REVIEWS

Replisome assembly and the direct

restart of stalled replication forks
Ryan C. Heller and Kenneth J. Marians
Abstract | Failure to reactivate either stalled or collapsed replication forks is a source of
genomic instability in both prokaryotes and eukaryotes. In prokaryotes, dedicated fork
repair systems that involve both recombination and replication proteins have been
identified genetically and characterized biochemically. Replication conflicts are solved
through several pathways, some of which require recombination and some of which
operate directly at the stalled fork. Some recent biochemical observations support models
of direct fork repair in which the removal of the blocking template lesion is not always
required for replication restart.

DNA-template lesion
Any alteration to either the
continuity of the DNA strands
or the chemical form of the
nucleotide bases and
phosphodiester backbone.
Replication fork
The branch point between the
two template DNA strands
where nascent DNA synthesis
is ongoing.
Replisome
The protein machine that
replicates DNA and that
comprises, at a minimum, the
replicative DNA polymerase,
the replication-fork DNA
helicase and the Okazaki-
fragment primase.
Molecular Biology Program,
Memorial Sloan-Kettering
Cancer Center, New York,
New York 10021, USA.
Correspondence to K.J.M.
e-mail:kmarians@
sloankettering.edu
doi:10.1038/nrm2058
Published online
8 November 2006
Cells have evolved specific repair mechanisms to remove
DNA-template lesions to restore a structurally sound tem-
plate for essential DNA transactions such as transcrip-
tion and replication. However, it is almost inevitable that
some damage will escape this primary layer of protection
long enough for it to collide with an active replication
fork1. Exogenous sources of DNA damage, such as ion-
izing radiation and chemical treatment, are well studied
methods of inducing a DNA-replication conflict, but
even during normal cell growth it has become appar-
ent that endogenous sources of damage can inactivate
a large proportion of replication forks. If a lesion pre-
vents further DNA polymerization, thereby causing the
replication fork to stall, the cell uses specialized restart
pathways to enable fork reactivation outside the origin
of replication2–7. The existence of multiple such pathways
reflects the importance of recovering from different
types of damage that affect replication-fork progression
in different ways in order to ensure that DNA replication
is completed faithfully.
Restart pathways encompass three basic steps, each
with alternative choices (FIG. 1). First, the stalled fork is
processed by specific enzymes such as DNA helicases,
nucleases and/or recombination proteins to generate
DNA structures with the proper strand configurations.
The Escherichia coli PriA, Rep and UvrD proteins are
examples of 3′→5′ DNA helicases that are involved in
restart pathways and that function by modulating the
structure of stalled forks. Second, the normal control
mechanisms that restrict replisome assembly to origins
of replication must be circumvented at the damaged
chromosomal regions to complete the DNA-replication
process. Therefore, it is DNA structures, and not DNA
sequences, that are targeted by enzymatic systems that
catalyse replicative helicase loading and replisome
reassembly. Third, the original block to replication must
be removed. Removal can either occur at the stalled
replication fork during the restart process with a com-
bination of fork-processing and nucleotide-excision repair
(NER) proteins, or it can be delayed by lesion bypass and
carried out later, behind the fork, allowing for replication
to proceed promptly. Lesion bypass can occur through a
switch from the high-fidelity replicative polymerase to
a translesion polymerase, through a template-switching
mechanism or through a lesion-skipping mechanism
that generates single-stranded gaps behind the fork.
Without the layers of protection afforded by the multiple
restart pathways, the consequences for genome stability
could range from a temporary delay in the completion
of chromosomal replication to gross chromosomal
rearrangements such as translocations, large deletions
or the loss of a chromosome arm8–10.
In recent years, it has become clear that the reactiv-
ation of stalled forks is intimately connected with the
recombination machinery, enabling repair or bypass
of the blocking lesion in a non-mutagenic manner2,7.
Recombination proteins process chromosomes that
have undergone breakage during the course of DNA
replication, allowing for the repair of the double-strand
break (DSB) and the resumption of DNA replication, in
a process called recombination-dependent replication (RDR).
In this process, the broken chromosome undergoes a
strand-invasion event with the intact duplex DNA to
form a D-loop recombination intermediate that is recog-
nized by a specific restart system to catalyse the assembly
of a new replisome.

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© 2006 Nature Publishing Group
 REVIEWS

Recent data provide evidence that, depending on how

the stalled fork is processed, the replisome-assembly
step of direct restart on a fork structure can occur by one
Stalled fork of two known enzymatic systems in E. coli, which are
defined by the involvement of one of two replication-
restart proteins, PriA or PriC12,13. The two assembly
mechanisms differ in the subset of restart proteins that
are involved and in their ability to recognize the nascent
DNA at the stalled fork. Using the knowledge of these
assembly mechanisms, here we examine existing models
of fork repair, genetic data and biochemical observa-
Nascent- tions of replication conflict and restart. We also discuss
strand
regression the role of recombination proteins, which can be used
a b c in the processing of a broken or stalled fork, as well as in
the repair of the gaps that are left behind after collision,
stalling and reassembly of the replisome.

Distinct replisome-loading systems

RuvABC-directed Extension of Degradation Because replication forks stall and collapse at a high
cleavage nascent leading of regressed frequency, there is an essential need for replisome
strand DNA
assembly outside the origin of replication, which
Lesion Lesion requires the restart proteins PriA, PriB, PriC and DnaT
excised excised (BOX 1). The accumulation of DNA damage is a stochastic
Lesion
bypassed process, so there is no guarantee that any particular
sequence will be present at the position where repli-
some assembly is required. To overcome this problem,
Strand invasion Fork reversal
instead of recognizing sequence elements at origins of
RDR Direct restart Direct restart replication, restart proteins have evolved to recognize
Figure 1 | Recombination-dependent replication and direct-restart mechanisms specific structural elements of DNA. The presence of
of reactivating forks that have been stalled by leading-strand-specific lesions. single-stranded-DNA-binding protein (SSB) would
When the replisome encounters a lesion (black triangle) in the leading-strand template, normally function as a barrier to the loading of the
the resulting stalled fork can have a nascent lagging strand that has progressed past the replicative helicase DnaB and to replisome assembly at
nascent leading strand if the leading-strand and lagging-strand polymerases become these structures, but restart systems have the capability
uncoupled. The stalled fork can undergo nascent-strand regression to form a four-way, to overcome this barrier.
Holliday-junction structure. From this point, the fork could be reactivated by one of
several mechanisms. a | A recombination-dependent replication (RDR) pathway in which PriA-directed replisome loading. PriA is a DEXH-
the Holliday junction is resolved by the branch-migration helicase and Holliday-junction type DNA helicase of the SF2 superfamily14 and has a
endonuclease RuvABC. After excision of the template lesion, the broken chromosome
capability to recognize branched DNA junctions with
can undergo recombination with the intact duplex to form a D-loop structure.
Replication restart then takes place. b | In the template-switching model, the nascent high affinity, including D-loop and stalled fork struc-
leading strand is extended, using the nascent lagging strand as a template. After reversal tures15,16. The PriA–DNA structure interaction induces
back to a fork structure and direct restart on the fork structure, the lesion is bypassed. the binding of PriB, which stabilizes the PriA–DNA
c | Nascent-strand regression allows for excision of the template lesion. The regressed interaction and facilitates the joining of DnaT to the
DNA of both the nascent leading and lagging strand is degraded by an exonuclease complex17. The crystal structure of PriB revealed two
(indicated in orange) and direct replication restart can take place. Black strands, parental oligonucleotide-binding (OB) folds18,19, which indicates
DNA; red strands, nascent leading-strand DNA; red dotted strands, extension past the that a PriB–single-stranded (ss)DNA interaction has
blocking lesion; blue strands, nascent lagging-strand DNA. The arrowheads at the end of a role in its replisome-assembly function. The multi-
the nascent DNA represent the 3′-OH termini. This figure has been adapted from REF. 11. protein complex is then competent for the loading
of DnaB at the specific DNA structures20,21, thereby
Alternative mechanisms have been proposed to nucleating replisome assembly13,22,23.
restart stalled replication forks directly, which can be The importance of PriA in the recombination-
accomplished without breakage of the replication fork3,11 dependent repair of DSBs and the restart of stalled forks
(FIG. 1). In these models, the stalled replication fork is is highlighted by the phenotype of cells that lack PriA;
processed through a four-way, Holliday-junction interme- they have poor viability, defects in homologous recom-
Nucleotide-excision repair
diate to allow for the removal or bypass of the blocking bination and sensitivity to DNA-damaging agents24–27.
A template-dependent process
by which modified nucleotides lesion. The fork structure is maintained and replication The proximity of the 3′-OH terminus of the nascent
are removed from the DNA by is restarted without a recombination event. The direct leading strand to the branch point of a stalled fork has a
the excision of a patch of restart of DNA replication might be advantageous for crucial role in the recognition by PriA and in replisome-
single-stranded DNA, including the cell, considering the potentially negative effects on loading activity. As the distance is increased, the bind-
nucleotides both upstream and
downstream of the affected
genome stability from illegitimate recombination or loss ing affinity of PriA for fork structures decreases16, as
base, leaving a gap that is of genetic material from the generation of a DSB for the does its capability to nucleate the assembly of active
subsequently filled in. purposes of RDR. replisomes13. Small gaps (3–5 nucleotides) are tolerated

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REVIEWS
Recombination-dependent
replication (RDR)
DNA replication that is
initiated from a recombinant
joint molecule formed by
homologous recombination.
D-loop
Displacement loop. Originally
referring to a region on
mitochondrial DNA where a
short RNA displaces one of the
template strands. In
homologous recombination, it
is the displacement of one
strand of the duplex by an
invading single strand of DNA
during a strand-pairing
reaction such as that catalysed
by RecA.
Holliday junction
The crossover point of
exchange of strands between
two sister chromosomes during
homologous recombination.
Leading strand
The nascent strand of DNA
that can be synthesized
continuously in the 5′→3′
direction at the replication
fork.
R-loop
A displacement loop that
contains an RNA strand
annealed to one of the
template strands.
Constitutive stable DNA
replication
A form of recombination-
dependent replication that is
induced by either rnhA or recG
mutations and that is DnaA
and RecBCD independent,
RecA dependent,
chloramphenicol resistant (that
is, it does not require protein
synthesis) and rifampicin
sensitive (that is, it does require
transcription).
Lagging strand
The nascent strand of DNA
that is synthesized
discontinuously in short (1–2
kilobase pair) pieces (Okazaki
fragments) at the replication
fork.
934 | DECEMBER 2006 | VOLUME 7
Box 1 | Building a replisome
In Escherichia coli, the set of replication-restart proteins was originally identified oriC
by biochemical reconstitution experiments that involved bacteriophage φX174 5′
replication. The PriA, PriB, PriC and DnaT proteins, along with DnaB, DnaC and 3′
DnaG, were found to be required for the initial steps of the conversion of the
single-stranded circular form of the viral DNA to the duplex replicative form 1
in vitro89,90. Although this mechanism was thought to mirror the workings of
lagging-strand Okazaki-fragment synthesis at the chromosomal replication fork,
the PriA, PriB, PriC and DnaT proteins were not required for normal chromosomal 5′
replication from the origin of replication, oriC91. By contrast, DnaB, DnaC and 3′
DnaG were required.
The initiation protein DnaA binds to specific sequences at the origin (step 1) and
catalyses the loading of two hexamers of the replicative helicase DnaB around 2
newly melted, single-stranded-DNA-binding protein (SSB)-coated regions of
single-stranded DNA92–94 (step 2). DnaB normally associates with DnaC in solution
5′
and is prevented from loading to SSB-coated single-stranded DNA95,96, which
3′
provides a control mechanism to prevent replication forks from initiating at
illegitimate chromosomal positions. DnaA allows for the SSB barrier to be
overcome, causing DnaB loading to occur at a defined time 3
and position. DnaC is required for this loading process, but DnaA
it dissociates afterwards. The DnaB hexamers move past
each other in the 5′→3′ direction97, and a short RNA primer DnaB
5′
is synthesized through a protein–protein interaction 3′
DnaC
between DnaB and the primase, DnaG98 (step 3). These
DnaG 4
primers are elongated by the DNA polymerase III
holoenzyme moving in the opposite direction of the DnaB SSB
on the same strand, and they become the leading strand of
the replisome. The formation of the replisome is complete Leading/
lagging 5′
when the polymerase and DnaB interact with each polymerase 3′
other 99,100
(step 4).
well, but larger ones severely reduce the replisome- PriC-directed replisome loading. The other known
loading activity. PriA contains a crucial 3′-terminal replisome-loading system, which is mediated by PriC,
binding pocket that is required for high-affinity bind- also has the capability to overcome the SSB barrier
ing to D-loops and stalled fork structures that contain in the loading of the replicative helicase DnaB to the
a nascent leading strand with a 3′-OH end near the fork lagging-strand template of specific stalled fork struc-
junction28. Expression of PriA-binding-pocket mutants tures for 5′→3′ unwinding at the fork and for repli-
was not sufficient to restore many of the phenotypes some assembly through protein–protein interactions13.
of a priA-null mutant to wild-type levels 28, which Evidence for the involvement of PriC, which was
indicates that the high-affinity binding interaction identified with the other restart components (BOX 1),
is biologically relevant.Taken together, the data from in distinct replication-restart pathways first came from
genetic and biochemical sources strongly point to a role genetic analyses of the restart components. Whereas
for PriA-directed replisome assembly during RDR at the priA mutation confers severe growth and recombi-
D-loops, which contain the crucially placed 3′-OH nation defects, cells that lack either PriB or PriC have
terminus from the invading strand that is necessary only relatively mild defects30. When the priB and priC
for recognition (FIG. 2a). mutations are combined, the cells are more severely
R-loops are another potential cellular restart sub- defective with respect to growth rate and/or viability
strate, considering that they have a similar structure than the priA mutant, which indicates a functional
to D-loops, with the optimally positioned 3′-OH redundancy between PriB and PriC in the process of
terminus and a requirement for PriA in constitutive replication restart30. Also, there is a synthetic lethality
stable DNA replication29 — a process in which replica- between priA and priC mutations and a requirement
tion is thought to initiate from transcription-generated for functional PriC in a putative altered restart pathway
R-loop structures in cells that lack RNase H activity. that occurs in a priA mutant with an extragenic suppres-
Without RNase H to degrade the RNA in DNA–RNA sor mutation in DnaC (dnaC809), but no requirement
hybrids, R-loops are generated more frequently and for PriC with an additional mutation (dnaC809,820)31.
tend to persist, allowing PriA-dependent replication These findings strongly point to a role for PriC in
initiation to take place. Last, only a subset of stalled replisome assembly in a wild-type background that is
replication forks are likely to be targeted by PriA for distinct from the PriA–PriB–DnaT-directed system.
replisome assembly, namely those in which the termi- This is in agreement with biochemical evidence13.
nus of the nascent leading strand is located near the However, the finding that a dnaT mutation confers an
branch point of the fork and the gap is small (FIG. 2a). equally high level of ultraviolet (UV) sensitivity as the
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© 2006 Nature Publishing Group

Inducible stable DNA
replication
An SOS-induced form of
recombination-dependent
replication that is DnaA
independent, RecA and
RecBCD dependent, and
chloramphenicol and
rifampicin resistant (that is,
neither protein synthesis nor
transcription are required).
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
priA mutation implies that DnaT is also involved in a
genetic pathway with PriC for replisome assembly32,
a finding that was not observed during reconstitution
of replication restart in vitro33.
Unlike PriA-directed replisome assembly, the PriC
system is inhibited by the presence of nascent leading-
strand DNA. A gap size of at least ~7 nucleotides
between the 3′ terminus of the nascent leading strand
and the fork junction is required for optimal activity.
Therefore, PriC will only target a subset of stalled forks:
those that harbour a gap that was probably generated by
the blockage of the nascent leading strand and continued
fork unwinding (FIG. 2b). This functional preference can
be used to argue against a role for PriC in recombination-
dependent restart at D-loops because replisome assem-
bly is inefficient on D-loop structures that lack a suitable
leading-strand gap.
Additional evidence that PriC-dependent replication
restart does not operate at recombination-generated
D-loop structures comes from genetic studies.
An examination of priA mutant strains, in which only
the PriC system is available to mediate replisome assem-
bly, reveals defects in DSB repair and RecA-mediated
and RecBCD-mediated homologous recombination26.
This indicates that there is no redundancy between the
PriC and PriA systems at D-loop recombination inter-
mediates. Also, PriA is required for an altered mode of
chromosomal replication, called inducible stable DNA
replication (iSDR), in which replication initiates from
D-loops29. And, both PriA and PriB, but not PriC, are
required for constitutive stable DNA replication at
R-loops29,34. Similarly, a priB-null mutant, which elimi-
nates the PriA–PriB pathway of replication restart, and
a dnaC1331 mutant, which carries an allele of dnaC that
is defective specifically in the same pathway, are compro-
mised in their capability to repair the increased quantity
of DSBs that are generated by growth in rich media in a
dam-mutant background35,36. Mutation of dam, the gene
encoding DNA adenine methyltransferase, sensitizes
cells to mismatch-repair-mediated breaks that lead to
DSBs in DNA.
The interplay of PriA and PriC. Whereas both bio-
chemical and genetic evidence are indicative that PriC-
directed restart does not function at D-loops during the
DSB repair process, the genetic data that point to a role
for PriC in the repair of stalled forks seem contradic-
tory. Because the dnaC1331 mutant and the priB-null
mutant are expected to eliminate PriA–PriB-dependent
replisome assembly and because neither mutation causes
UV sensitivity, the forks that are stalled in these strains
by strand-specific damage, such as UV-induced lesions,
are presumably reactivated in a PriC-dependent man-
ner. However, cells that contain a mutation in priC have
almost no defects in either growth rate or sensitivity
to UV compared with wild-type cells30. Prima facie, this
indicates that PriC has no function in the reactivation
of UV-stalled forks, but it is possible that PriA has a
redundant role. The fork structure that would nor-
mally be recognized by PriC could be converted to a
PriA substrate, either by processing the stalled fork into
© 2006 Nature Publishing Group
REVIEWS
a PriA substrates
D-loop
RDR and direct restart
R-loop
DNA–RNA
hybrid
Stalled fork
b PriC substrate
Direct restart
Stalled fork
with gap
Figure 2 | PriA and PriC recognize different structures
for replication restart. a | The replication-restart protein
PriA targets those DNA structures for replisome reassembly
that possess a 3′-OH terminus in close proximity to the
branch junction, such as a D-loop, an R-loop or a stalled
fork that contains nascent leading-strand DNA. Replication
restart by PriA can take place by recombination-dependent
replication (RDR) or by a direct-restart mechanism.
b | By contrast, PriC-dependent direct restart works most
efficiently on stalled forks with a large single-stranded
DNA gap, in which lagging-strand DNA synthesis has
proceeded past the nascent leading strand. Black strands,
parental DNA; red strands, nascent leading-strand DNA or
invading-strand DNA; blue strands, nascent lagging-strand
DNA; green strand, RNA of DNA–RNA hybrid. The
arrowheads at the end of the nascent DNA represent the
3′-OH termini.
one that contains nascent leading-strand DNA at the
fork junction, or by breaking of the chromosome and
allowing DSB repair to take place, generating a D-loop
structure (FIG. 1). The conversion of restart substrates
would proceed in one direction only because once the
chromosome is broken a gapped fork for PriC cannot
be regenerated.
In contrast to priC mutants, priA mutants have a
high degree of UV sensitivity, which indicates that the
PriA system is the primary means of restoring forks
that were stalled by UV-induced damage and that the
PriC system, at best, has a minor role. However, PriA
has multiple activities: priA mutation abrogates not only
PriA-directed replisome assembly, but also its 3′→5′ hel-
icase activity that is important for the processing of the
stalled fork in a pathway that requires PriC-dependent
replisome assembly 33,37 (see below). This indicates that
the complete elimination of PriA might inactivate both the
PriA and PriC systems. In support of this idea, the priB
mutation alone has no UV-sensitivity phenotype30,
VOLUME 7 | DECEMBER 2006 | 935
REVIEWS
a the replisome encounters strand-specific damage such as
Lagging-strand lesion
b ment, leaving leading-strand synthesis unaffected39,40.
Leading-strand lesion
Or
DnaB
Leading/lagging
polymerase
Figure 3 | Replisome collisions with polymerase-blocking lesions have different
consequences depending on the location of the damage. a | Lesions (black triangle)
on the lagging-strand template do not cause fork stalling. The synthesis of a single
Okazaki fragment is blocked, but the lagging-strand polymerase remains associated
with the replisome and simply extends the next primer. The lesion is bypassed and a gap
is left behind. b | A polymerase-blocking lesion on the leading-strand template arrests
leading-strand synthesis, but unwinding of the fork and lagging-strand synthesis
continues, generating single-stranded DNA on the leading strand. It is unknown
whether the polymerases remain physically associated (top) or whether complete
uncoupling takes place (bottom). Once the replisome proteins have either been
removed or have dissociated, the stalled fork can be restarted by either one of the
pathways outlined in FIG. 1, which are all PriA dependent, or by the PriC system, as
outlined in FIG. 4b. Black strands, parental DNA; red strands, nascent leading-strand
DNA; blue strands, nascent lagging-strand DNA. The arrowheads at the end of the
nascent DNA represent the 3′-OH termini.
presumably because the PriC system is proficient at
restoring UV-stalled forks. But when the priB mutation
is combined with the priA300 allele, which encodes a
helicase-defective PriA protein, the result is a high
degree of UV sensitivity37. This implies that much of the
UV sensitivity in the priA mutant is due to the loss in
helicase activity and not the loss in PriA–PriB-dependent
replisome assembly. When the 3′→5′ helicase activity
of PriA is eliminated, the choice of restart pathway
might be shifted away from one that uses PriC towards
PriA–PriB-dependent restart. Furthermore, the expres-
sion of priA300 creates a strong need for RuvABC,
which is thought to catalyse the breakage of reversed
stalled forks38 (FIG. 1a) and therefore requires PriA–PriB-
dependent restart.
Polymerase uncoupling
The concept that the replisome
can become functionally Forks stalled at strand-specific lesions
uncoupled, with the leading- At stalled replication forks, two known replisome-loading
strand and lagging-strand systems operate, and they differ in their recognition
polymerases working
independently of one another
of the nascent DNA at the fork junction. How do the
and of the replication-fork structural preferences of the replisome-loading systems
helicase. compare with the structure of the replication fork after
936 | DECEMBER 2006 | VOLUME 7
© 2006 Nature Publishing Group
that caused by UV irradiation? An encounter between
the replisome and a DNA-template lesion has different
consequences depending on which strand contains the
damage.
On the lagging-strand template, a polymerase-blocking
lesion probably does not cause the replisome to stall or
otherwise affect replication-fork progression (FIG. 3a). In
two studies of plasmid replication in vitro with purified
E. coli components, a lagging-strand block by an abasic
lesion or a dideoxy NTP incorporation caused only
blockage in the synthesis of the current Okazaki frag-
The blocked lagging-strand polymerase remained asso-
ciated with the replisome and simply extended the next
primer, leaving the lesion and a small single-stranded
gap behind. Evidence for the same process also comes
from SV40-origin-initiated replication by a human
cell extract of plasmid DNA that contained a thymine
dimer41. Presumably, for effective lesion bypass to take
place on the lagging strand in prokaryotes, the size of
the lesion must be small enough to allow for uninhib-
ited unwinding of the DNA duplex by the replicative
helicase.
On the other hand, evidence is accumulating that after
the replisome encounters a lesion in the leading-strand
template, unwinding of the replication fork and lagging-
strand synthesis might continue for some distance before
the replisome stalls, even though the leading-strand
polymerase is blocked. A single thymine dimer or bulky
N-2-acetylaminofluorene (AAF) adduct in the leading-
strand template during SV40-plasmid replication in
human cell extracts resulted in replication-fork stalling
with preferential DNA synthesis on the lagging-strand
template past the lesion, which indicated that the two
polymerases had become uncoupled41–43. Visualization
of blocked intermediates by electron microscopy showed
that the replication forks had indeed moved past the
leading-strand-specific lesion and revealed the presence
of extended single-stranded DNA regions in one of the
branches of the replication fork44. During oriC-plasmid
replication in vitro, lagging-strand DNA synthesis was
observed to proceed for ~1 kilobase pair past the leading
strand, which had been blocked by an abasic lesion39.
Further evidence for polymerase uncoupling comes from
studies of E. coli plasmid replication in vivo, in which
the presence of an AAF leading-strand block caused
lagging-strand products to accumulate with faster
kinetics than leading-strand products45. More recently,
uncoupled replication intermediates could be detected
as long single-stranded DNA stretches on the leading-
strand side of forks in UV-irradiated, NER-deficient
S. cerevisiae cells46.
Based on findings that leading-strand-specific
blocks, such as those caused by UV irradiation, cause
replication-fork stalling in vitro, and the observation
of stalled fork intermediates in vivo, it is reasonable
to conclude that there are mechanisms to process
and reactivate these structures to restore processive
replication. However, the fate of the existing repli-
some proteins at the stalled fork is not clear. Although
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 REVIEWS

the leading-strand polymerase is blocked, the two
functionally uncoupled polymerases might remain
physically associated (FIG. 3b), blocking access to the
DNA. So, a disassembly process might be required to
license the stalled fork for reactivation. Do some or
all of the components disassemble from the blocked
fork? Is the putative disassembly process a passive or an
active one? If replisome disassembly is an active proc-
ess, which proteins are involved in clearing the DNA?
These questions have not yet been answered, although
it has recently been proposed that the chaperone
DnaK is required to remodel stalled replisomes dur-
ing a replication-fork repair pathway that involves a
replication template switch47.

Polymerase uncoupling Replisome dissociates

a b c

Nascent-strand
lagging
Nascent lagging-strand
unwinding

Breakage by RuvABC,
processing to D-loop via
recombination proteins;
see FIG. 1a.

Leading strand reinitiated DnaB loaded by PriC and

nascent leading strand
reinitiated Or

HJ processing back to fork:

extension of the nascent
leading strand and fork
resetting (FIG. 1b), or lesion
removal and nascent-strand
degradation (FIG. 1c).
Gap le behind Gap le behind PriA restart

Leading/lagging DnaB DnaG 3′→5′ helicase

polymerase (PriA or Rep)

Figure 4 | Replication-fork reactivation after encounter with leading-strand-specific lesions using nascent
leading-strand reinitiation. a | At a replication fork where polymerase uncoupling has occurred because of a blockage
in the leading-strand template, it is possible that DnaB can continue to unwind the template downstream of the lesion
(black triangle). The lagging-strand polymerase might also still be present. Under these circumstances, DnaG primase-
directed re-priming of the nascent leading strand could result in the re-establishment of the replisome with a resulting
gap left behind in the nascent leading strand. b | On the other hand, if the fork stalls completely and the replisome
components dissociate, unwinding of nascent lagging-strand DNA by a 3′→5′ helicase, such as PriA or Rep, provides
sufficient single-stranded DNA for the loading of DnaB by the PriC-directed system. Through a protein–protein
interaction between DnaB and DnaG, a RNA primer is synthesized on the leading-strand template, allowing reinitiation of
the nascent leading strand. The fork progresses past the blocking lesion, leaving a single-stranded gap on the opposite
strand. c | For models that involve nascent-strand regression, the nascent leading-strand and lagging-strand DNA anneals
together, and the fork reverses to form a four-way Holliday junction (HJ). The RuvABC branch-migration/Holliday-junction
endonuclease can resolve the structure, allowing the recombination proteins to process the double-strand end and
catalyse the formation of a D-loop (FIG. 1a). In the template-switching model (FIG. 1b), the nascent leading strand is
extended using the nascent lagging strand as a template. By resetting the fork, the lesion is effectively bypassed.
Alternatively, the lesion could be excised and the four-way junction reset to a fork by an exonuclease (FIG. 1c). All these
processes would generate a structure that is recognized by PriA for restart. Black strands, parental DNA; red strands,
nascent leading-strand DNA; blue strands, nascent lagging-strand DNA. The arrowheads at the end of the nascent DNA
represent the 3′-OH termini.

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© 2006 Nature Publishing Group
REVIEWS
Daughter-strand gap repair
Repair of gaps in the nascent
DNA using the complementary
strands of the sister duplex
and catalysed by the RecF-
dependent pathway of
homologous recombination.
938 | DECEMBER 2006 | VOLUME 7
Restart with reinitiation
In Okazaki’s classic studies on the mechanisms of DNA
replication, cells were briefly pulse-labelled by the
incorporation of [3H]thymidine into growing strands
of DNA. It was observed that essentially all of the nas-
cent DNA at the replication fork was present as small
fragments, as if both leading-strand and lagging-strand
DNA were synthesized in a discontinuous manner48.
Similar findings came from studies with temperature-
sensitive DNA-ligase mutants and with DNA polymer-
ase I mutants, which were used to prevent the sealing
of nascent DNA at the fork, even after ruling out the
possibility that breaks were not caused by known DNA-
repair processes49,50. Without a means to reinitiate the
nascent leading strand outside the origin of replication,
these in vivo findings were difficult to reconcile with
the mechanistically continuous nature of leading-
strand synthesis in vitro. The recent finding that the
nascent leading strand can in fact be reinitiated outside
the origin of replication during the process of replica-
tion restart51 helps explain the inconsistency between
the observations in vivo and the known biochemical
mechanisms of replication.
We have shown that at artificially engineered model
replication forks that reflect stalling from a leading-
strand-specific lesion, the loading of DnaB onto the
lagging-strand template by both the PriA and PriC
systems is sufficient to coordinate the priming de novo
of both the nascent leading and lagging strands through
association with the primase DnaG51 (FIG. 4a,b). With a
primed template and the DnaB helicase at the fork, DNA
polymerase III holoenzyme joins the complex to assem-
ble an active replisome. This mechanism of leading-
strand priming raises the possibility that reinitiation
events might also occur through a DnaB hexamer that
has not dissociated from the template DNA and has
managed to pass the blocking lesion. However, the fre-
quency of such events is not known and this proposed
mechanism cannot be the only way to restart replication
(FIG. 4a).
Leading-strand reinitiation might also occur dur-
ing the RDR process, in which the RecA recombinase
coats ssDNA of a broken chromosome, forming a fila-
ment, and catalyses a strand-invasion event to form a
D-loop structure (FIG. 1a). Because PriA targets D-loop
recombination intermediates for replisome assembly
during RDR, and because the RecA filament can act
as a block to the extension of the invading strand, lead-
ing-strand reinitiation might allow for replication to
proceed quickly and without disassembly of the RecA
filament. Allowing for RecA to remain on the DNA
would also provide added protection and stability to the
recombination joint.
If the PriC-dependent fork-restart mechanism that
involves nascent leading-strand reinitiation operates
at forks in which the nascent leading strand has been
blocked, the result would be the replicative bypass of the
lesion and the creation of a single-stranded gap behind
the fork (FIG. 4b). We propose that this restart mecha-
nism is responsible in part for the observation in vivo
of discontinuities in the chromosomal DNA generated
© 2006 Nature Publishing Group
by replicating through UV-induced lesions in NER-
deficient strains that are repaired with further post-UV
incubation52. An analysis of the single-stranded regions
showed gaps in the nascent DNA that were estimated
to be approximately the size of an Okazaki fragment,
~1,000 nucleotides in length53. Even though it could not
be determined whether these daughter-strand gaps were
present on both strands, the finding that all nascent
DNA was reduced in size following UV irradiation52
indicates that both strands are affected. Because the
lesions did not function as a complete block in NER-
deficient strains 52,54, and because the lesions could
remain in the DNA template after the passage of the
replication fork55, removal of the lesions did not seem
to be necessary for further replication-fork progres-
sion, in line with the findings that blocking lesions on
either strand can be bypassed in vitro40,51. Under optimal
growth conditions, the E. coli chromosome is replicated
in ~40 minutes, between cell divisions, which means
that each fork must travel at nearly 1,000 nucleotides
per second, a rate that is roughly equivalent to that
measured in vitro56. Considering that replication restart
is common under normal growth conditions (as shown
by the extreme inviability of priA-null cells24 and the
synthetic lethality between priA and priC mutations31),
NER would contribute a long delay if it was required
for restart. By contrast, at other chromosomal positions,
such as behind the replication fork, lesion excision by
NER would not be the limiting factor for a 40-minute
cell division.
Gap repair and replication recovery
Although UV-induced strand-specific lesions can be
skipped over by a colliding replisome without concur-
rent lesion removal, secondary lesions are left behind
in the form of single-stranded gaps that must be filled
by specialized repair mechanisms to maintain genomic
integrity. The primary means of repairing these gaps is
by homologous recombination, which uses complemen-
tary DNA from the sister chromatid57,58. Even though
there is a risk of illegitimate recombination, this proc-
ess is inherently error free compared with the frequent
misincorporation that occurs through error-prone
translesion polymerases59. Note that it is not possible
to remove the lesion from the DNA template until the
gap has been repaired because the NER process requires
that double-stranded DNA be present on either side of
the lesion.
Daughter-strand gap repair involves RecF-mediated
homologous recombination, a pathway that includes
a number of important proteins such as: RecA; RecF,
RecO and RecR (RecFOR); RecG; RecJ; RecN; RecQ;
RuvA, RuvB and RuvC (RuvABC); and SSB60 (FIG. 5). It is
thought that the RecFOR proteins enable the loading of
the RecA recombinase to ssDNA at the gap. This allows
strand exchange to take place with the sister chromatid
and provides an unobstructed template for the filling
of the gap. Alone, recF, recO and recR mutations have
little effect on cell viability, but in the absence of PriA,
the RecFOR recombination proteins are crucial for
cell survival, which implies that the PriC-dependent
www.nature.com/reviews/molcellbio

Branch migration
Movement of the Holliday
junction in a recombinant joint
molecule axially along the
length of the DNA molecules. It
results in the exchange of
strands between, for example,
two sister chromosomes.
Crossover formation
Resolution of the Holliday
junction in recombinant joint
molecules in a manner that
results in the exchange of
flanking genetic markers.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
REVIEWS
replication-restart pathway generates single-stranded
gaps that must be repaired by homologous recombina-
tion61. Either the absence of PriA causes an increase in the
formation of gapped DNA, or the gaps can be repaired
differently in the presence of PriA by being converted to
DSBs and undergoing DSB repair with PriA.
Further examination of recombination mutations RecA loaded to gap by RecFOR
in the priA background highlights the importance of
gap repair in these strains. If RecFOR-mediated homo-
logous recombination is blocked in the priA mutant,
the unrepaired gaps lead to chromosome breakage61.
During the later stages of daughter-strand gap repair
by homologous recombination, the Holliday-junction
intermediate is acted upon by the branch migration
and resolution proteins RuvABC and RecG62. As with Strand invasion (RecA)
RecFOR, the synergistic negative effects on cell viabil-
ity between priA and recG61,63,64 and between priA and
ruvABC or ruvC61,63,65 are consistent with a role for the
PriC-dependent replication-restart pathway in generating
gaps that are repaired by homologous recombination
through a Holliday-junction intermediate, which
requires specific enzymes to resolve the junctions.
Holliday-junction resolution with crossing over
Branch migration (RuvAB, RecG)
leads to a dimeric chromosome for those species that
have a circular chromosome. Based on the findings that
Holliday-junction resolution is required in priA mutants,
but that chromosome dimer resolution is not, it was con-
cluded that the resolution of Holliday junctions in priA
mutants is strongly biased towards non-crossovers63.
How this bias is mediated with respect to the Holliday-
junction branch-migration and branch-resolving
enzymes RecG and RuvABC is not known. However, Gap-filling DNA synthesis (DNA poymerase I)
reverse branch migration by RecG can dissociate joint
molecules that contain Holliday junctions in the presence
of RecA, a mechanism of junction resolution that avoids
crossover formation66. Because the PriC replication-restart
pathway is prone to generating gaps in the nascent DNA,
this bias in junction resolution towards non-crossovers
probably reflects gap-repair events, which is in line with
the finding that gaps that are produced by the replication Holliday-junction resolution (RuvC, RecG)
of palindromes or UV-induced damage lead primarily
to RecFOR-repaired non-crossovers67. Albeit through Figure 5 | Daughter-strand gap repair. The gaps that
different mechanisms, both leading-strand-specific and are left behind by restart mechanisms involving the
lagging-strand-specific lesions would produce gaps with initiation of the nascent leading strand are repaired by
the RecA-dependent and RecFOR-dependent
similar characteristics, except that on the lagging strand,
homologous-recombination pathway. With the aid of
the gap size would have an upper limit of the size of an RecFOR, RecA is loaded onto the daughter-strand gap.
Okazaki fragment. Nevertheless, the RecFOR gap-repair RecA-catalysed strand invasion provides an undamaged
machinery would target both types of damage-induced template for the filling of the gap. The template lesion
gap for repair throughout the genome, and in the pro- can be repaired at this or any of the subsequent steps.
cess would minimize the frequency of crossovers and Branch migration, which is catalysed by RuvAB and/or
the formation of chromosome dimers. RecG, and repair DNA synthesis, which is catalysed by
DNA polymerase I, allows the formerly blocked nascent
Replication recovery. A number of observations seem strand to be extended. Resolution of the double
to contradict the notion that the replisome can bypass Holliday junctions by RuvC then yields two intact sister
chromosome arms. Alternatively, reverse branch
UV-induced lesions and that recombination proteins
migration of the Holliday junctions by RecG leads to
are used for subsequent gap repair. In wild-type cells, resolution without crossing over. Black strands, parental
a moderate dose of UV irradiation induces a transient DNA; black triangle, lesion; red strands, nascent
inhibition in the rate of DNA synthesis, and the delay
leading-strand DNA; dotted red strand, repair
in replication recovery correlates with the time that is synthesis; blue strands, nascent lagging-strand DNA.
required for the repair of the UV-induced lesions68. The arrowheads at the end of the nascent DNA
Furthermore, cells that lack RecA and RecFOR fail represent the 3′-OH termini.
VOLUME 7 | DECEMBER 2006 | 939
© 2006 Nature Publishing Group
REVIEWS
SOS response
The prokaryotic DNA-damage
response.
Replication-fork collapse
The disjunction of the two
partially replicated sister
duplexes at the replication
fork, such as when the
replisome encounters a nick in
one of the template strands.
940 | DECEMBER 2006 | VOLUME 7
to recover wild-type rates of replication following
UV-irradiation and nascent DNA becomes extensively
degraded69. It was proposed that RecFOR enables RecA
to promote replication restart by protecting or main-
taining the stalled fork, allowing the lesion to be excised
or bypassed by an alternative polymerase69. In this way,
the recombination proteins could aid in replication
recovery without a recombination event actually tak-
ing place. Even though it is clear that NER-defective
mutants fail to recover normal rates of DNA synthe-
sis68, lesion removal cannot be a requirement for stalled
fork reactivation given that UV-induced lesions do
not function as permanent blocks to DNA synthesis52.
However, higher UV doses do have a significant
effect on total nascent-DNA synthesis. Furthermore,
DNA-strand exchanges have been detected after UV
irradiation of cells that undergo replication55,70, which
indicates that recombination frequently takes place in
conjunction with lesion excision, although it does not
prove that recombination was initiated from gaps in
both strands.
An alternative hypothesis with regard to the inhibi-
tion, delay and recovery of the DNA-synthesis rate after
UV irradiation in wild-type cells is that recovery simply
reflects less frequent replication-fork stalling, because
increasing numbers of lesions are removed from the
DNA duplex ahead of the fork by SOS-response-induced
NER. The SOS response would be induced upon the
first collision and exposure of the first gap, and could
facilitate both lesion bypass and allow increased
excision repair to reduce subsequent collisions.
The UV-induced lesions might not be permanent blocks,
but if gaps are generated on replication restart and they are
left unrepaired in the recombination mutants, they
could be preventing the recovery of DNA synthesis.
If there was a signal to suspend DNA replication, the
nascent-strand gaps could themselves be a source of
DNA-strand breakage and/or degradation. An active
replisome that encounters such a gap would probably
lead to replication-fork collapse, generating a double-
strand end, surely promoting DNA degradation and
preventing continued DNA synthesis. Evidence that
a second replication fork is initiated and undergoes
collapse after it encounters damage from a previous
fork comes from a study in which forks were blocked
in vivo by ectopic terminator sequences71. However, in
opposition to a role for the recombination proteins
in the gap-repair process is the finding that RuvAB and
RecG are not essential for DNA-synthesis recovery72,
unless another protein — perhaps a helicase — can
compensate for their branch-migration activity to
dissolve the Holliday junction.
The processing of stalled forks by 3′→5′ helicases
Although the exact means by which the 3′→5′ helicases
PriA, Rep and UvrD target and function at stalled
forks is not entirely clear, a common theme is emerg-
ing that they have an important role in maintaining
genome integrity by avoiding unnecessary fork breakage
and recombination by promoting accurate and timely
replisome reactivation.
© 2006 Nature Publishing Group
The PriA 3′→5′ helicase activity. The helicase activity of
PriA is dispensable for replisome assembly, but it might
have an important function at stalled replication forks.
A role for PriA helicase activity at stalled forks was orig-
inally proposed on the basis of a study of bacteriophage
Mu DNA replication by transposition12. Because PriA
helicase activity was required for Mu replication in vivo,
it was thought that PriA removes nascent DNA at the
lagging-strand arm of the fork in Mu strand-transfer
intermediates to allow the loading of DnaB for repli-
some assembly12. It was noted that a similar mechanism
could operate at chromosomal forks that were stalled
by a leading-strand-specific lesion where uncoupled
DNA replication allowed lagging-strand synthesis to
proceed past the blocked leading strand. If there were
an insufficient stretch of single-stranded DNA available
on the lagging-strand template for the loading of DnaB,
PriA could function to unwind some of the obstructing
nascent lagging-strand DNA (FIG. 4b).
This idea is supported by a number of studies12,33,64,73.
The helicase activity of PriA at stalled forks is modulated
by two factors: the presence of nascent leading-strand
DNA at the fork junction and the presence of SSB. This
point is illustrated nicely by the finding of a specific
interaction between the acidic C terminus of SSB and
PriA that stimulates the helicase activity of PriA in the
unwinding of nascent lagging-strand DNA at forks74.
The stimulation of PriA by SSB requires that SSB be
bound to DNA, so the presence of nascent leading-strand
DNA at the fork junction abrogates the stimulatory
effect. Besides eliminating SSB-mediated stimulation,
the nascent leading strand also has been shown to
inhibit unwinding by PriA in vitro under various con-
ditions12,33,73,75. An interaction between the 3′-terminal
binding pocket of PriA and the nascent leading strand
3′-OH causes PriA to become more stably bound28,
potentially allowing for more efficient replisome assembly
to the stalled fork75. In the presence of SSB and a nascent
lagging strand that needs to be unwound, the helicase
activity of PriA is equally effective in removing enough of
the obstruction for subsequent DnaB loading, regardless
of the presence of a nascent leading strand33.
Studies of specific mutations in PriA that affect heli-
case activity offer more insight into its role in replication-
fork repair. Cells in which the ATPase and helicase activ-
ities of PriA are impaired have a near wild-type pheno-
type, but they are severely affected when combined
with a priB, but not priC, mutation37. These data were
interpreted to mean that the PriA helicase is strongly
required for a restart pathway that involves PriC, but
not the pathway that is mediated by PriA and PriB.
If PriA does unwind nascent lagging-strand DNA at
stalled replication forks as a precursor to PriC-directed
replisome assembly, it would only happen at those forks
that had been stalled by a leading-strand-specific block,
because PriC does not tolerate well the presence of nas-
cent leading-strand DNA at the fork13. These forks could
potentially be reactivated by a combination of lagging-
strand unwinding by PriA and PriA–PriB-dependent
replisome assembly, but this scheme does not fit well
with the genetic requirements.
www.nature.com/reviews/molcellbio

Fork regression
Pairing of the nascent strands
of DNA at a replication fork. It
results in the rewinding of
duplex template DNA (that is,
the position of the replication
fork regresses, moving
backwards along the template)
and the formation of a Holliday
junction.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
Other mutations in priA that map in or near con-
served helicase motifs were identified as extragenic
suppressors of the recG UV-sensitivity phenotype
(srgA mutations)76. Many of these mutant proteins are
defective in the unwinding of nascent lagging-strand
DNA at forks that lack a nascent leading strand64. This
fork models the situation in which a lesion blocks the
leading-strand polymerase, and continued template
unwinding generates ssDNA on the leading-strand
template. It was therefore proposed that the unwind-
ing of the nascent lagging-strand DNA by PriA at
a fork with a blocked nascent leading strand would
be a deleterious event in the absence of RecG. RecG
could normally counteract the inappropriate unwind-
ing by catalysing fork regression, a process in which the
nascent DNA is annealed and the fork is converted to
a Holliday junction. The blocking lesion could then
be removed and the fork restored by exonucleolytic
digestion of the regressed strands, or the extension
of the leading strand by template switching could allow
the lesion to be bypassed for later repair (FIG. 4c). In
either case, RecG activity will have provided an acces-
sible nascent leading strand to serve as a primer for
replication restart at the restored fork.
However, a recent finding is that the loading of
DnaB to an unprimed fork is fully capable of nucleating
a replication-restart event through priming on the
leading strand de novo51. This challenges the notion
that the fork must be processed through a Holliday
junction intermediate to provide a primer for leading-
strand synthesis. In the absence of RecG, if PriA-
catalysed unwinding of the nascent lagging strand at
leading-strand blocked forks is not deleterious because
of inappropriate DnaB loading, then how can the sup-
pression of recG by srgA mutants be explained? Either
the altered PriA helicase activity has some unknown
role in branch migration that would normally be
accomplished by RecG, or the lack of helicase activ-
ity prevents the accumulation of an intermediate that
requires RecG processing. If the PriA helicase activity
on stalled forks does lead to replication restart, the
daughter-strand gaps that are produced would require
recombinational repair, perhaps creating a need for
branch migration or resolution by RecG for full UV
resistance.
The Rep and UvrD 3′→5′ helicases. Rep is another
protein with helicase activity that is potentially
involved in ensuring that progression of the replication
fork proceeds smoothly. Replication forks in cells that
lack Rep move slowly77, which implies frequent paus-
ing or stalling during chromosomal replication. Based
on the evidence that Rep has a capability to displace
bound proteins from the DNA template in vitro78, it has
been proposed that Rep functions as an accessory to
the replicative helicase DnaB and functions to suppress
fork stalling by clearing DNA-bound proteins ahead
of the advancing replication fork79. Alternatively, Rep
participates actively in the recovery of stalled forks by
unwinding nascent lagging-strand DNA in a manner
that is analogous to that proposed for PriA.
© 2006 Nature Publishing Group
REVIEWS
Like PriA, Rep possesses intrinsic 3′→5′ helicase
activity and has been proposed to function in a rep-
lication-restart pathway with PriC31. Rep also could
remove the nascent lagging strand from model fork
structures in vitro, but unlike PriA, its helicase activity
is stimulated by PriC, which means that an interac-
tion between the two proteins targets Rep helicase
activity to those stalled forks that PriC recognizes33.
PriC must therefore carry out multiple roles in replica-
tion restart by recognizing stalled forks of a specific
structure and coordinating the activity of Rep helicase
for fork processing and DnaB loading for replisome
assembly. Because the helicase activities of either PriA
or Rep have the potential to enable replisome assembly
and fork restart in vitro33, it is unclear which of the
two proteins targets the majority of cellular restart
substrates.
Compared with the loss of only a single compo-
nent, cells that lack both Rep and the helicase activity
of PriA exhibit a number of phenotypes, including a
loss in viability, which implies that stalled-fork reacti-
vation is compromised80. In these cells, the loading of
RecA to the stalled fork in a RecFOR-dependent man-
ner drives fork reversal such that recombination pro-
teins become necessary to catalyse a strand-invasion
event to allow replication restart to take place 80.
The helicase activities of Rep and PriA might there-
fore serve to limit the loading or activity of RecA
by unwinding nascent DNA, thereby adjusting the
structure of the fork.
Complicating matters, another 3′→5′ helicase,
UvrD, shares homology with Rep and is involved in
countering futile RecA-dependent reactions at forks
stalled by replisome inactivation81. Unlike Rep, UvrD
has been reported to disrupt a RecA nucleoprotein
filament directly in vitro82, an activity that might com-
plement nascent-strand unwinding by Rep or PriA, by
directly removing RecA filaments from stalled forks to
prevent unnecessary recombination.
Restart mechanisms in eukaryotes
Although best studied in bacteria, the processes
that generate and repair daughter-strand gaps are
not limited to prokaryotes. After UV irradiation,
gaps in the nascent DNA have been detected in vari-
ous higher organisms, including human cells 46,83,84,
which implies that similar and universal repair
mechanisms are shared among different species.
The yeast Saccharomyces cerevisiae proteins of the
Rad6 and Rad18 epistasis group accomplish the repair
of UV-generated gaps in the nascent DNA85. Even
though the precise mechanism of error-free gap repair
is unclear, the process is distinct from the DSB-repair
homologous-recombination pathway. The gene encod-
ing Srs2, a 3′→5′ helicase and homologue of the E. coli
Rep and UvrD proteins, was identified in a screen for
suppressors of the UV-sensitivity phenotype of rad6
and rad18 mutants86. Based on genetic evidence, it was
proposed that Srs2 channels lesions into the Rad6-
dependent damage-tolerance pathway, preventing
recombinational repair from inappropriately resolving
VOLUME 7 | DECEMBER 2006 | 941
REVIEWS

stalled replication forks87. Srs2 is targeted to stalled Concluding remarks

replication forks through an interaction with a post-
translationally modified form of proliferating cell
nuclear antigen (PCNA), the DNA-replication proces-
sivity clamp, and under conditions in which Srs2 is
recruited, the levels of Rad51 were associated with
DNA decreases 88. The precise mechanism by which
Srs2 either inhibits inappropriate recombination or
promotes the damage-tolerance pathway, as well as
whether any similarity exists with the mechanisms by
which E. coli Rep or UvrD suppress recombination at
stalled forks, is not clear. Although no other obvious
homologues of replication-restart components have
been identified in eukaryotes, further study of the
conserved, non-mutagenic, replicative bypass path-
ways of polymerase-blocking lesions should provide
additional insight into mechanisms of fork arrest and
reactivation, given that the two processes are linked
in E. coli.
The capability of the replication and restart machinery
to effectively bypass polymerase-blocking DNA-template
lesions without concomitant excision repair would allow
for efficient replication recovery and might promote over-
all fitness at the cost of the generation of single-stranded
gaps. The risk of instability that results from illegitimate
recombinational repair of the gaps might be tempered by
the ability to ensure complete and accurate duplication
of the genome, averting the need for a means to protect the
DNA during prolonged stalling, which would make
the fork prone to breakage and degradation.
With the findings and descriptions in recent years of
several stalled-fork processing and restart pathways that
are available to the cell, an important aspect of future
research will be to more clearly associate specific lesion
types with mechanisms of replisome deactivation and to
determine how they relate to the use of these reactivation
pathways.

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Acknowledgements
We thank S. Keeney, J. Petrini and R. Rothstein for their com-
ments on the manuscript. Studies from the authors’ labora-
tory were supported by the National Institutes of Health.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
The following terms in this article are linked online to:
UniProtKB: http://ca.expasy.org/sprot
DnaC | DnaT | PriA | PriB | PriC | RecF | RecO | RecR | Rep |
RuvA | RuvB | RuvC | SSB | UvrD
FURTHER INFORMATION
Kenneth J. Marians’s homepage: http://www.mskcc.org/
mskcc/html/10294.cfm
Access to this links box is available online.
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