molecules. (FROM R. OKAZAKI ET AL., COLD
SPRING HARBOR SYMP. QUANT. BIOL. 33:130, 1968.)
4 Strand sealed
by DNA ligase
FIGURE 13.11 The use of short RNA fragments as removable primers
in initiating synthesis of each Okazaki fragment of the lagging
strand. The major steps are indicated in the drawing and discussed in
the text. The role of various accessory proteins in these activities is indicated in the following figures.
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Chapter 13 DNA REPLICATION AND REPAIR
Primase
DNA
helicase
5'
3'
Lagging strand
RNA Primer
5'
Movement of helicase
Single-stranded
DNA binding protein
(SSB)
DNA Duplex
Leading strand
3'
(a)
A
DNA Helicase
FIGURE 13.12 The role of the DNA helicase, single-stranded
DNA-binding proteins, and primase at the replication fork.
(a) The helicase moves along the DNA, catalyzing the ATP-driven
unwinding of the duplex. As the DNA is unwound, the strands are prevented from reforming the duplex by single-stranded DNA-binding
proteins (SSBs). The primase associated with the helicase synthesizes
the RNA primers that begin each Okazaki fragment. The RNA primers,
which are about 10 nucleotides long, are subsequently removed. (b) A
metabolism. One of these helicases—the product of the dnaB
gene—serves as the major unwinding machine during replication. The DnaB helicase consists of six subunits arranged to
form a ring-shaped protein that encircles a single DNA strand
(Figure 13.12a). The DnaB helicase is first loaded onto the
DNA at the origin of replication (with the help of the protein DnaC) and translocates in a 5⬘ → 3⬘ direction along the
lagging-strand template, unwinding the helix as it proceeds
(Figure 13.12). A three-dimensional model of a similar
shaped bacteriophage helicase engaged in strand separation
during replication is depicted on page 533. DNA unwinding by
the helicase is aided by the attachment of SSB proteins to the
separated DNA strands (Figure 13.12). These proteins bind
selectively to single-stranded DNA, keeping it in an extended
state and preventing it from becoming rewound or damaged.
A visual portrait of the combined action of a DNA helicase
and SSB proteins on the structure of the DNA double helix is
illustrated in the electron micrographs of Figure 13.12b.
Recall that an enzyme called primase initiates the synthesis of each Okazaki fragment. In bacteria, the primase and the
helicase associate transiently to form what is called a “primosome.” Of the two members of the primosome, the helicase moves along the lagging-strand template processively
(i.e., without being released from the template strand during
the lifetime of the replication fork). As the helicase “motors”
along the lagging-strand template, opening the strands of the
duplex, the primase periodically binds to the helicase and
synthesizes the short RNA primers that begin the formation
of each Okazaki fragment. As noted above, the RNA primers
are subsequently extended as DNA by a DNA polymerase,
specifically DNA polymerase III.
A body of evidence suggests that the same DNA polymerase III molecule synthesizes successive fragments of the lagging strand. To accomplish this, the polymerase III molecule is
recycled from the site where it has just completed one Okazaki
fragment to the next site along the lagging-strand template
closer to the site of DNA unwinding. Once at the new site, the
(b)
Unwound DNA Strand
with SSB Proteins
200 nm
series of five electron micrographs showing DNA molecules incubated
with a viral DNA helicase (T antigen, page 550) and E. coli SSB proteins. The DNA molecules are progressively unwound from left to right.
The helicase appears as the round particle at the fork, and the SSB proteins are bound to the single-stranded ends, giving them a thickened appearance. (B: FROM RAINER WESSEL, JOHANNES SCHWEIZER, AND HANS
STAHL, J. VIROL. 66:807, 1992; COPYRIGHT © 1992, AMERICAN SOCIETY FOR
MICROBIOLOGY.)
polymerase attaches to the 3⬘ OH of the RNA primer that has
just been laid down by a primase and begins to incorporate deoxyribonucleotides onto the end of the short RNA.
How does a polymerase III molecule move from one site
on the lagging-strand template to another site that is closer to
the replication fork? The enzyme does this by “hitching a ride”
with the DNA polymerase that is moving in that direction
along the leading-strand template. Thus even though the two
polymerases are moving in opposite directions with respect to
the linear axis of the DNA molecule, they are, in fact, part of a
single protein complex (Figure 13.13). The two tethered polymerases can replicate both strands by looping the DNA of the
lagging-strand template back on itself, causing this template to
have the same orientation as the leading-strand template. Both
polymerases then can move together as part of a single replicative complex without violating the “5⬘ → 3⬘ rule” for synthesis of a DNA strand (Figure 13.13). Once the polymerase
assembling the lagging strand reaches the 5⬘ end of the Okazaki
fragment synthesized during the previous round, the laggingstrand template is released and the polymerase begins work at
the 3⬘ end of the next RNA primer toward the fork. The model
depicted in Figure 13.13 is often referred to as the “trombone
model” because the looping DNA repeatedly grows and shortens during the replication of the lagging strand, reminiscent of
the movement of the brass “loop” of a trombone as it is played.
The Structure and Functions
of DNA Polymerases
DNA polymerase III, the enzyme that synthesizes DNA
strands during replication in E. coli, is part of a large “replication machine” called the DNA polymerase III holoenzyme
(Figure 13.14). One of the noncatalytic components of the holoenzyme, called the ␤ clamp, keeps the polymerase associated
with the DNA template. DNA polymerases (like RNA polymerases) possess two somewhat contrasting properties: (1)
they must remain associated with the template over long
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13.1 DNA REPLICATION
β subunit
DNA polymerase III
SSB
Lagging-strand template
Leading-strand template
Unreplicated parental DNA
5'
5'
3'
3'
Leading strand
DNA Helicase
5′
3′
5'
RNA primer
#2
(a)
543
RNA primer
#1
Growing Okazaki
fragment of lagging strand
Lagging strand
5'
3'
5'
3'
3' OH
5'
RNA primer
RNA primer
#2
#3
5'
Polymerase released from template
3'
5'
Completed Okazaki fragment
RNA primer #1 to be replaced
with DNA by DNA Polymerase I;
nick sealed by DNA ligase
(b)
5'
5'
3'
3'
RNA primer
#2
RNA primer
#3
5'
(c)
FIGURE 13.13 Replication of the leading and lagging strands in E. coli
is accomplished by two DNA polymerases working together as part
of a single complex. (a) The two DNA polymerase III molecules travel
together, even though they are moving toward the opposite ends of their
respective templates. This is accomplished by causing the lagging-strand
template to form a loop. (b) The polymerase releases the lagging-strand
template when it encounters the previously synthesized Okazaki fragment.
3'
5'
Newly
initiated
Okazaki fragment
Old Okazaki
fragment
(c) The polymerase that was involved in the assembly of the previous
Okazaki fragment has now rebound the lagging-strand template farther
along its length and is synthesizing DNA onto the end of RNA primer
#3 that has just been constructed by the primase. (AFTER D. VOET AND
J. G. VOET, BIOCHEMISTRY, 2D ED.; COPYRIGHT © 1995, JOHN WILEY AND
SONS, INC. REPRINTED BY PERMISSION OF JOHN WILEY AND SONS, INC.)
β clamp
stretches if they are to synthesize a continuous complementary
strand, and (2) they must be attached loosely enough to the
template to move from one nucleotide to the next. These contrasting properties are provided by the doughnut-shaped ␤
Leading
strand
Core
polymerase
τ
τ
FIGURE 13.14 Schematic representation of DNA polymerase III holoenzyme. The holoenzyme contains ten different subunits organized into
several distinct components. Included as part of the holoenzyme are
(1) two core polymerases which replicate the DNA, (2) two or more ␤
clamps, which allow the polymerase to remain associated with the
DNA, and (3) a clamp loading (␥) complex, which loads each sliding
clamp onto the DNA. The clamp loader of an active replication fork
contains two t subunits, which hold the core polymerases in the complex
and also bind the helicase. Another term, the replisome, is often used to
refer to the entire complex of proteins that are active at the replication
fork, including the DNA polymerase III holoenzyme, the helicase,
SSBs, and primase. (BASED ON DRAWINGS BY M. O’DONNELL.)
5'
γ-clamp loader
(ready to load β
clamp for next
Okazaki fragment)
Lagging
strand
DNA helicase
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Chapter 13 DNA REPLICATION AND REPAIR
clamp that encircles the DNA (Figure 13.15a) and slides freely
along it. As long as it is attached to a ␤ “sliding clamp,” a DNA
polymerase can move processively from one nucleotide to the
next without diffusing away from the template. The polymerase on the leading-strand template remains tethered to a
single ␤ clamp during replication. In contrast, when the polymerase on the lagging-strand template completes the synthesis
of an Okazaki fragment, it disengages from the ␤ clamp and is
cycled to a new ␤ clamp that has been assembled at an RNA
primer–DNA template junction located closer to the replication fork (Figure 13.15b). But how does a highly elongated
DNA molecule get inside of a ring-shaped clamp as in Figure
13.15a? The assembly of the ␤ clamp around the DNA requires a multisubunit clamp loader that is also part of the DNA
polymerase III holoenzyme (Figures 13.14, 13.15c). In the
ATP-bound state, the clamp loader binds to a primer-template
junction while holding the ␤ clamp in an open conformation
as illustrated in Figure 13.15c. Once the DNA has squeezed
through the opening in the clamp wall, the ATP bound to the
clamp loader is hydrolyzed, causing the release of the clamp,
which closes around the DNA. The ␤ clamp is then ready to
bind polymerase III as depicted in Figure 13.15b.
DNA polymerase I, which consists of only a single subunit, is involved primarily in DNA repair, a process by which
damaged sections of DNA are corrected (page 552). DNA
polymerase I also removes the RNA primers at the 5⬘ end of
each Okazaki fragment during replication and replaces them
with DNA. The enzyme’s ability to accomplish this feat is discussed in the following section.
Exonuclease Activities of DNA Polymerases Now that
we have explained several of the puzzling properties of DNA
polymerase I, such as the enzyme’s inability to initiate strand
synthesis, we can consider another curious observation. Kornberg
found that DNA polymerase I preparations always contained
exonuclease activities; that is, they were able to degrade DNA
5' 3'
5' 3'
β clamp
β clamp
Polymerase III
Leading
strand
template
(a)
FIGURE 13.15 The ␤ sliding clamp and clamp loader. (a) Spacefilling model showing the two subunits that make up the
doughnut-shaped ␤ sliding clamp in E. coli. Double-stranded DNA is
shown in blue within the ␤ clamp. (b) Schematic diagram of polymerase
cycling on the lagging strand. The polymerase is held to the DNA by the
␤ sliding clamp as it moves along the template strand and synthesizes
the complementary strand. Following completion of the Okazaki fragment, the enzyme disengages from its ␤ clamp and cycles to a recently
assembled clamp “waiting” at an upstream RNA primer–DNA template
junction. The original ␤ clamp is left behind for a period on the finished
Okazaki fragment, but it is eventually disassembled and reutilized. (c) A
model of a complex between a sliding clamp and a clamp loader from an
archaean prokaryote based on electron microscopic image analysis. The
clamp loader (shown with red and green subunits) is bound to the sliding clamp (blue), which is held in an open, spiral conformation resembling a lock-washer. The DNA has squeezed through the gap in the
clamp. The primer strand of the DNA terminates within the clamp
loader whereas the template strand extends through an opening at the
top of the protein. The clamp loader has been described as a “screw-cap”
that fits onto the DNA in such a way that the subdomains of the protein
form a spiral that can thread onto the helical DNA backbone. (A: FROM
JOHN KURIYAN, CELL, 69:427, 1992; © CELL PRESS; B: AFTER P. T. STUKENBERG,
Lagging
strand
template
(b)
Previously
synthesized
Okazaki fragment
(c)
J. TURNER, AND M. O’DONNELL, CELL 78:878, 1994; BY PERMISSION OF
CELL PRESS; C: FROM T. MIYATA ET AL., PROC. NAT ’L. ACAD. SCI. U.S.A.
102:13799, 2005; COURTESY OF K. MORIKAWA)
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13.1 DNA REPLICATION
polymers by removing one or more nucleotides from the end
of the molecule. At first, Kornberg assumed this activity was
due to a contaminating enzyme because the action of exonucleases is so dramatically opposed to that of DNA synthesis.
Nonetheless, the exonuclease activity could not be removed
from the polymerase preparation and was, in fact, a true activity of the polymerase molecule. It was subsequently shown
that all of the bacterial DNA polymerases possess exonuclease
activity. Exonucleases can be divided into 5 → 3 and 3 → 5
exonucleases, depending on the direction in which the strand
is degraded. DNA polymerase I has both 3 → 5 and 5 → 3
exonuclease activities, in addition to its polymerizing activity
(Figure 13.16). These three activities are found in different
domains of the single polypeptide. Thus, remarkably, DNA
polymerase I is three different enzymes in one. The two exonuclease activities have entirely different roles in replication.
We will consider the 5 → 3 exonuclease activity first.
Most nucleases are specific for either DNA or RNA, but
the 5 → 3 exonuclease of DNA polymerase I can degrade
either type of nucleic acid. Initiation of Okazaki fragments
by the primase leaves a stretch of RNA at the 5 end of each
fragment (see RNA primer #1 of Figure 13.13b), which is
removed by the 5 → 3 exonuclease activity of DNA polymerase I (Figure 13.16a). As the enzyme removes ribonu5'
3' Exonuclease
hydrolysis site
5'
C
T
X
C
3'
T
C
T
A
C
G
T
A
...
G
G
T
...
G
5'
G
C A
...
...
...
...
A
...
...
A
p
...
...
...
...
G
C A
T
A
cleotides of the primer, its polymerase activity simultaneously
fills the resulting gap with deoxyribonucleotides. The last deoxyribonucleotide incorporated is subsequently joined covalently to the 5 end of the previously synthesized DNA
fragment by DNA ligase. The role of the 3 → 5 exonuclease
activity will be apparent in the following section.
Ensuring High Fidelity during DNA Replication The survival of an organism depends on the accurate duplication of the
genome. A mistake made in the synthesis of a messenger RNA
molecule by an RNA polymerase results in the synthesis of defective proteins, but an mRNA molecule is only one shortlived template among a large population of such molecules;
therefore, little lasting damage results from the mistake. In
contrast, a mistake made during DNA replication results in a
permanent mutation and the possible elimination of that cell’s
progeny. In E. coli, the chance that an incorrect nucleotide will
be incorporated into DNA during replication and remain
there is less than 109, or fewer than 1 out of 1 billion nucleotides. Because the genome of E. coli contains approximately 4 106 nucleotide pairs, this error rate corresponds to
fewer than 1 nucleotide alteration for every 100 replication cycles. This represents the spontaneous mutation rate in this bacterium. Humans are thought to have a similar spontaneous
mutation rate for replication of protein-coding sequences.
Incorporation of a particular nucleotide onto the end of a
growing strand depends on the incoming nucleoside triphosphate being able to form an acceptable base pair with the nucleotide of the template strand (see Figure 13.8b). Analysis of
the distances between atoms and bond angles indicates that
A-T and G-C base pairs have nearly identical geometry (i.e.,
size and shape). Any deviation from those pairings results in a
different geometry, as shown in Figure 13.17. At each site
along the template, DNA polymerase must discriminate
3'
Single-strand nick
(a)
CH3
O
A
A
C
C
A
G
C
T
C
...
...
...
...
T
G
T
...
...
...
T
C G
...
...
...
...
G
C
A
T
O
T
H
p
OH
3'
5'
Exonuclease
hydrolysis site
3'
5 → 3 exonuclease function removes nucleotides from the 5 end of a
single-strand nick. This activity plays a key role in removing the RNA
primers. (b) The 3 → 5 exonuclease function removes mispaired nucleotides from the 3 end of the growing DNA strand. This activity plays
a key role in maintaining the accuracy of DNA synthesis. (FROM D. VOET
J. G. VOET, BIOCHEMISTRY, 2D
ED.; COPYRIGHT
O
H N
N
© 1995, JOHN WILEY
AND SONS, INC. REPRINTED BY PERMISSION OF JOHN WILEY AND SONS, INC.)
C
N
C 1'
N
C 1'
54˚
10.8
CH3
H
N
H N
O
H N+ A
O
T
O
N H
N
N
N
C 1'
N
46˚
10.3
H
52˚
N H
68˚
G
N
H N
O
11.1
FIGURE 13.16 The exonuclease activities of DNA polymerase I. (a) The
AND
N
C 1' C 1'
51˚
5'
(b)
N H
N
C
N
N
H
50˚
C
G
G
N A
N H
C 1'
A
T
T
5'
G
H
H
N
Mismatched
bases
3'
H N
C 1'
O
69˚
H N G
N
N
H N
H
C 1'
42˚
10.3
FIGURE 13.17 Geometry of proper and mismatched base pairs.
(FROM
H. ECHOLS AND M. F. GOODMAN, REPRODUCED WITH PERMISSION FROM
THE ANNUAL REVIEW OF BIOCHEMISTRY, VOLUME 60; © 1991, BY ANNUAL
REVIEWS INC.)
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Chapter 13 DNA REPLICATION AND REPAIR
among four different potential precursors as they move in and
out of the active site. Among the four possible incoming nucleoside triphosphates, only one forms a proper geometric fit
with the template, producing either an A-T or a G-C base
pair that can fit into a binding pocket within the enzyme. This
is only the first step in the discrimination process. If the incoming nucleotide is “perceived” by the enzyme as being correct, a conformational change occurs in which the “fingers” of
the polymerase rotate toward the “palm” (Figure 13.18a),
gripping the incoming nucleotide. This is an example of an
induced fit as discussed on page 98. If the newly formed base
pair exhibits improper geometry, the active site cannot achieve
the conformation required for catalysis and the incorrect
nucleotide is not incorporated. In contrast, if the base pair
exhibits proper geometry, the incoming nucleotide is covalently linked to the end of the growing strand.
On occasion, the polymerase incorporates an incorrect
nucleotide, resulting in a mismatched base pair, that is, a base
pair other than A-T or G-C. It is estimated that an incorrect
pairing of this sort occurs once for every 105–106 nucleotides
incorporated, a frequency that is 103–104 times greater than
the spontaneous mutation rate of approximately 109. How is
the mutation rate kept so low? Part of the answer lies in the
second of the two exonuclease activities mentioned above, the
3 → 5 activity (Figure 13.16b). When an incorrect nucleotide is incorporated by DNA polymerase I, the enzyme
stalls and the end of the newly synthesized strand has an increased tendency to separate from the template and form a
single-stranded 3 terminus. When this occurs, the frayed end
of the newly synthesized strand is directed into the 3 → 5
exonuclease site (Figure 13.18), which removes the mismatched nucleotide. This job of “proofreading” is one of the
most remarkable of all enzymatic activities and illustrates
the sophistication to which biological molecular machinery
has evolved. The 3 → 5 exonuclease activity removes approximately 99 out of every 100 mismatched bases, raising the
fidelity to about 107–108. In addition, bacteria possess a
mechanism called mismatch repair that operates after replication (page 554) and corrects nearly all of the mismatches that
escape the proofreading step. Together these processes reduce
the overall observed error rate to about 109. Thus the fidelity
of DNA replication can be traced to three distinct activities:
(1) accurate selection of nucleotides, (2) immediate proofreading, and (3) postreplicative mismatch repair.
Another remarkable feature of bacterial replication is its
rate. The replication of an entire bacterial chromosome in approximately 40 minutes at 37C requires that each replication
fork move about 1000 nucleotides per second, which is equivalent to the length of an entire Okazaki fragment. Thus the
entire process of Okazaki fragment synthesis, including formation of an RNA primer, DNA elongation and simultaneous proofreading by the DNA polymerase, excision of the
RNA, its replacement with DNA, and strand ligation, occurs
within a few seconds. Although it takes E. coli approximately
40 minutes to replicate its DNA, a new round of replication
can begin before the previous round has been completed.
Consequently, when these bacteria are growing at their maximal rate, they double their numbers in about 20 minutes.
Fingers Palm
Thumb Newly
synthesized
strand
3' OH
3'
3'
5'
5'
Template
strand
Mismatched
base
3' 5'
Exonuclease
site
(a)
Mismatched base
to be removed
(b)
FIGURE 13.18 Activation of the 3 → 5 exonuclease of DNA polymerase I. (a) A schematic model of a portion of DNA polymerase I
known as the Klenow fragment, which contains the polymerase and
3 → 5 exonuclease active sites. The 5 → 3 exonuclease activity is located in a different portion of the polypeptide, which is not shown here.
The regions of the Klenow fragment are often likened to the shape of a
partially opened right hand, hence the portions labeled as “fingers,”
“palm,” and “thumb.” The catalytic site for polymerization is located in
the central “palm” subdomain. The 3 terminus of the growing strand
can be shuttled between the polymerase and exonuclease active sites.
Addition of a mismatched base to the end of the growing strand produces
a frayed (single-stranded) 3 end that enters the exonuclease site, where it
is removed. (The polymerase and exonuclease sites of polymerase III operate similarly but are located on different subunits.) (b) A molecular
model of the Klenow fragment complexed to DNA. The template DNA
strand being copied is shown in blue, and the primer strand to which
the next nucleotides would be added is shown in red. (A: AFTER T. A.
BAKER AND S. P. BELL, CELL 92:296, 1998; AFTER A DRAWING BY C. M. JOYCE
CELL PRESS; B: COURTESY OF THOMAS
A. STEITZ.)
AND T. A. STEITZ, BY PERMISSION OF
Replication in Eukaryotic Cells
As noted in Chapter 10, the nucleotide letters of the human
genome sequence would fill a book roughly one million pages in
length. While it took several years for hundreds of researchers
to sequence the human genome, a single cell nucleus of approximately 10 m diameter can copy all of this DNA within a few
hours. Given the fact that eukaryotic cells have large genomes
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13.1 DNA REPLICATION
547
and complex chromosomal structure, our understanding of
replication in eukaryotes has lagged behind that in bacteria.
This imbalance has been addressed by the development of eukaryotic experimental systems that parallel those used for
decades to study bacterial replication. These include
■
■
■
The isolation of mutant yeast and animal cells unable to
produce specific gene products required for various aspects
of replication.
Analysis of the structure and mechanism of action of replication proteins from archaeal species (as in Figure 13.15c).
Replication in these prokaryotes begins at multiple origins
and requires proteins that are homologous to those of eukaryotic cells but are less complex and easier to study.
The development of in vitro systems where replication can
occur in cellular extracts or mixtures of purified proteins.
The most valuable of these systems has utilized Xenopus,
an aquatic frog that begins life as a huge egg stocked with
all of the proteins required to carry it through a dozen or
so very rapid rounds of cell division. Extracts can be prepared from these frog eggs that will replicate any added
DNA, regardless of sequence. Frog egg extracts will also
support the replication and mitotic division of mammalian
nuclei, which has made this a particularly useful cell-free
system. Antibodies can be used to deplete the extracts of
particular proteins, and the replication ability of the extract
can then be tested in the absence of the affected protein.
Replication in
E. coli begins at only one site along the single, circular chromosome (Figure 13.5). Cells of higher organisms may have a
thousand times as much DNA as this bacterium, yet their
polymerases incorporate nucleotides into DNA at much slower
rates. To accommodate these differences, eukaryotic cells
replicate their genome in small portions, termed replicons.
Each replicon has its own origin from which replication forks
proceed outward in both directions (see Figure 13.24a). In a
human cell, replication begins at about 10,000 to 100,000
different replication origins. The existence of replicons was
first demonstrated in autoradiographic experiments in which
single DNA molecules were shown to be replicated simultaneously at several sites along their length (Figure 13.19).
Approximately 10 to 15 percent of replicons are actively
engaged in replication at any given time during the S phase of
the cell cycle (see Figure 14.1). Replicons located close together in a given chromosome tend to undergo replication simultaneously (as evident in Figure 13.19). Moreover, those
replicons active at a particular time during one round of DNA
synthesis tend to be active at a comparable time in succeeding
rounds. In mammalian cells, the timing of replication of a
chromosomal region is roughly correlated with the activity of
the genes in the region and/or its state of compaction. The
presence of acetylated histones, which is closely correlated with
gene transcription (page 516), is a likely factor in determining
the early replication of active gene loci. The most highly compacted, least acetylated regions of the chromosome are packaged into heterochromatin (page 485), and they are the last
regions to be replicated. This difference in timing of replica-
Initiation of Replication in Eukaryotic Cells
FIGURE 13.19 Experimental demonstration that replication in eukaryotic
chromosomes begins at many sites along the DNA. Cells were incubated in [3H] thymidine for a brief period before preparation of DNA
fibers for autoradiography. The lines of black silver grains indicate sites
that had incorporated the radioactive DNA precursor during the labeling
period. It is evident that synthesis is occurring at separated sites along the
same DNA molecule. As indicated in the accompanying line drawing,
initiation begins in the center of each site of thymidine incorporation,
forming two replication forks that travel away from each other until they
meet a neighboring fork. (MICROGRAPH COURTESY OF JOEL HUBERMAN.)
tion is not related to DNA sequence because the inactive, heterochromatic X chromosome in the cells of female mammals
(page 486) is replicated late in S phase, whereas the active, euchromatic X chromosome is replicated at an earlier stage.
The mechanism by which replication is initiated in eukaryotes has been a focus of research over the past decade. The
greatest progress in this area has been made with budding
yeast because the origins of replication can be removed from
the yeast chromosome and inserted into bacterial DNA
molecules, conferring on them the ability to replicate either
within a yeast cell or in cellular extracts containing the
required eukaryotic replication proteins. Because these
sequences promote replication of the DNA in which they
are contained, they are referred to as autonomous replicating
sequences (ARSs). Those ARSs that have been isolated and
analyzed share several distinct elements. The core element of
an ARS consists of a conserved sequence of 11 base pairs,
which functions as a specific binding site for an essential multiprotein complex called the origin recognition complex (ORC)
(see Figure 13.20). If the ARS is mutated so that it is unable
to bind the ORC, initiation of replication cannot occur.
Replication origins have proven more difficult to study in
vertebrate cells than in yeast. Part of the problem stems from
the fact that virtually any type of purified, naked DNA is suitable for replication using extracts from frog eggs. These studies
suggested that, unlike yeast, vertebrate DNA does not possess
specific sequences (e.g., ARSs) at which replication is initiated.
However, studies of replication of intact mammalian chromosomes in vivo suggest that replication does begin within defined regions of the DNA, rather than by random selection as
occurs in the amphibian egg extract. It is thought that a DNA
molecule contains many sites where DNA replication can be
initiated, but only a subset of these sites are actually used at a
given time in a given cell. Cells that reproduce via shorter cell
cycles, such as those of early amphibian embryos, utilize a
greater number of sites as origins of replication than cells with
longer cell cycles. The actual selection of sites for initiation of
replication is thought to be governed by local epigenetic factors
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Chapter 13 DNA REPLICATION AND REPAIR
(page 496), such as the positions of nucleosomes, the types of
histone modifications, the state of DNA methylation, the degree of supercoiling, and the level of transcription.
Restricting Replication to Once Per Cell Cycle It is essential that each portion of the genome is replicated once, and
only once, during each cell cycle. Consequently, some mechanism must exist to prevent the reinitiation of replication at a
site that has already been duplicated. The initiation of replication at a particular origin requires passage of the origin through
several distinct states. Some of the steps that occur at an origin
of replication in a yeast cell are illustrated in Figure 13.20.
Similar steps requiring homologous proteins take place in
plants and animals, suggesting that the basic mechanism of
initiation of replication is conserved among eukaryotes.
1. In step 1 (Figure 13.20), the origin of replication is bound
by an ORC protein complex, which in yeast cells remains
associated with the origin throughout the cell cycle. The
ORC has been described as a “molecular landing pad”
because of its role in binding the proteins required in subsequent steps.
2. Proteins referred to as “licensing factors” bind to the ORC
(step 2, Figure 13.20) to assemble a protein–DNA complex, called the prereplication complex (pre-RC), that is “licensed” (competent) to initiate replication. Studies of the
molecular nature of the licensing factors have focused on a
set of six related Mcm proteins (Mcm2–Mcm7). The Mcm
proteins are loaded onto the replication origin at a late
stage of mitosis, or soon thereafter. Studies indicate that
the Mcm2–Mcm7 proteins are capable of associating into a
ring-shaped complex that possesses helicase activity (as in
step 4, Figure 13.20). Most evidence suggests that the
Mcm2–Mcm7 complex is the eukaryotic replicative helicase; that is, the helicase responsible for unwinding DNA
at the replication fork (analogous to DnaB in E. coli).
3. Just before the beginning of S phase of the cell cycle, the
activation of key protein kinases leads to the activation of
the Mcm2–Mcm7 helicase and the initiation of replication (step 3, Figure 13.20). One of these protein kinases
is a cyclin-dependent kinase (Cdk) whose function is discussed at length in Chapter 14. Cdk activity remains high
from S phase through mitosis, which suppresses the formation of new prereplication complexes. Consequently,
1
ARS
ORC
Licensing factors bind
soon after mitosis
Requires Cdc6 and Cdt1
Licensing factors
(Mcm2-Mcm7)
Prereplication complex
2
Protein kinases (Cdk and
DDK) phosphorylate and
activate pre-RC complex
at beginning of S phase
3
Initiation of replication
FIGURE 13.20 Steps leading to the replication of a yeast replicon. Yeast
origins of replication contain a conserved sequence (ARS) that binds the
multisubunit origin recognition complex (ORC) (step 1). The presence
of the bound ORC is required for initiation of replication. The ORC is
bound to the origin throughout the yeast cell cycle. In step 2, licensing
factors (identified as Mcm proteins) bind to the origin during or following mitosis, establishing a prereplication complex that is competent to
initiate replication, given the proper stimulus. Loading of Mcm proteins
at the origin requires additional proteins (Cdc6 and Cdt1, not shown). In
step 3, DNA replication is initiated following the activation of specific
protein kinases, including a cyclin-dependent kinase (Cdk). Step 4
shows a stage where replication has proceeded a short distance in both
directions from the origin. In this model, the Mcm proteins form a
replicative DNA helicase that unwinds DNA of the oppositely directed
replication forks. The other proteins required for replication are not
shown in this illustration but are indicated in the next figure. In step 5,
the two strands of the original duplex have been replicated, an ORC is
present at both origins, and the replication proteins, including the Mcm
helicases, have been displaced from the DNA. In yeast, the Mcm proteins are exported from the nucleus, and reinitiation of replication cannot occur until the cell has passed through mitosis. [In vertebrate cells,
several events appear to prevent reinitiation of replication, including
(1) continued Cdk activity from S phase into mitosis, (2) phosphorylation
of Cdc6 and its subsequent export from the nucleus, and (3) inactivation
of Cdt1 by a bound inhibitor.]
Newly synthesized
DNA strand
Mcm2-Mcm7
(helicase?)
4
ORC
5
+
Mcm proteins
Nuclear envelope
Daughter DNAs
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TABLE 13.1 Some of the Proteins Required for Replication
E. coli
protein
Eukaryotic
protein
DnaA
Gyrase
DnaB
DnaC
SSB
-complex
pol III core
ORC proteins
Topoisomerase I/II
Mcm
Cdc6, Cdt1
RPA
RFC
pol /
clamp
PCNA
Primase
———
DNA ligase
pol I
Primase
pol DNA ligase
FEN-1
Function
Recognition of origin of replication
Relieves positive supercoils ahead of replication fork
DNA helicase that unwinds parental duplex
Loads helicase onto DNA
Maintains DNA in single-stranded state
Subunits of the DNA polymerase holoenzyme that load the clamp onto the DNA
Primary replicating enzymes; synthesize entire leading strand and Okazaki fragments; have
proofreading capability
Ring-shaped subunit of DNA polymerase holoenzyme that clamps replicating polymerase to
DNA; works with pol III in E. coli and pol or in eukaryotes
Synthesizes RNA primers
Synthesizes short DNA oligonucleotides as part of RNA–DNA primer
Seals Okazaki fragments into continuous strand
Removes RNA primers; pol I of E. coli also fills gap with DNA
each origin can only be activated once per cell cycle. Cessation of Cdk activity at the end of mitosis permits the
assembly of a pre-RC for the next cell cycle.
4. Once replication is initiated at the beginning of S phase,
the Mcm proteins move with the replication fork (step 4)
and are essential for completion of replication of a replicon.
The fate of the Mcm proteins after replication depends on
the species studied. In yeast, the Mcm proteins are displaced from the chromatin and exported from the nucleus
(step 5). In contrast, the Mcm proteins in mammalian cells
are displaced from the DNA but apparently remain in the
nucleus. Regardless, Mcm proteins cannot reassociate with
an origin of replication that has already “fired.”
Overall, the activities
that occur at replication forks are quite similar, regardless
of the type of genome being replicated—whether viral, bacterial, archaeal, or eukaryotic. The various proteins in the replication “tool kit” of eukaryotic cells are listed in Table 13.1
and depicted in Figure 13.21. All replication systems require
helicases, single-stranded DNA-binding proteins, topoisomerases, primase, DNA polymerase, sliding clamp and clamp
The Eukaryotic Replication Fork
PCNA
Pol ε
RPA
Leading-strand template
DNA Ligase
PCNA
PCNA
Pol ε
RFC
RNA primer
Pol α
RFC
RFC
Helicase (T antigen)
Helicase (T antigen)
Pol δ
Topoisomerase
Topoisomerase
Primase
RPA
FEN-I
Pol δ
Lagging-strand template
(a)
FIGURE 13.21 A schematic view of the major components at the eukaryotic replication fork. (a) The proteins required for eukaryotic replication. The viral T antigen is drawn as the replicative helicase in this
figure because it is prominently employed in in vitro studies of DNA
replication. DNA polymerases and are thought to be the primary
DNA synthesizing enzymes of the lagging and leading strands, respectively. PCNA acts as a sliding clamp for both polymerases and . The
sliding clamp is loaded onto the DNA by a protein called RFC (replication factor C), which is similar in structure and function to the -clamp
loader of E. coli. RPA is a trimeric single-stranded DNA-binding protein comparable in function to that of SSB utilized in E. coli replication.
Primase
(b)
The RNA-DNA primers of the lagging strand that are synthesized by
the polymerase -primase complex are displaced by the continued
movement of polymerase , generating a flap of RNA-DNA that is
removed by the FEN-1 endonuclease. The gap is sealed by a DNA
ligase. As in E. coli, a topoisomerase is required to remove the positive
supercoils that develop ahead of the replication fork. (b) A proposed version of events at the replication fork illustrating how the replicative
polymerases on the leading- and lagging-strand templates might act together as part of a replisome. To date there is no firm evidence that the
leading and lagging strands are replicated by a single replicative complex
as in E. coli.
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Chapter 13 DNA REPLICATION AND REPAIR
loader, and DNA ligase. When studying the initiation of eukaryotic replication in vitro, researchers often combine mammalian replication proteins with a viral helicase called the large
T antigen, which is encoded by the SV40 genome. The large T
antigen induces strand separation at the SV40 origin of replication and unwinds the DNA as the replication fork progresses (as in Figure 13.21a).
As in bacteria, the DNA of eukaryotic cells is synthesized
in a semidiscontinuous manner, although the Okazaki fragments of the lagging strand are considerably smaller than
in bacteria, averaging about 150 nucleotides in length. Like
DNA polymerase III of E. coli, the eukaryotic replicative DNA
polymerase is present as a dimer, suggesting that the leading
and lagging strands are synthesized in a coordinate manner by
a single replicative complex, or replisome (Figure 13.21b).
To date, five “classic” DNA polymerases have been isolated from eukaryotic cells, and they are designated ␣, ␤, ␥, ␦,
and ␧. Of these enzymes, polymerase ␥ replicates mitochondrial DNA, and polymerase ␤ functions in DNA repair. The
other three polymerases have replicative functions. Polymerase
␣ is tightly associated with the primase, and together they initiate the synthesis of each Okazaki fragment. Primase initiates
synthesis by assembly of a short RNA primer, which is then extended by the addition of about 20 deoxyribonucleotides by
polymerase ␣. Polymerase ␦ is thought to be the primary
DNA-synthesizing enzyme during replication of the lagging
strand, whereas polymerase ␧ is thought to be the primary
DNA-synthesizing enzyme during replication of the leading
strand. Like the major replicating enzyme of E. coli, both polymerase ␦ and ␧ require a “sliding clamp” that tethers the enzyme to the DNA, allowing it to move processively along a
template. The sliding clamp of eukaryotic cells is very similar
in structure and function to the ␤ clamp of E. coli polymerase
III illustrated in Figure 13.14. In eukaryotes, the sliding clamp
is called PCNA. The clamp loader that loads PCNA onto the
DNA is called RFC and is analogous to the E. coli polymerase
III clamp loader complex. After synthesizing an RNA-DNA
primer, polymerase ␣ is replaced at each template–primer
junction by the PCNA–polymerase ␦ complex, which completes synthesis of the Okazaki fragment. When polymerase ␦
reaches the 5⬘ end of the previously synthesized Okazaki fragment, the polymerase continues along the lagging-strand template, displacing the primer (shown as a green flap in Figure
13.21a). The displaced primer is cut from the newly synthesized DNA strand by an endonuclease (FEN-1) and the resulting nick in the DNA is sealed by a DNA ligase. FEN-1 and
DNA ligase are thought to be recruited to the replication fork
through an interaction with the PCNA sliding clamp. In fact,
PCNA is thought to play a major role in orchestrating events
that occur during DNA replication, repair, and recombination.
Because of its ability to bind a diverse array of proteins, PCNA
has been referred to as a “molecular toolbelt.”
Like bacterial polymerases, all of the eukaryotic polymerases elongate DNA strands in the 5⬘ → 3⬘ direction by the
addition of nucleotides to a 3⬘ hydroxyl group, and none of
them is able to initiate the synthesis of a DNA chain without
a primer. Polymerases ␥, ␦, and ␧ possess a 3⬘ → 5⬘ exonuclease, whose proofreading activity ensures that replication oc-
curs with very high accuracy. Several other DNA polymerases
(including ␩, ␬ and ␫) have a specialized function that allows
cells to replicate damaged DNA as described on page 558.
Up to this point in the
chapter, the illustrations of replication depict a replicating
polymerase moving like a locomotive along a stationary DNA
track. But the replication apparatus consists of a huge complex
of proteins that operates within the confines of a structured
nucleus. Considerable evidence suggests that the replication
machinery is present in association with both the nuclear lamina
(page 477) and the nuclear matrix (page 499). When cells are
given very short pulses of radioactive DNA precursors, over
80 percent of the incorporated label is associated with the nuclear matrix. If, instead of fixing the cells immediately after a
pulse, the cells are allowed to incorporate unlabeled DNA precursors for an hour or so before fixation, most of the radioactivity is chased from the matrix into the surrounding DNA
loops. This latter finding suggests that rather than remaining stationary, replicating DNA moves like a conveyer belt
through an immobilized replication apparatus (Figure 13.22).
Further studies suggest that replication forks that are active at a given time are not distributed randomly throughout
the cell nucleus, but instead are localized within 50 to 250
sites, called replication foci (Figure 13.23). It is estimated
that each of the bright red regions indicated in Figure 13.23
contains approximately 40 replication forks incorporating nucleotides into DNA strands simultaneously. The clustering of
replication forks may provide a mechanism for coordinating
Replication and Nuclear Structure
3'
5'
3'
5'
3'
5'
FIGURE 13.22 The involvement of the nuclear matrix in DNA replication. The origins of replication are indicated by the black dots, and the
arrows indicate the direction of elongation of growing strands. According to this schematic model, it isn’t the replication machinery that moves
along stationary DNA tracks, but the DNA that is spooled through the
replication apparatus, which is firmly attached to the nuclear matrix.
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13.1 DNA REPLICATION
551
the replication of adjacent replicons on individual chromosomes (as in Figure 13.19).
The chromosomes of
eukaryotic cells consist of DNA tightly complexed to regular
arrays of histone proteins that are present in the form of nucleosomes (page 481). Movement of the replication machinery along
the DNA is thought to displace nucleosomes that reside in its
path. Yet, examination of a replicating DNA molecule with the
electron microscope reveals nucleosomes on both daughter duplexes very near the replication fork (Figure 13.24a), indicating
that the reassembly of nucleosomes is a very rapid event. Collectively, the nucleosomes that form during the replication
process are comprised of a roughly equivalent mixture of histone molecules that are inherited from parental chromosomes
and histone molecules that have been newly synthesized. Recall
from page 482 that the core histone octamer of a nucleosome
consists of an (H3H4)2 tetramer together with a pair of
H2A/H2B dimers. The way in which parental nucleosomes are
distributed during replication has been an area of recent debate.
According to one line of research, the (H3H4)2 tetramers present prior to replication remain intact and are distributed randomly between the two daughter duplexes. As a result, old and
new (H3H4)2 tetramers are thought to be intermixed on each
daughter DNA molecule as indicated in the model shown in
Figure 13.24b. According to this model, the two H2A/H2B
dimers of each parental nucleosome fail to remain together as
the replication fork moves through the chromatin. Instead, the
H2A-H2B dimers of a nucleosome separate from one another
and bind randomly to the new and old (H3H4)2 tetramers
already present on the daughter duplexes (Figure 13.24b).
According to another viewpoint, the (H3H4)2 tetramer from
parental nucleosomes can be split into two H3-H4 dimers, each
Chromatin Structure and Replication
FIGURE 13.23 Demonstration that replication activities do not occur
randomly throughout the nucleus but are confined to distinct sites.
Prior to the onset of DNA synthesis at the start of S phase, various factors required for the initiation of replication are assembled at discrete sites
within the nucleus, forming prereplication centers. These sites appear as
discrete red objects in the micrograph, which has been stained with a fluorescent antibody against replication factor A (RPA), which is a singlestranded DNA-binding protein required for the initiation of replication.
Other replication factors, such as PCNA and the polymerase–primase
complex, are also localized to these foci. (FROM YASUHISA ADACHI AND
ULRICH K. LAEMMLI, EMBO J. VOL. 13, COVER NO. 17, 1994.)
5'
3'
3'
5'
(H3H4)2
Nucleosome
(b)
H2A/H2B
H2A/H2B
FIGURE 13.24 The distribution of histone core complexes to daughter strands following replication. (a) Electron micrograph of chromatin isolated from the nucleus of a rapidly cleaving Drosophila embryo showing a pair
of replication forks (arrows) moving away from each other in opposite directions. Between the two forks one sees
regions of newly replicated DNA that are already covered by nucleosomal core particles to the same approximate
density as the parental strands that have not yet undergone replication. (b) Schematic model showing the distribution of core histones after DNA replication. Each nucleosome core particle is shown schematically to be composed of a central (H3H4)2 tetramer flanked by two H2A/H2B dimers. Histones that were present in parental
nucleosomes prior to replication are indicated in blue; newly synthesized histones are indicated in red. According to this model, the parental (H3H4)2 tetramers remain intact and are distributed randomly to both daughter
duplexes. In contrast, the pairs of H2A/H2B dimers present in parental nucleosomes separate and recombine
randomly with the (H3H4)2 tetramers on the daughter duplexes. Other models have been presented in which the
parental (H3H4)2 tetramer is split in half by a histone chaperone, and the two resulting H3-H4 dimers are distributed to different DNA strands (discussed in Cell 128:721, 2007 and Trends Cell Biol. 19:29, 2009). (A: COUR(a)
TESY OF
STEVEN L. MCKNIGHT AND OSCAR L. MILLER, JR.)
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Chapter 13 DNA REPLICATION AND REPAIR
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
The original Watson-Crick proposal for DNA replication envisioned the continuous synthesis of DNA
strands. How and why has this concept been modified
over the intervening years?
What does it mean that replication is semiconservative?
How was this feature of replication demonstrated in
bacterial cells? in eukaryotic cells?
Why are there no heavy bands in the top three centrifuge tubes of Figure 13.3a?
How is it possible to obtain mutants whose defects lie in
genes that are required for an essential activity such as
DNA replication?
Describe the events that occur at an origin of replication
during the initiation of replication in yeast cells. What is
meant by replication being bidirectional?
Why do the DNA molecules depicted in Figure 13.7a
fail to stimulate the polymerization of nucleotides by
DNA polymerase I? What are the properties of a DNA
molecule that allow it to serve as a template for nucleotide incorporation by DNA polymerase I?
Describe the mechanism of action of DNA polymerases
operating on the two template strands and the effect
this has on the synthesis of the lagging versus the leading strand.
Contrast the role of DNA polymerases I and III in bacterial replication.
Describe the role of the DNA helicase, the SSBs, the
␤ clamp, the DNA gyrase, and the DNA ligase during
replication in bacteria.
What is the consequence of having the DNA of the
lagging-strand template looped back on itself as in Figure 13.13a?
How do the two exonuclease activities of DNA polymerase I differ from one another? What are their respective roles in replication?
Describe the factors that contribute to the high fidelity
of DNA replication.
What is the major difference between bacteria and eukaryotes that allows a eukaryotic cell to replicate its
DNA in a reasonable amount of time?
5'
3'
OH
O
O
O
O
OH
3'
O
1.
?
Life on Earth is subject to a relentless onslaught of destructive forces that originate in both the internal and external environments of an organism. Of all the molecules in a
cell, DNA is placed in the most precarious position. On one
hand, it is essential that the genetic information remain mostly
unchanged as it is passed from cell to cell and individual to individual. On the other hand, DNA is one of the molecules in a
cell that is most susceptible to environmental damage. When
struck by ionizing radiation, the backbone of a DNA molecule
is often broken; when exposed to a variety of reactive chemicals, many of which are produced by a cell’s own metabolism,
the bases of a DNA molecule may be altered structurally; when
subjected to ultraviolet radiation, adjacent pyrimidines on a
DNA strand have a tendency to interact with one another to
form a covalent complex, that is, a dimer (Figure 13.25). Even
the absorption of thermal energy generated by metabolism is
sufficient to split adenine and guanine bases from their attachment to the sugars of the DNA backbone. The magnitude
of these spontaneous alterations, or lesions, can be appreciated
from the estimate that each cell of a warm-blooded mammal
loses approximately 10,000 bases per day! Failure to repair such
lesions produces permanent alterations, or mutations, in the
DNA. If the mutation occurs in a cell destined to become a gamete, the genetic alteration may be passed on to the next generation. Mutations also have effects in somatic cells (i.e., cells
that are not in the germ line): they can interfere with transcription and replication, lead to the malignant transformation of a
cell, or speed the process by which an organism ages.
Considering the potentially drastic consequences of alterations in DNA molecules and the high frequency at which they
occur, it is essential that cells possess mechanisms for repairing
DNA damage. In fact, cells have a bewildering arsenal of repair
O
REVIEW
13.2 DNA REPAIR
O
of which may combine with a newly synthesized H3-H4 dimer
to form a “mixed” (H3H4)2 tetramer, which then assembles
with H2A-H2B dimers. Regardless of the pattern by which it
occurs, the stepwise assembly of nucleosomes and their orderly
spacing along the DNA is facilitated by a network of accessory
proteins. Included among these proteins are a number of histone chaperones that are able to accept either newly synthesized
or parental histones and transfer them to the daughter strands.
The best studied of these histone chaperones, CAF-1, is recruited to the advancing replication fork through an interaction
with the sliding clamp PCNA.
O
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5'
FIGURE 13.25 A pyrimidine dimer that has formed within a DNA duplex
following UV irradiation.
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13.2 DNA REPAIR
systems that correct virtually any type of damage to which a
DNA molecule is vulnerable. It is estimated that less than one
base change in a thousand escapes a cell’s repair systems. The
existence of these systems provides an excellent example of the
molecular mechanisms that maintain cellular homeostasis. The
importance of DNA repair can be appreciated by examining the
effects on humans that result from DNA repair deficiencies, a
subject discussed in the Human Perspective on page 556.
Both prokaryotic and eukaryotic cells possess a variety of
proteins that patrol vast stretches of DNA, searching for subtle
chemical modifications or distortions of the DNA duplex. In
some cases, damage can be repaired directly. Humans, for example, possess enzymes that can directly repair damage from
cancer-producing alkylating agents. Most repair systems, however, require that a damaged section of the DNA be excised, that
is, selectively removed. One of the great virtues of the DNA duplex is that each strand contains the information required for
constructing its partner. Consequently, if one or more nucleotides is removed from one strand, the complementary
strand can serve as a template for reconstruction of the duplex.
The repair of DNA damage in eukaryotic cells is complicated
by the relative inaccessibility of DNA within the folded chromatin fibers of the nucleus. As in the case of transcription,
DNA repair involves the participation of chromatin-reshaping
machines, such as the histone modifying enzymes and nucleosome remodeling complexes discussed on page 517. Although
presumably important in DNA repair, the roles of these proteins will not be considered in the following discussion.
553
on the Web at www.wiley.com/college/karp). Included among
the various subunits of TFIIH are two subunits (XPB and XPD)
that possess helicase activity; these enzymes separate the two
strands of the duplex (step 2, Figure 13.26) in preparation for removal of the lesion. The damaged strand is then cut on both sides
of the lesion by a pair of endonucleases (step 3), and the segment
of DNA between the incisions is released (step 4). Once excised,
the gap is filled by a DNA polymerase (step 5), and the strand is
sealed by DNA ligase (step 6).
CSB
RNA polymerase
T=T
1
RNA
XPC
Transcription-coupled pathway
T=T
Global pathway
2
T=T
3
3'OH 5' P
3'OH 5'P
T=T
Nucleotide Excision Repair
4
3'OH
5' P
T
T=
Nucleotide excision repair (NER) operates by a cut-andpatch mechanism that removes a variety of bulky lesions, including pyrimidine dimers and nucleotides to which various
chemical groups have become attached. Two distinct NER
pathways can be distinguished:
1. A transcription-coupled pathway in which the template
strands of genes that are being actively transcribed are
preferentially repaired. Repair of a template strand is
thought to occur as the DNA is being transcribed, and
the presence of the lesion may be signaled by a stalled
RNA polymerase. This preferential repair pathway ensures that those genes of greatest importance to the cell,
which are the genes the cell is actively transcribing, receive the highest priority on the “repair list.”
2. A slower, less efficient global genomic pathway that corrects DNA strands in the remainder of the genome.
Although recognition of the lesion is probably accomplished
by different proteins in the two NER pathways (step 1, Figure
13.26), the steps that occur during repair of the lesion are thought
to be very similar, as indicated in steps 2–6 of Figure 13.26. One
of the key components of the NER repair machinery is TFIIH, a
huge protein that also participates in the initiation of transcription. The discovery of the involvement of TFIIH established a
crucial link between transcription and DNA repair, two processes
that were previously assumed to be independent of one another
(discussed in the Experimental Pathways, which can be accessed
5
DNA polymerase δ/ε
3'OH
5' P
6
FIGURE 13.26 Nucleotide excision repair. The following steps are depicted
in the drawing and discussed in the text: (1) damage recognition in the
global pathway is mediated by an XPC-containing protein complex,
whereas damage recognition in the transcription-coupled pathway is
thought to be mediated by a stalled RNA polymerase in conjunction
with a CSB protein; (2) DNA strand separation (by XPB and XPD
proteins, two helicase subunits of TFIIH); (3) incision (by XPG on the
3 side and the XPF–ERCC1 complex on the 5 side); (4) excision,
(5) DNA repair synthesis (by DNA polymerase and/or ); and (6) ligation
(by DNA ligase I).
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Base Excision Repair
A separate excision repair system operates to remove altered
nucleotides generated by reactive chemicals present in the diet
or produced by metabolism. The steps in this repair pathway
in eukaryotes, which is called base excision repair (BER), are
shown in Figure 13.27. BER is initiated by a DNA glycosylase
that recognizes the alteration (step 1, Figure 13.27) and removes the base by cleavage of the glycosidic bond holding the
base to the deoxyribose sugar (step 2). A number of different
DNA glycosylases have been identified, each more-or-less
specific for a particular type of altered base, including uracil
(formed by the hydrolytic removal of the amino group of cytosine), 8-oxoguanine (caused by damage from oxygen free
radicals, page 34), and 3-methyladenine (produced by transfer
of a methyl group from a methyl donor, page 431).
Structural studies of the DNA glycosylase that removes
the highly mutagenic 8-oxoguanine (oxoG) indicate that this
enzyme diffuses rapidly along the DNA “inspecting” each of
the G-C base pairs within the DNA duplex (Figure 13.28,
step 1). In step 2, the enzyme has come across an oxoG-C
base pair. When this occurs, the enzyme inserts a specific
amino acid side chain into the DNA helix, causing the nucleotide to rotate (“flip”) 180 degrees out of the DNA helix
and into the body of the enzyme (step 2). If the nucleotide
does, in fact, contain an oxoG, the base fits into the active site
of the enzyme (step 3) and is cleaved from its associated sugar.
In contrast, if the extruded nucleotide contains a normal guanine, which only differs in structure by two atoms from oxoG,
it is unable to fit into the enzyme’s active site (step 4) and it is
returned to its appropriate position within the stack of bases.
Once an altered purine or pyrimidine is removed by a glycosylase, the “beheaded” deoxyribose phosphate remaining in
the site is excised by the combined action of a specialized
(AP) endonuclease and a DNA polymerase. AP endonuclease cleaves the DNA backbone (Figure 13.27, step 3) and a
phosphodiesterase activity of polymerase ␤ removes the
sugar–phosphate remnant that had been attached to the excised base (step 4). Polymerase ␤ then fills the gap by inserting a nucleotide complementary to the undamaged strand
(step 5), and the strand is sealed by DNA ligase III (step 6).
The fact that cytosine can be converted to uracil may explain why natural selection favored the use of thymine, rather
than uracil, as a base in DNA, even though uracil was presumably present in RNA when it served as genetic material
during the early evolution of life (page 448). If uracil had been
retained as a DNA base, it would have caused difficulty for repair
systems to distinguish between a uracil that “belonged” at a particular site and one that resulted from an alteration of cytosine.
5'
It was noted earlier that cells can remove mismatched bases that
are incorporated by the DNA polymerase and escape the enzyme’s proofreading exonuclease. This process is called mismatch repair (MMR). A mismatched base pair causes a
distortion in the geometry of the double helix that can be recognized by a repair enzyme. But how does the enzyme “recognize”
P
P
P
P
G
U
C
A
C
G
G
T
P
3'
1
3'
P
P
P
P
P
P
5'
Uracil–DNA
glycosylase
5'
P
P
P
P
G
P
C
A
G
T
P
3'
2
C
3'
P
G
P
P
P
P
P
5'
AP endonuclease
OH P
5'
P
P
P
G
P
C
A
G
T
P
3'
3
C
3'
P
G
P
P
P
P
P
5'
Phosphodiesterase
activity of DNA
polymerase β
OH
5'
P
P
P
G
P
C
A
G
T
P
3'
4
G
C
3'
P
P
P
P
P
P
5'
Polymerase activity of
DNA polymerase β
OH
5'
P
P
P
P
P
G
C
C
A
C
G
G
T
P
3'
5
3'
P
P
P
P
P
P
5'
DNA ligase III
5'
Mismatch Repair
P
P
P
P
P
P
G
C
C
A
C
G
G
T
P
3'
6
3'
P
P
P
P
P
P
5'
FIGURE 13.27 Base excision repair. The steps are described in the text.
Other pathways for BER are known, and BER also has been shown to
have distinct transcription-coupled and global repair pathways.
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13.2 DNA REPAIR
1
2
h0GG1
o
G
C
G
C
3
4
o
G
o
G
C
555
G
C
C
G
G
C
C
C
FIGURE 13.28 Detecting damaged bases during BER. In step 1, a DNA
glycosylase (named hOGG1) is inspecting a nucleotide that is paired
to a cytosine. In step 2, the nucleotide is flipped out of the DNA duplex.
In this case, the base is an oxidized version of guanine, 8-oxoguanine,
and it is able to fit into the active site of the enzyme (step 3) where it is
cleaved from its attached sugar. The subsequent steps in BER were
shown in Figure 13.27. In step 4, the extruded base is a normal guanine,
which is unable to fit into the active site of the glycosylase and is
returned to the base stack. Failure to remove oxoG would have resulted
in a G-to-T mutation. (BASED ON S. S. DAVID, WITH PERMISSION FROM
which member of the mismatched pair is the incorrect nucleotide? If it were to remove one of the nucleotides at random,
it would make the wrong choice 50 percent of the time, creating
a permanent mutation at that site. Thus, for a mismatch to be repaired after the DNA polymerase has moved past a site, it is important that the repair system distinguish the newly synthesized
strand, which contains the incorrect nucleotide, from the
parental strand, which contains the correct nucleotide. In E. coli,
the two strands are distinguished by the presence of methylated
adenosine residues on the parental strand. DNA methylation
does not appear to be utilized by the MMR system in eukaryotes, and the mechanism of identification of the newly synthe-
sized strand remains unclear. Several different MMR pathways
have been identified and will not be discussed.
1
Ku
2
DNA-PKcs
NATURE 434:569, 2005; ©
LIMITED.)
COPYRIGHT
2005,
BY
MACMILLAN MAGAZINES
Double-Strand Breakage Repair
X-rays, gamma rays, and particles released by radioactive atoms
are all described as ionizing radiation because they generate ions
as they pass through matter. Millions of gamma rays pass
through our bodies every minute. When these forms of radiation collide with a fragile DNA molecule, they often break both
strands of the double helix. Double-strand breaks (DSBs) can
also be caused by certain chemicals, including several (e.g.,
bleomycin) used in cancer chemotherapy, and free radicals produced by normal cellular metabolism (page 34). DSBs are also
introduced during replication of damaged DNA. A single double-strand break can cause serious chromosome abnormalities,
which can have grave consequences for the cell. DSBs can be
repaired by several alternate pathways. The predominant pathway in mammalian cells is called nonhomologous end joining
(NHEJ), in which a complex of proteins bind to the broken
ends of the DNA duplex and catalyze a series of reactions that
rejoin the broken strands. The major steps that occur during
NHEJ are shown in Figure 13.29a and described in the accompanying legend. Figure 13.29b shows the nuclei of human fi-
3
DNA Ligase IV
4
(a)
FIGURE 13.29 Repairing double-strand breaks (DSBs) by nonhomologous end joining. (a) In this simplified model of double-strand break
repair, the lesion (step 1) is detected by a heterodimeric, ring-shaped
protein called Ku, that binds to the broken ends of the DNA (step 2).
The DNA-bound Ku recruits another protein, called DNA-PKcs, which
is the catalytic subunit of a DNA-dependent protein kinase (step 3).
Most of the substrates phosphorylated by this protein kinase have not
been identified. These proteins bring the ends of the broken DNA
together in such a way that they can be joined by DNA ligase IV to regenerate an intact DNA duplex (step 4). The NHEJ pathway may also
(b)
involve the activities of nucleases and polymerases (not shown) and is
more error prone than is the homologous recombination pathway of
DSB repair. (b) Time course analysis of Ku localization at sites of DSB
formation induced by laser microbeam irradiation at a site indicated by
the arrowheads. The NHEJ protein Ku becomes localized at the damage
site immediately following irradiation but remains there just briefly as
the damage is presumably repaired. Micrographs were taken (1) immediately, (2) 2 hours, and (3) 8 hours after irradiation. (B: FROM JONG-SOO
KIM ET AL, COURTESY OF KYOKO YOKOMORI, J. CELL BIOL. 170:344, 2005; BY
THE ROCKEFELLER UNIVERSITY PRESS.)
COPYRIGHT PERMISSION OF
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Chapter 13 DNA REPLICATION AND REPAIR
broblasts that had been treated with a laser to induce a localized
cluster of double-strand breaks and then stained for the presence of the protein Ku at various times after laser treatment.
This NHEJ repair protein is seen to localize at the site of the
DSBs immediately following their appearance. Another DSB
repair pathway known as homologous recombination requires a
homologous chromosome to serve as a template for repair of
the broken strand. The steps that occur during homologous recombination are similar to those of genetic recombination depicted in Figure 14.47. Defects in both repair pathways have
been linked to increased cancer susceptibility.
REVIEW
?
Contrast the events of nucleotide excision repair and base
excision repair.
2. Why is it important in mismatch repair that the cell distinguish the parental strands from the newly synthesized
strands? How is this accomplished?
1.
THE HUMAN PERSPECTIVE
The Consequences of DNA Repair Deficiencies
We owe our lives to light from the sun, which provides the energy
captured during photosynthesis. But the sun also emits a constant
stream of ultraviolet rays that ages and mutates the cells of our skin.
The hazardous effects of the sun are most dramatically illustrated by
the rare recessive genetic disorder, xeroderma pigmentosum (XP). Patients with XP possess a deficient nucleotide excision repair system that
cannot remove segments of DNA damaged by ultraviolet radiation.
As a result, persons with XP are extremely sensitive to sunlight; even
very limited exposure to the direct rays of the sun can produce large
numbers of dark-pigmented spots on exposed areas of the body (Figure 1) and a greatly elevated risk of developing disfiguring and fatal
skin cancers. Some help for XP patients may be on the way in the form
FIGURE 1 Darkly pigmented regions of the skin are evident in this boy
with xeroderma pigmentosum. The area of skin below the chin, which is
protected from the sun, is relatively devoid of the lesions. (KEN GREER/
VISUALS UNLIMITED.)
of a skin cream (Dimericine) that contains a bacterial DNA repair enzyme. The enzyme is contained in liposomes that can apparently
penetrate the outer layer of the skin and participate in DNA repair.
XP is not the only genetic disorder characterized by nucleotide
excision repair deficiency. Cockayne syndrome (CS) is an inherited
disorder characterized by acute sensitivity to light, neurological dysfunction due to demyelination of neurons, and dwarfism, but no evident increase in the frequency of skin cancer. Cells from persons
with CS are deficient in the pathway by which transcriptionally active DNA is repaired (page 553). The remainder of the genome is repaired at the normal rate, presumably accounting for the normal
levels of skin cancer. But why are persons with a defective repair
mechanism subject to specific abnormalities such as dwarfism? Most
cases of CS can be traced to a mutation in one of two genes, either
CSA or CSB, which are thought to be involved in coupling transcription to DNA repair (see Figure 13.26). Mutations in these genes, in
addition to impacting DNA repair, may also disturb the transcription of certain genes, leading to growth retardation and abnormal
development of the nervous system. This possibility is strengthened
by the finding that, in rare cases, the symptoms of CS can also occur
in persons with XP who carry specific mutations in the XPD gene.
As noted on page 553, XPD encodes a subunit of the transcription
factor TFIIH required for transcription initiation. Mutations in
XPD could lead to defects in both DNA repair and transcription.
Certain other mutations in the XPD gene are responsible for another
disease, trichothiodystrophy (TTD), which also combines symptoms suggestive of both DNA repair and transcription defects. Like
CS patients, individuals with TTD exhibit increased sun sensitivity
without the increased risk of development of cancer. TTD patients
have additional symptoms, including brittle hair and scaly skin.
These findings indicate that three distinct disorders—XP, CS, and
TTD—can be caused by defects in a single gene, with the particular
disease outcome determined by the specific mutation present in that
gene. Structural studies of mutant XPD molecules suggest that these
different mutations affect different functions of the protein.
Elsewhere in this text, we have described circumstances that lead
to premature (or accelerated) aging in humans or animal models: as
the result of (1) increased free radicals (page 34), (2) increased mitochondrial DNA mutations (page 202), and (3) mutations in a protein
of the nuclear envelope (page 477). In 2006, a 15-year-old boy who
suffered from frequent sunburns and certain characteristics of prema-
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13.3 BETWEEN REPLICATION AND REPAIR
ture aging came to the attention of clinical researchers. Genetic
analysis determined that the boy carried a mutation in the XPF gene
whose encoded protein makes one of the cuts during the NER pathway (Figure 13.26). Patients with mild mutations in XPF develop XP
and have impaired NER. This individual had a more severe mutation
in the XPF gene, causing his cells to be unable to repair covalent
cross-links that form occasionally between the two strands of a DNA
duplex. Studies on the cells of this individual, and on mice with a corresponding mutation, suggested that the unrepaired cross-links lead
to increased cell death (apoptosis), which either directly or indirectly
promotes premature aging. According to one hypothesis, defects in
DNA repair systems that result primarily in an increased mutation
rate in the body’s cells are associated with an increased susceptibility
to cancer, whereas defects in DNA repair systems that result primarily in cell death are associated with accelerated aging.a Whether any
of these premature-aging syndromes provides insight into the mechanisms of normal aging remains a matter of debate.
Persons with DNA-repair disorders are not the only individuals
who should worry about exposure to the sun. Even in a skin cell
whose repair enzymes are functioning at optimal levels, a small fraction of the lesions fail to be excised and replaced. Alterations in
DNA lead to mutations that can cause a cell to become malignant.
Thus, one of the consequences of the failure to correct UV-induced
damage is the risk of skin cancer. Consider the following statistics:
more than one million persons develop one of three forms of skin
cancer every year in the United States, and most of these cases are attributed to overexposure to the sun’s ultraviolet rays. Fortunately, the
two most common forms of skin cancer—basal cell carcinoma and
squamous cell carcinoma—rarely spread to other parts of the body
and can usually be excised in a doctor’s office. Both of these types of
cancer originate from the skin’s epithelial cells.
a
It has not been mentioned in this discussion, for a number of reasons, that two
of the genes most often responsible for premature aging syndromes encode members of a particular type of DNA helicase family called RecQ helicases. The
genes in question are WRN and BLM which, when mutated, are responsible
for the inherited diseases Werner Syndrome and Bloom Syndrome, respectively,
which are characterized by both increased cancer risk and features of accelerated
aging. It is suggested that these helicases are involved in certain types of base
excision and DSB repair pathways. They appear to be particularly important in
resolving situations where a replicative DNA polymerase becomes stalled at a
lesion and the replication fork “collapses” (disassembles). The subject is discussed
in Trends Biochem. Sci. 33:609, 2008.
13.3 BETWEEN REPLICATION AND REPAIR
The human perspective describes an inherited disease—
xeroderma pigmentosum (XP)—that leaves patients with an
inability to repair certain lesions caused by exposure to ultraviolet radiation. Patients described as having the “classical”
form of XP have a defect in one of seven different genes involved in nucleotide excision repair (page 553). These genes
are designated XPA, XPB, XPC, XPD, XPE, XPF, and XPG,
and some of their roles in NER are indicated in the legend of
Figure 13.26. Another group of patients were identified that,
like those with XP, were highly susceptible to developing skin
cancer as the result of sun exposure. However, unlike the cells
from XP sufferers, cells from these patients were capable of
557
However, malignant melanoma, the third type of skin cancer, is
a potential killer. Unlike the others, melanomas develop from pigment cells (melanocytes) in the skin. The number of cases of
melanoma diagnosed in the United States is climbing at the alarming rate of 4 percent per year due to the increasing amount of time
people have spent in the sun over the past few decades. Studies suggest that one of the greatest risk factors to developing melanoma as
an adult is the occurrence of a severe, blistering sunburn as a child or
adolescent. Individuals at greatest risk are Caucasians with extremely
light skin. Many of these individuals have pigment cells whose surfaces lack a functioning receptor (called MC1R) for a hormone that
is secreted by nearby epithelial cells of the skin in response to ultraviolet radiation. Melanocytes respond to MC1R activation by producing the dark pigment melanin, thereby providing the individual
with a tan. Tanned skin is more protected from UV rays than is light,
untanned skin, even though it is UV radiation that is responsible for
triggering the tanning response. What if it were possible to develop
a tanned skin without having to suffer UV exposure? A number of
research groups are working on such an approach by using various
means other than exposure to UV-containing sunlight to stimulate
the tanning response in pigment cells. Whether any of these approaches will prove safe and effective remains to be seen.
Skin cancer is not the only disease that is promoted by deficient
or overworked DNA repair systems. It is estimated that up to 15
percent of colon cancer cases can be attributed to mutations in the
genes that encode the proteins required for mismatch repair. Mutations that cripple the mismatch repair system inevitably lead to a
higher mutation rate in other genes because mistakes made during
replication are not corrected.
Cancer is also one of the consequences of double-strand DNA
breaks that have either gone unrepaired or been repaired incorrectly.
Breaks in DNA can be caused by a variety of environmental agents
to which we are commonly exposed, including X-rays, gamma rays,
and radioactive emissions. The most serious environmental hazard
in this regard is probably radon (specifically 222Rn), a radioactive isotope formed during the disintegration of uranium. Some areas of the
planet contain relatively high levels of uranium in the soil, and
houses built in these regions can contain dangerous levels of radon
gas. When the gas is breathed into the lungs, it can lead to doublestrand DNA breaks that increase the risk of lung cancer. A significant fraction of lung-cancer deaths in nonsmokers is probably due to
radon exposure.
nucleotide excision repair and were only slightly more sensitive
to UV light than normal cells. This heightened UV sensitivity
revealed itself during replication, as these cells often produced
fragmented daughter strands following UV irradiation. Patients
in this group were classified as having a variant form of XP,
designated XP-V. We will return to the basis of the XP-V
defect in a moment.
We have seen in the previous section that cells can repair
a great variety of DNA lesions. On occasion, however, a DNA
lesion is not repaired by the time that segment of DNA is
scheduled to undergo replication. On these occasions, the
replication machinery arrives at the site of damage on the
template strand and becomes stalled there. When this happens, some type of signal is emitted that leads to the recruit-
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Chapter 13 DNA REPLICATION AND REPAIR
ment of a specialized polymerase that is able to bypass the lesion.3 Suppose the lesion in question is a thymidine dimer
(Figure 13.25) in a skin cell that was caused by exposure to
UV radiation. When the replicative polymerase (pol ) reaches
the obstacle, the enzyme is temporarily replaced by a “specialized” DNA polymerase designated pol , which is able to
insert two A residues into the newly synthesized strand across
from the two T residues that are covalently linked as part of
the dimer. Once this “damage bypass” is accomplished, the cell
switches back to the normal replicative polymerase and DNA
synthesis continues without leaving any trace that a serious
problem had been resolved. As you might have guessed from
the juxtaposition of topics, patients afflicted with XP-V have
3
A cell has other options to deal with a stalled replication fork, but they are
more complex and poorly understood, and will not be discussed.
a mutation in the gene encoding pol and thus have difficulty
replicating past thymidine dimers.
Discovered in 1999, polymerase is a member of a family of DNA polymerases in which each member is specialized
for incorporating nucleotides opposite particular types of
DNA lesions in the template strand. The polymerases of this
family are said to engage in translesion synthesis (TLS). X-ray
crystallographic studies reveal that the TLS polymerases have
an unusually spacious active site that is able to physically accommodate altered nucleotides that would not fit in the active
site of a replicative polymerase. These TLS polymerases are
only capable of incorporating one to a few nucleotides into a
DNA strand (they lack processivity); they have no proofreading capability; and they are much more likely to incorporate
an incorrect (i.e., noncomplementary) nucleotide when copying undamaged DNA than the classic polymerases.
SYNOPSIS
DNA replication occurs semiconservatively, which indicates that
one intact strand of the parent duplex is transmitted to each of the
daughter cells during cell division. This mechanism of replication
was first suggested by Watson and Crick as part of their model of
DNA structure. They suggested that replication occurred by gradual
separation of the strands by means of hydrogen bond breakage, so
that each strand could serve as a template for the synthesis of a complementary strand. This model was soon confirmed in both bacterial
and eukaryotic cells by showing that cells transferred to labeled media for one generation produce daughter cells whose DNA has one
labeled strand and one unlabeled strand. (p. 534)
The mechanism of replication is best understood in bacterial
cells. Replication begins at a single origin on the circular bacterial
chromosome and proceeds outward in both directions as a pair of
replication forks. Replication forks are sites where the double helix is
unwound and nucleotides are incorporated into both newly synthesized strands. (p. 537)
DNA synthesis is catalyzed by a family of DNA polymerases. The
first of these enzymes to be characterized was DNA polymerase I of
E. coli. To catalyze the polymerization reaction, the enzyme requires
all four deoxyribonucleoside triphosphates, a template strand to
copy, and a primer containing a free 3 OH to which nucleotides can
be added. The primer is required because the enzyme cannot initiate
the synthesis of a DNA strand. Rather, it can only add nucleotides to
the 3 hydroxyl terminus of an existing strand. Another unexpected
characteristic of DNA polymerase I is that it only polymerizes a
strand in a 5 → 3 direction. It had been presumed that the two new
strands would be synthesized in opposite directions by polymerases
moving in opposite directions along the two parental template
strands. This finding was explained when it was shown that the two
strands were synthesized quite differently. (p. 538)
One of the newly synthesized strands (the leading strand) grows
toward the replication fork and is synthesized continuously. The
other newly synthesized strand (the lagging strand) grows away
from the fork and is synthesized discontinuously. In bacterial
cells, the lagging strand is synthesized as fragments approximately
1000 nucleotides long, called Okazaki fragments, that are covalently
joined to one another by a DNA ligase. In contrast, the leading
strand is synthesized as a single continuous strand. Neither the con-
tinuous strand nor any of the Okazaki fragments can be initiated by
the DNA polymerase but instead begin as a short RNA primer that
is synthesized by a type of RNA polymerase called primase. After
the RNA primer is assembled, DNA polymerase continues to synthesize the strand or fragment as DNA. The RNA is subsequently
degraded, and the gap is filled in as DNA. (p. 540)
Events at the replication fork require a variety of different types of
proteins having specialized functions. These include a DNA gyrase, which is a type II topoisomerase required to relieve the tension
that builds up ahead of the fork as a result of DNA unwinding; a
DNA helicase that unwinds the DNA by separating the strands;
single-stranded DNA-binding proteins that bind selectively to
single-stranded DNA and prevent reassociation; a primase that synthesizes the RNA primers; and a DNA ligase that seals the fragments of the lagging strand into a continuous polynucleotide. DNA
polymerase III is the primary DNA-synthesizing enzyme that adds
nucleotides to each RNA primer, whereas DNA polymerase I is responsible for removing the RNA primers and replacing them with
DNA. Two molecules of DNA polymerase III are thought to move
together as a complex along their respective template strands. This
is accomplished as the lagging-strand template loops back on itself.
(p. 541)
DNA polymerases possess separate catalytic sites for polymerization and degradation of nucleic acid strands. Most DNA polymerases possess both 5 → 3 and 3 → 5 exonuclease activities. The
first acts to degrade the RNA primers that begin each Okazaki fragment, and the second removes inappropriate nucleotides following
their mistaken incorporation, thus contributing to the fidelity of
replication. It is estimated that approximately one in 109 nucleotides
is incorporated incorrectly during replication in E. coli. (p. 544)
Replication in eukaryotic cells follows a similar mechanism and
employs similar proteins to those of prokaryotes. All of the DNA
polymerases involved in replication elongate DNA strands in the
5 → 3 direction. None of them initiates the synthesis of a chain
without a primer. Most possess a 3 → 5 exonuclease activity, ensuring that replication occurs with high fidelity. Unlike bacteria, replication in eukaryotes is initiated simultaneously at many sites along a
chromosome, with replication forks proceeding outward in both directions from each site of initiation. Studies on yeast indicate that
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ANALYTIC QUESTIONS
origins of replication contain a specific binding site for an essential
multiprotein complex called ORC. Events at the origin ensure that
replication of each DNA segment occurs once and only once per cell
cycle. (p. 546)
Replication in eukaryotic cells is intimately associated with nuclear structures. Evidence indicates that much of the machinery required for replication is associated with the nuclear matrix. In
addition, replication forks that are active at any given time are localized within about 50 to 250 sites called replication foci. Newly synthesized DNA is rapidly associated with nucleosomes. According to
one model, (H3H4)2 tetramers present prior to replication remain intact and are passed on to the daughter duplexes, whereas H2A/H2B
dimers separate from one another and bind randomly to new and old
(H3H4)2 tetramers on the daughter duplexes. (p. 550)
DNA is subject to damage by many environmental influences, including ionizing radiation, common chemicals, and ultraviolet radiation. Cells possess a variety of systems to recognize and repair
the resulting damage. It is estimated that less than one base change in
a thousand escapes a cell’s repair systems. Four major types of DNA
repair systems are discussed. Nucleotide excision repair (NER) systems operate by removing a small section of a DNA strand containing a bulky lesion, such as a pyrimidine dimer. During NER, the
strands of DNA containing the lesion are separated by a helicase;
559
paired incisions are made by endonucleases; the gap is filled by a
DNA polymerase; and the strand is sealed by a DNA ligase. The
template strands of genes that are actively transcribed are preferentially repaired by NER. Base excision repair removes a variety of altered nucleotides that produce minor distortions in the DNA helix.
Cells possess a variety of glycosylases that recognize and remove various types of altered bases. Once the base is removed, the remaining
portion of the nucleotide is removed by an endonuclease, the gap is
enlarged by a phosphodiesterase activity, and the gap is filled and
sealed by a polymerase and ligase. Mismatch repair is responsible for
removing incorrect nucleotides incorporated during replication that
escape the proofreading activity of the polymerase. In bacteria, the
newly synthesized strand is selected for repair by virtue of its lack of
methyl groups compared to the parental strand. Double-strand
breaks are repaired as proteins bind to the broken strands and join
the ends together. (p. 552)
In addition to the classic DNA polymerases involved in DNA
replication and repair, cells also possess an array of DNA polymerases that facilitate replication at sites of DNA lesions or misalignments. These polymerases, which engage in translesion
synthesis, lack processivity and proofreading capability and are more
error-prone than classic polymerases. (p. 557)
ANALYTIC QUESTIONS
1. Suppose that Meselson and Stahl had carried out their experi-
2.
3.
4.
5.
6.
ment by growing cells in medium with 14N and then transferring the cells to medium containing 15N. How would the bands
within the centrifuge tubes have appeared if replication were
semiconservative? If replication were conservative? If replication
were dispersive?
Suppose you isolated a mutant strain of yeast that replicated its
DNA more than once per cell cycle. In other words, each gene
in the genome was replicated several times between successive
cell divisions. How might you explain such a phenomenon?
How would the chromosomes from the experiment on eukaryotic cells depicted in Figure 13.4 have appeared if replication
occurred by a conservative or a dispersive mechanism?
We have seen that cells possess a special enzyme to remove
uracil from DNA. What do you suppose would happen if the
uracil groups were not removed? (You might consider the information presented in Figure 11.44 on the pairing properties of
uracil.)
Draw a partially double-stranded DNA molecule that would not
serve as a template for DNA synthesis by DNA polymerase I.
Some temperature-sensitive bacterial mutants stop replication
immediately following elevation of temperature, whereas others
continue to replicate their DNA for a period of time before they
cease this activity, and still others continue until a round of
replication is completed. How might these three types of mutants differ?
7. Suppose the error rate during replication in human cells were
8.
9.
10.
11.
12.
13.
the same as that of bacteria (about 10⫺9). How would this impact the two cells differently?
Figure 13.19 shows the results from an experiment in which
cells were incubated with [3H]thymidine for less than 30 minutes prior to fixation. How would you expect this photograph to
appear after a one-hour labeling period? Can you conclude that
the entire genome is replicated within an hour? If not, why not?
Origins of replication tend to have a region that is very rich in
A-T base pairs. What function do you suppose these sections
might serve?
What advantages might you expect for DNA replication to occur in conjunction with the nuclear matrix as opposed to the nucleoplasm? What are the advantages of replication occurring in
a small number of replication foci?
What are some of the reasons you might expect human cells to
have more efficient repair systems than those of a frog?
Suppose you were to compare autoradiographs of two cells that
had been exposed to [3H]thymidine, one that was engaged in
DNA replication (S phase) and another that was not. How
would you expect autoradiographs of these cells to differ?
Construct a model that would explain how transcriptionally active DNA is repaired preferentially over transcriptionally silent
DNA.
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14
Cellular Reproduction
14.1
14.2
14.3
The Cell Cycle
M Phase: Mitosis and Cytokinesis
Meiosis
The Human Perspective:
Meiotic Nondisjunction
and Its Consequences
Experimental Pathways:
The Discovery
and Characterization of MPF
A
ccording to the third tenet of the cell theory, new cells originate only
from other living cells. The process by which this occurs is called cell
division. For a multicellular organism, such as a human or an oak tree,
countless divisions of a single-celled zygote produce an organism of astonishing cellular complexity and organization. Cell division does not stop with the formation of
the mature organism but continues in certain tissues throughout life. Millions of
cells residing within the marrow of your bones or the lining of your intestinal tract
are undergoing division at this very moment. This enormous output of cells is
needed to replace cells that have aged or died.
Although cell division occurs in all organisms, it takes place very differently in
prokaryotes and eukaryotes. We will restrict discussion to the eukaryotic version. Two
distinct types of eukaryotic cell division will be discussed in this chapter. Mitosis
leads to production of cells that are genetically identical to their parent, whereas
meiosis leads to production of cells with half the genetic content of the parent. Mitosis serves as the basis for producing new cells, meiosis as the basis for producing new
sexually reproducing organisms. Together, these two types of cell division form the
links in the chain between parents and their offspring and, in a broader sense,
between living species and the earliest eukaryotic life forms present on Earth. ■
Fluorescence micrograph of a mitotic spindle that had assembled in a cell-free extract
prepared from frog eggs, which are cells that lack a centrosome. The red spheres consist of
chromatin-covered beads that were added to the extract. It is evident from this micrograph
that a bipolar spindle can assemble in the absence of both chromosomes and centrosomes. In
this experiment, the chromatin-covered beads served as nucleating sites for the assembly of the
microtubules that subsequently formed this spindle. The mechanism by which cells construct
mitotic spindles in the absence of centrosomes is discussed on page 575. (REPRINTED WITH PERMISSION
FROM
560
R. HEALD
ET AL.,
NATURE
VOL.
382,
COVER OF
8/1/96; ©
COPYRIGHT
1996, MACMILLAN MAGAZINES LIMITED.)