the dna damage response during dna replication

The DNA damage response during DNA replicationDana Branzei1,2 and Marco Foiani1

Eukaryotic chromosome replication is mediated by multiple

replicons and is coordinated with sister chromatid cohesion,

DNA recombination, transcription and cell cycle progression.

Replication forks stall or collapse at DNA lesions or problematic

genomic regions, and these events have often been

associated with recombination and chromosomal

rearrangements. Stalled forks generate single-stranded DNA

that activates the replication checkpoint, which in turn

functions to protect the stability of the fork until the replication

can resume. Recombination-mediated and damage-bypass

processes are themainmechanisms responsible for replication

restart. New findings have helped to unmask the molecular

mechanisms that sense replication stress, control the

stability of replication forks, and regulate the mechanisms

that promote replication restart, thereby giving us a better

understanding of how genome integrity is preserved during

replication.

Addresses1 FIRC Institute of Molecular Oncology Foundation and DSBB-University

of Milan, Via Adamello 16, 20139, Milan, Italy2Genetic Dynamics Research Unit Laboratory, RIKEN Research

Institute, Wako, Saitama, 351-0198, Japan

Corresponding author: Foiani, Marco ([email protected])

Current Opinion in Cell Biology 2005, 17:568–575

This review comes from a themed issue on

Cell division, growth and death

Edited by Scott H Kaufmann and Michael Tyers

Available online 13th October 2005

0955-0674/$ – see front matter

# 2005 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.ceb.2005.09.003

IntroductionMost of the chromosomal abnormalities arising in cancer

cells are caused by faulty chromosome replication [1].

DNA replication represents a dangerous moment in the

life of the cell as endogenous and exogenous events

challenge genome integrity by interfering with the pro-

gression, stability and restart of the replication fork.

To deal with this responsibility, replication forks are

endowed with an extraordinary potential to coordinate

fork stalling with fork resumption processes. Failure to

protect stalled forks or to process the replication fork

appropriately for replication restart results in the accu-

mulation of mutations and genomic aberrations. Indeed,

a variety of human genetic syndromes that lead to

cancer predisposition are caused by mutations in genes


that protect the genome integrity during chromosome

replication.

In this review we will comment on the recent findings

that helped to elucidate how stalled forks signal to the

replication checkpoint, how the checkpoint mechanisms

contribute to the stability of the fork, the mechanisms

that assist and coordinate fork restart, and the enzymatic

activities that process stalled or collapsed forks.

Endogenous and exogenous events causingreplication fork stalling and collapseReplication fork progression is slowed down at several

genomic sites, such as tRNA genes [2], specialized pro-

tein-mediated replication fork barriers [3,4], replication

slow zones [5] and inverted repeats [6��]. These chromo-

somal loci are known as fragile sites and induce fork

pausing, which is often associated with chromosome

breakage and genomic rearrangements [5]. Fork pausing

can also be caused by intra-S DNA damage through

several mechanisms: by causing uncoupling between

the replisome and the helicase at the fork; by uncoupling

the leading and lagging strand synthesis; or by blocking

the replicative helicase progression and therefore inhibit-

ing template unwinding (Figure 1).

In most cases, the replisome remains stably associated

with the stalled fork [7,8], and then resumption of repli-

cation can occur once the block is relieved. During this

process, specialized proteins, such as the yeast Rrm3

DNA helicase, have been proposed to assist the restart

of DNA synthesis by removing the impediments at

specific replication pausing sites [9,10].

In certain situations, however, replication forks may

experience replisome dissociation and collapse. Fork

collapse may be caused by protein–DNA complexes

that cannot be efficiently removed [11�,12��], templates

that have been exposed to the action of cross-linking

agents, DNA breaks [13] or by the run off of the repli-

some at telomeres. In most cases, fork collapse should

not represent a problem in eukaryotes using multiple

replicons, as forks converging from adjacent replicons

can complete replication [14]. Furthermore, considering

that only a fraction of replication origins are fired at each

round of DNA synthesis, it is reasonable to assume that

the excess origins represent a reserve of replicons for

cells experiencing extensive fork collapse. However, if

forks collapse at sub-telomeric regions, where there are

no converging forks, then completion of replication

under these circumstances will require the restart of

the collapsed forks.

www.sciencedirect.com

The DNA damage response during DNA replication Branzei and Foiani 569

Sensing replication stress: signals, thresholdsand limitations of the checkpoint mechanismStalled forks promote checkpoint activation by exposing

significant amounts of single-stranded DNA (ssDNA)

coated by replication protein A (RPA) [15–17,18��]. Itis becoming clear that it is not the damaged DNA per sethat generates the checkpoint signal but rather the colli-

sion of the fork with the lesion (Figure 1). Recent

observations show that functional uncoupling of the

MCM helicase and polymerase activities at the fork is

required for generation of RPA–ssDNA and checkpoint

signalling in Xenopus extracts [18��]. Alternatively,

ssDNA can result from transient uncoupling between

leading and lagging strand synthesis [15] or may be

generated at collapsed forks processed by Exo1 exonu-

clease and perhaps by other as yet unidentified factors

[19��] (Figure 2).

However, it is still unclear whether the RPA filaments

represent the primary signal or whether they are just

required to boost the checkpoint response. This could

explain why the rfa1 mutants isolated so far are only

partially checkpoint-defective [8,20]. Besides, a two-step

mechanism would have the advantage of preventing

Figure 1

Stalling of replication forks. (a) Replication forks encountering genomic pau

checkpoint signals represented by long stretches of ssDNA coated by RPA

replicative helicase or (c) uncoupling of leading and lagging strand synthesi

prevents the helicase progression. The red and blue circles indicate the lea

circle, the RPA complex; and the triangle, the helicase.


futile activation of the pathway when events cause only

transient chromosomal stress. The idea that a threshold is

indeed required for checkpoint activation was indicated

by findings showing that an amount of ssDNA above a

certain level or threshold must be produced in order to

activate the checkpoint response [15,18��,21,22].

The Mec1/ATR checkpoint kinase is recruited to stalled

forks, and once activated it phosphorylates Mrc1, a pro-

tein required for replisome stabilization and intra-S

checkpoint activation [23]. Mrc1 phosphorylation was

proposed to stabilize the MCM complex, stop the pro-

gression of the MCM-mediated DNA unwinding, and

allow replication fork restart after the replication block is

relieved [24�].

One important implication of these results is that lesions

that inhibit the helicase from unwinding may not gen-

erate a checkpoint response (Figure 1d). In support of this

view, a recent study shows that blockage of fork progres-

sion in S. pombe by DNA–protein complexes at a specific

genomic location does not cause checkpoint activation

[12��]. In addition, in certain situations or mutation con-

texts, the checkpoint signal might be muffled; perhaps

sing sites or lesions on the template may stall, accumulating

that result from (b) uncoupling between the replisome and the

s; (d) checkpoint activation does not occur when the replication block

ding and the lagging strand DNA polymerases, respectively; the green


570 Cell division, growth and death

Figure 2

Processing of collapsed replication forks. Stalled forks deprived of the replisome (in box, left) rapidly collapse and undergo alternative processing

through (a) Exo1-mediated resection of nascent chains, (b) run-off of hemicatenanes that leads to the formation of cruciform structures that can be

further resected by Exo1 or (c) double-strand break formation, possibly mediated by the Mus81 complex. The red shape indicates the Exo1

exonuclease and the blue shape the Mus81 complex.

this is the case with MMS-treated sgs1 mutants, in which

the ssDNA formed at stalled replication forks is engaged

into Rad51 filaments, thus diminishing the checkpoint

signal represented by the RPA–ssDNA filaments [25��].

Recent findings suggest that several events, probably

resulting from replication accidents, might escape check-

point surveillance or be dealt with in an inappropriate

manner either because of adaptation or because of faulty

repair. This would explain the genomic instability of cells

operating with a limited pool of replication proteins [6��],the absence of checkpoint system activation during cer-

tain events that generate fork collapse [12��], and the

segmental duplications of chromosomes that occur spon-

taneously, preferentially in the slow, late replicating

zones of the chromosomes [26�].

Checkpoint-mediated stabilization ofstalled forksOne of the most important and so far best-studied

mechanisms guarding genomic integrity during S phase

is provided by replication checkpoints. Electron micro-

scopic analysis of the budding yeast rad53mutant showed

that an important function of the replication checkpoint is

to protect the stability of stalled forks [15]. In checkpoint

mutants, stalled forks rapidly degenerate, accumulating


gapped and hemireplicated molecules [15]. These long

ssDNA regions seem to result from lagging strand defects,

probably due to the unscheduled dissociation of the

lagging strand polymerases and to the erosion of nascent

chains mediated by the Exo1 exonuclease [19��](Figure 2). Furthermore, a large fraction of forks accu-

mulate four-branched molecules resembling reversed

forks. Recent observations have suggested that these

four-way junctions do not form as a consequence of fork

reversal but rather as a result of an active process causing

the run off of specialized sister chromatid junctions (SCJs)

resembling hemicatenanes at stalled forks deprived of the

replisome [27] (Figure 2).

Replication checkpoints are involved in modulating the

replication fork response to intra-S damage by stabilizing

the stalled fork and by preventing the firing of late origins

and the unscheduled firing of dormant origins [28–30]

(Figure 3). The abnormal replicon firing occurring in

checkpoint-defective cells accelerates the completion

of replication in response to intra-S DNA damage

[31,32], and could be the best option that checkpoint

mutants have to complete replication after extensive fork

collapse. It is important to note that the abnormal origin

firing only modestly contributes to cell viability and can be

genetically uncoupled from replication fork collapse [16].



Figure 3

Replication stress response in eukaryotes. Schematic representation of the cellular pathways activated in response to replication stress.

Dashed arrows indicate partial dependencies.

Stabilization of stalled replication forks by replication

checkpoints is thought to occur through stabilizing the

association of the replisome with the fork [7,8] and

through restraining the activity of recombination

enzymes at stalled forks [33��,34] (Figure 3). In both

cases, physical interaction with the checkpoint or check-

point-dependent phosphorylation of replication or recom-

bination/repair enzymes has been invoked. The DNA

polymerase a-primase complex [35], RPA [36] and Mrc1

[23,24�] phosphorylation are thought to be implicated in

stabilizing the replisome–fork association. In fission yeast,

Cds1-dependent phosphorylation of Mus81 [33��] and

Rad60 [34] occurs after replication stalling induced by

hydroxyurea (HU) treatment, and is associated with

delocalization of Rad60 from the nucleus and a reduction

in the chromatin-binding ability of the Mus81–Eme1

endonuclease complex. These findings agree with the

observations that in fission yeast recombination repair foci

are rare in S phase and peak in G2 after intra-S damage

[37], and that in budding yeast recombination proteins are

not recruited to HU-arrested replication forks unless the

forks collapse [38��]. These results can also explain why

attempts to visualize the Rad51 and Rad52 recombination

proteins at stalled forks by chromatin immunoprecipita-

tion have failed [8] (K Shirahige, personal communica-

tion). The concept emerging from these results is that

stalled forks do not accumulate breaks or recombinogenic

intermediates [15] unless the forks are prone to collapse,

as is the case with replication checkpoint mutants and


with cells that operate with an altered replication pro-

gram. In this respect, it is interesting that cells experien-

cing slow replication due to limiting levels of DNA

polymerases accumulate hot-spots for recombination at

specific chromosomal regions that resemble mammalian

fragile sites [6��].

In addition to protecting the stalled forks from collapsing,

replication checkpoints are also thought to mediate the

damage response that promotes replication resumption

following fork collapse. Although numerous details

remain to be worked out, fork restart is also thought to

be largely mediated through phosphorylation of targets

and their subsequent recruitment to sites of damage. In

the following section we will discuss the mechanisms

implicated in restarting replication and those that might

modulate the choice of the pathway responsible for

processing DNA lesions during replication.

Fork restart mechanismsUnlikeE. coli, which rely on a single replication origin andneed to engage the collapsed forks into replication-

coupled recombination processes in order to complete

replication, eukaryotic organisms are endowed with sev-

eral routes to restart the fork (Figure 3).

Recombination-mediated replication-restart and damage-

bypass mechanisms are frequently used by eukaryotic

organisms to replicate damaged DNA or to resume repli-



cation after fork collapse. Recombination mechanisms

assist completion of replication when forks collapse in

regions where there are no converging forks that could

complete replication, or when the DNA lesions or the

stalled replication forks are processed to double-strand

breaks (DSBs). Unlike S phase replication, however,

recombination-mediated replication does not appear to

require MCM or replication initiation functions [39�],consistent with the view that MCM loading is restricted

to G1, which ensures that origin firing and replication

occur just once per cell cycle [40]. Damage bypass repli-

cation mechanisms either are assisted by specialized

translesion synthesis polymerases or utilize the genetic

information encoded by the undamaged sister chromatid

to overcome intra-S damage and replication impedi-

ments. Template-switch-mediated damage bypass might

be assisted by joint structures resembling hemicatenanes,

in which one newly synthesized strand is coiled around

the other newly synthesized strand, and which were

shown to form after origin firing and to migrate chasing

the forks [27].

Recent studies have unmasked several proteins and post-

translational modification mechanisms capable of coordi-

nating or modulating the choice of the pathway used to

process DNA lesions during replication, and we will

briefly note them here.

PCNA, a key signal integrator at the replication fork, is

target for both ubiquitin and SUMO modifications [41].

Monoubiquitination of PCNA promotes translesion

synthesis and damage-induced mutagenesis by error-

prone polymerases [42–44], and its polyubiquitination

promotes an error-free damage-bypass mechanism to

overcome DNA damage [41], or to promote replication

resumption in response to replication perturbations

induced by replication mutants [45]. Sumoylation of

PCNA contributes to spontaneous mutagenesis [42]

and prevents recombination repair from occurring during

S phase by promoting recruitment of Srs2 to replication

forks [46–48]. Previous studies on budding yeast Srs2

have suggested that Srs2 is involved in the metabolism of

single-strand gaps to prepare a substrate for lesion bypass

or damage avoidance and that it inhibits recombination

by disrupting Rad51 filaments [49,50]. Since replication

checkpoints have also been associated with suppression of

recombination in S phase and mutation suppression (see

the above section) and the 9-1-1 checkpoint complex in

fission yeast was suggested to promote mutagenesis in

response to replication stress [51] (Figure 3), it would be

of great interest to determine whether there is coordina-

tion between the DNA damage response modulated by

checkpoint activation and ubiquitin/SUMO-mediated

mutagenesis and recombination suppression.

Post-translational modification of several other proteins in

addition to PCNA was shown to regulate their function in


the DNA damage response. In this respect, UV-DDB-

dependent ubiquitination of XPC upon UV irradiation

appears to be critical for nucleotide excision repair of UV-

induced lesions [52]; monoubiquitination of FANCD2

requires ATR function [53] and mediates its targeting to

DNA repair foci, where it co-localizes with BRCA1 and

RAD51; and the repair proteins yku70 and Smc5 are

modified with SUMOby a newly identified SUMO ligase,

Mms21/Nse2 [54�].

RecQ helicases are also targets for checkpoint and post-

translational modifications, and have been implicated in

maintaining genome stability, probably through their role

in the recombination events that occur in response to

replication damage. Genetic and in vitro studies indicatedthat the RecQ–Top3 complex functions to resolve recom-

bination intermediates such as double Holliday junctions

(HJs), resulting in non-crossover products [55,56].

Recently, analysis of the replication intermediates

formed during chromosomal replication of damaged tem-

plates has implicated the Sgs1/Top3 complex in the

resolution of pseudo double HJs, which probably result

from replication-related SCJs or arise during replication

termination when replication forks converge [25��]. This

study also helps to explain how Sgs1 and Srs2 helicases

function to counteract fork-induced recombination

events, showing that they probably act in a coordinated

manner via two different mechanisms, Srs2 preventing

their formation, and Sgs1/Top3 promoting their resolu-

tion [25��]. With regard to how the RecQ/Top3 complex

might be recruited to such recombination structures, fresh

hints came from the identification of a newmember of the

RecQ/Top3 complex. The RMI1 gene encodes a DNA-

binding protein with preference for cruciform structures

that is an integral component of the RecQ/Top3 complex

in both budding yeast and human, and whose depletion

leads to phenotypes reminiscent of BLM-syndrome cells

or sgs1, top3mutant cells in budding yeast with respect to

genomic instability and the intra-S damage checkpoint

response [57–59].

ConclusionsThe past few years have brought significant contributions

in understanding the molecular basis of replication initia-

tion, how replication problems are sensed and how the

replication forks are appropriately stabilized or restored in

order to prevent genomic aberrations. Important issues,

however, such as mitochondrial replication and the coor-

dination between replication progression and recombina-

tion induction in the meiotic cycle, still await elucidation.

Recent advances in imaging, microarray analysis, proteo-

mic technologies, mouse modelling and genome-wide

techniques havemade it possible to determine replication

profiles and the temporal coordination between different

processes, as well as to study gene and protein expression

in different conditions or in individual tumours and to

address the role of different DNA replication checkpoints



and DNA repair proteins in physiological as well as

pathological situations. Single-molecule techniques are

being developed to assess replication at the level of single

replication forks, as opposed to whole cell populations,

and should help us understand the replication response

induced by DNA damage, chemotherapeutic drugs or

mutations in the replication checkpoint. These efforts are

expected to give us a better understanding of the intricate

mechanism that preserves genome integrity.

AcknowledgementsThe authors apologize for the many interesting articles that they were notable to discuss or acknowledge due to space limitations. We thank allmembers of Foiani’s laboratory and Joel A. Huberman for usefuldiscussions, and Katsuhiko Shirahige for communicating unpublishedresults. The work carried out in Foiani’s laboratory was supported by grantsof the Italian Association for Cancer Research and the EuropeanCommunity, and D. Branzei was supported by the RIKEN SpecialPostdoctoral Research Program in Japan and the European CommunityGrant LSHC-CT-2005-512113.

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54.�

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the dna damage response during dna replication

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