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DNA Replication,
Repair, and
1 DNA Replication: An Overview 1 DNA REPLICATION: AN OVERVIEW
A. Replication Forks
B. Role of DNA Gyrase Watson and Crick’s seminal paper describing the DNA
C. Semidiscontinuous Replication double helix ended with the statement: “It has not escaped
D. RNA Primers our notice that the specific pairing we have postulated
2 Enzymes of Replication
immediately suggests a possible copying mechanism for
A. DNA Polymerase I the genetic material.” In a succeeding paper they expanded
B. DNA Polymerase III on this rather cryptic remark by pointing out that a DNA
C. Unwinding DNA: Helicases and Single-Strand Binding Protein strand could act as a template to direct the synthesis of its
D. DNA Ligase complementary strand. Although Meselson and Stahl
E. Primase demonstrated, in 1958, that DNA is, in fact, semiconserva-
3 Prokaryotic Replication tively replicated (Section 5-3B), it was not until some 20 years
A. Bacteriophage M13 later that the mechanism of DNA replication in prokary-
B. Bacteriophage ␾X174 otes was understood in reasonable detail. This is because,
C. Escherichia coli as we shall see in this chapter, the DNA replication process
D. Fidelity of Replication rivals translation in its complexity but is mediated by often
4 Eukaryotic Replication loosely associated protein assemblies that are present in
A. The Cell Cycle only a few copies per cell. The surprising intricacy of DNA
B. Eukaryotic Replication Mechanisms replication compared to the chemically similar transcription
C. Reverse Transcriptase process (Section 31-2) arises from the need for extreme
D. Telomeres and Telomerase accuracy in DNA replication so as to preserve the integrity
5 Repair of DNA of the genome from generation to generation.
A. Direct Reversal of Damage
B. Excision Repair A. Replication Forks
C. Mismatch Repair
D. The SOS Response DNA is replicated by enzymes known as DNA-directed
E. Double-Strand Break Repair DNA polymerases or simply DNA polymerases. These en-
F. Identification of Carcinogens zymes utilize single-stranded DNA as templates on which
6 Recombination and Mobile Genetic Elements to catalyze the synthesis of the complementary strand from
A. Homologous Recombination the appropriate deoxynucleoside triphosphates (Fig. 30-1).
B. Transposition and Site-Specific Recombination The incoming nucleotides are selected by their ability to
7 DNA Methylation and Trinucleotide Repeat form Watson–Crick base pairs with the template DNA so
Expansions that the newly synthesized DNA strand forms a double
helix with the template strand. Nearly all known DNA
polymerases can only add a nucleotide donated by a nucle-
oside triphosphate to the free 3¿-OH group of a base paired
polynucleotide so that DNA chains are extended only in the
5¿ S 3¿ direction. DNA polymerases are discussed further
in Sections 30-2A, 30-2B, and 30-4B.

Here we begin a three-chapter series on the basic processes a. Duplex DNA Replicates Semiconservatively
of gene expression: DNA replication (this chapter), tran- at Replication Forks
scription (Chapter 31), and translation (Chapter 32). These John Cairns obtained the earliest indications of how
processes have been outlined in Section 5-4. We shall now chromosomes replicate through the autoradiography of
discuss them in greater depth with an emphasis on how we replicating DNA. Autoradiograms of circular chromo-
have come to know what we know. somes grown in a medium containing [3H]thymidine show
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1174 Chapter 30. DNA Replication, Repair, and Recombination

3⬘ ... 5⬘ ...
p p p p p p p p p p p p p p

... ...



OH + OH + OH + etc. OH + OH + etc.
p p p ppp ppp p p p p ppp

... ...
5⬘ Primer 3⬘ dCTP dTTP
Figure 30-1 Action of DNA polymerase. DNA polymerases single-stranded DNA templates such that the growing strand is
assemble incoming deoxynucleoside triphosphates on elongated in its 5¿ S 3¿ direction.

the presence of replication “eyes” or “bubbles” (Fig. 30-2). B. Role of DNA Gyrase
These so-called ␪ structures (after their resemblance to the
The requirement that the parent DNA unwind at the repli-
Greek letter theta) indicate that double-stranded DNA
cation fork (Fig. 30-3) presents a formidable topological
(dsDNA) replicates by the progressive separation of its two
obstacle. For instance, E. coli DNA is replicated at a rate of
parental strands accompanied by the synthesis of their com-
⬃1000 nucleotides/s. If its 1300-␮m-long chromosome
plementary strands to yield two semiconservatively repli-
were linear, it would have to flail around within the con-
cated duplex daughter strands (Fig. 30-3). DNA replication
fines of a 3-␮m-long E. coli cell at ⬃100 revolutions/s
involving ␪ structures is known as ␪ replication.
A branch point in a replication eye at which DNA syn-
thesis occurs is called a replication fork. A replication bub-
ble may contain one or two replication forks (unidirec-
tional or bidirectional replication). Autoradiographic
studies have demonstrated that ␪ replication is almost
Parent DNA
always bidirectional (Fig. 30-4). Moreover, such experi-
ments, together with genetic evidence, have established
that prokaryotic and bacteriophage DNAs have but one
replication origin (point where DNA synthesis is initiated).

Replication eye

Figure 30-2 Autoradiogram and its interpretive drawing of a

replicating E. coli chromosome. The bacterium had been grown
for somewhat more than one generation in a medium containing
[3H]thymidine, thereby labeling the subsequently synthesized
DNA so that it appears as a line of dark grains in the
photographic emulsion (red lines in the interpretive drawing). The Replica DNAs
size of the replication eye indicates that the circular chromosome
is about one-sixth duplicated in the present round of replication.
[Courtesy of John Cairns, Cold Spring Harbor Laboratory,
New York.] Figure 30-3 Replication of DNA.
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Section 30-1. DNA Replication: An Overview 1175

(a) C. Semidiscontinuous Replication

The low-resolution images provided by autoradiograms
Unidirectional such as Figs. 30-2 and 30-4b suggest that dsDNA’s two
replication antiparallel strands are simultaneously replicated at an ad-
Heavily Lightly vancing replication fork. Yet, all known DNA polymerases
labeled DNA labeled DNA can only extend DNA strands in the 5¿ S 3¿ direction. How,
then, does DNA polymerase copy the parent strand that
extends in the 5¿ S 3¿ direction past the replication fork?
This question was answered in 1968 by Reiji Okazaki
through the following experiments. If a growing E. coli cul-
ture is pulse-labeled for 30 s with [3H]thymidine, much of
the radioactive and hence newly synthesized DNA has a
sedimentation coefficient in alkali of 7S to 11S. These so-
called Okazaki fragments evidently consist of only 1000 to
2000 nucleotides (nt; 100–200 nt in eukaryotes). If, how-
(b) ever, following the 30 s [3H]thymidine pulse, the E. coli are
transferred to an unlabeled medium (a pulse–chase exper-
iment), the resulting radioactively labeled DNA sediments
at a rate that increases with the time that the cells had
grown in the unlabeled medium. The Okazaki fragments
must therefore become covalently incorporated into larger
Figure 30-4 Autoradiographic differentiation of unidirectional DNA molecules.
and bidirectional ␪ replication of DNA. (a) An organism is Okazaki interpreted his experimental results in terms of
grown for several generations in a medium that is lightly labeled the semidiscontinuous replication model (Fig. 30-5). The
with [3H]thymidine so that all of its DNA will be visible in an two parent strands are replicated in different ways. The
autoradiogram. A large amount of [3H]thymidine is then added newly synthesized DNA strand that extends 5¿ S 3¿ in the di-
to the medium for a few seconds before the DNA is isolated rection of replication fork movement, the so-called leading
(pulse labeling) in order to label only those bases near the strand, is essentially continuously synthesized in its 5¿ S 3¿
replication fork(s). Unidirectional DNA replication will exhibit
direction as the replication fork advances. The other newly
only one heavily labeled branch point (above), whereas
synthesized strand, the lagging strand, is also synthesized in
bidirectional DNA replication will exhibit two such branch
points (below). (b) An autoradiogram of E. coli DNA so treated, its 5¿ S 3¿ direction but discontinuously as Okazaki frag-
demonstrating that it is bidirectionally replicated. [Courtesy of ments. The Okazaki fragments are only covalently joined to-
David M. Prescott, University of Colorado.] gether sometime after their synthesis in a reaction catalyzed
by the enzyme DNA ligase (Section 30-2D).
The semidiscontinuous model of DNA replication is
corroborated by electron micrographs of replicating
DNA showing single-stranded regions on one side of the

(recall that B-DNA has ⬃10 bp per turn). But since the
E. coli chromosome is, in fact, circular, even this could not
occur. Rather, the DNA molecule would accumulate ⫹100
Motion of
supercoils/s (see Section 29-3A for a discussion of super-
coiling) until it became too tightly coiled to permit further fork
unwinding. Naturally occurring DNA’s negative supercoil- 3′
ing promotes DNA unwinding but only to the extent of 5′ 3′ 5′
⬃5% of its duplex turns (recall that naturally occurring Leading strand
DNAs are typically underwound by one supercoil per ⬃19
Lagging strand (Okazaki fragments)
duplex turns; Section 29-3Bb). In prokaryotes, however,
3′ 5′ Parental strands
negative supercoils may be introduced into DNA through
the action of a type IIA topoisomerase (DNA gyrase; Sec- 5′
tion 29-3Cd) at the expense of ATP hydrolysis. This process Figure 30-5 Semidiscontinuous DNA replication. In DNA
is essential for prokaryotic DNA replication as is demon- replication, both daughter strands (leading strand red, lagging
strated by the observation that DNA gyrase inhibitors, strand blue) are synthesized in their 5¿ S 3¿ directions. The
such as novobiocin, arrest DNA replication except in mu- leading strand is synthesized continuously, whereas the lagging
tants whose DNA gyrase does not bind these antibiotics. strand is synthesized discontinuously.
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1176 Chapter 30. DNA Replication, Repair, and Recombination

that their 5¿ ends consist of RNA segments of 1 to 60 nt (a

length that is species dependent) that are complementary to
the template DNA chain (Fig. 30-7). E. coli has two enzymes
that can catalyze the formation of these RNA primers:
RNA polymerase, the ⬃459-kD multisubunit enzyme that
mediates transcription (Section 31-2), and the much
smaller primase (60 kD), the monomeric product of the
dnaG gene.
Primase is insensitive to the RNA polymerase inhibitor
rifampicin (Section 31-2Bb). The observation that ri-
fampicin inhibits only leading strand synthesis therefore
indicates that primase initiates the Okazaki fragment
primers. The initiation of leading strand synthesis in E. coli,
a much rarer event than that of Okazaki fragments, can be
Figure 30-6 Electron micrograph of a replication eye in mediated in vitro by either RNA polymerase or primase
Drosophila melanogaster DNA. Note that the single-stranded alone but is greatly stimulated when both enzymes are
regions (arrows) near the replication forks have the trans present. It is therefore thought that these enzymes act syn-
configuration consistent with the semidiscontinuous model of ergistically in vivo to prime leading strand synthesis.
DNA replication. [From Kreigstein, H.J. and Hogness, D.S., Proc. Mature DNA does not contain RNA. The RNA primers
Natl. Acad. Sci. 71, 173 (1974).] are eventually removed and the resulting single-strand
gaps are filled in with DNA by a mechanism described in
Section 30-2Aj.

replication fork (Fig. 30-6). In bidirectionally replicating

DNA, moreover, the two single-stranded regions occur,
as expected, on diagonally opposite sides of the replica- 2 ENZYMES OF REPLICATION
tion bubble. DNA replication is a complex process involving a great
variety of enzymes. It requires, to list only its major actors
D. RNA Primers in their order of appearance: (1) DNA topoisomerases,
(2) enzymes known as helicases that separate the DNA
DNA polymerases’ all but universal requirement for a free strands at the replication fork, (3) proteins that prevent
3¿-OH group to extend a DNA chain poses a question that them from reannealing before they are replicated, (4) en-
was emphasized by the establishment of the semidiscontin- zymes that synthesize RNA primers, (5) a DNA poly-
uous model of DNA replication: How is DNA synthesis merase, (6) an enzyme to remove the RNA primers, and
initiated? Careful analysis of Okazaki fragments revealed (7) an enzyme to covalently link successive Okazaki frag-
ments. In this section, we describe the properties and func-
tions of many of these proteins.

A. DNA Polymerase I
5′ 3′
3′ In 1957, Arthur Kornberg reported that he had discovered
RNA primer an enzyme that catalyzes the synthesis of DNA in extracts
of E. coli through its ability to incorporate the radioactive
label from [14C]thymidine triphosphate into DNA. This en-
5′ zyme, which has since become known as DNA polymerase
I or Pol I, consists of a monomeric 928-residue polypeptide.
Pol I couples deoxynucleoside triphosphates on DNA
templates (Fig. 30-1) in a reaction that occurs through the
nucleophilic attack of the growing DNA chain’s 3¿-OH
5′ 3′ 3′ group on the ␣-phosphoryl of an incoming nucleoside
5′ triphosphate. The reaction is driven by the resulting elimina-
Okazaki fragment tion of PPi and its subsequent hydrolysis by inorganic py-
5′ rophosphatase. The overall reaction resembles that cat-
alyzed by RNA polymerase (Fig. 5-23) but differs from it
by the strict requirement that the incoming nucleoside be
Figure 30-7 Priming of DNA synthesis by short RNA linked to a free 3¿-OH group of a polynucleoside that is
segments. base paired to the template (RNA polymerase initiates