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The DNA damage response during DNA replication

Dana 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 the main mechanisms 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.
Addresses
1
FIRC Institute of Molecular Oncology Foundation and DSBB-University
of Milan, Via Adamello 16, 20139, Milan, Italy
2
Genetic Dynamics Research Unit Laboratory, RIKEN Research
Institute, Wako, Saitama, 351-0198, Japan
Corresponding author: Foiani, Marco (marco.foiani@ifom-ieo-campus.it)

Current Opinion in Cell Biology 2005, 17:568575


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

Introduction
Most 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 progression, 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 accumulation of mutations and genomic aberrations. Indeed,
a variety of human genetic syndromes that lead to
cancer predisposition are caused by mutations in genes
Current Opinion in Cell Biology 2005, 17:568575

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 causing


replication fork stalling and collapse
Replication fork progression is slowed down at several
genomic sites, such as tRNA genes [2], specialized protein-mediated replication fork barriers [3,4], replication
slow zones [5] and inverted repeats [6]. These chromosomal 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 inhibiting template unwinding (Figure 1).
In most cases, the replisome remains stably associated
with the stalled fork [7,8], and then resumption of replication 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 proteinDNA 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 replisome 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.
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The DNA damage response during DNA replication Branzei and Foiani 569

Sensing replication stress: signals, thresholds


and limitations of the checkpoint mechanism
Stalled forks promote checkpoint activation by exposing
significant amounts of single-stranded DNA (ssDNA)
coated by replication protein A (RPA) [1517,18]. It
is becoming clear that it is not the damaged DNA per se
that generates the checkpoint signal but rather the collision 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 RPAssDNA 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 exonuclease 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

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 protein required for replisome stabilization and intra-S
checkpoint activation [23]. Mrc1 phosphorylation was
proposed to stabilize the MCM complex, stop the progression 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 generate a checkpoint response (Figure 1d). In support of this
view, a recent study shows that blockage of fork progression in S. pombe by DNAprotein complexes at a specific
genomic location does not cause checkpoint activation
[12]. In addition, in certain situations or mutation contexts, the checkpoint signal might be muffled; perhaps

Figure 1

Stalling of replication forks. (a) Replication forks encountering genomic pausing sites or lesions on the template may stall, accumulating
checkpoint signals represented by long stretches of ssDNA coated by RPA that result from (b) uncoupling between the replisome and the
replicative helicase or (c) uncoupling of leading and lagging strand synthesis; (d) checkpoint activation does not occur when the replication block
prevents the helicase progression. The red and blue circles indicate the leading and the lagging strand DNA polymerases, respectively; the green
circle, the RPA complex; and the triangle, the helicase.
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Current Opinion in Cell Biology 2005, 17:568575

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 RPAssDNA filaments [25].
Recent findings suggest that several events, probably
resulting from replication accidents, might escape checkpoint 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 certain events that generate fork collapse [12], and the
segmental duplications of chromosomes that occur spontaneously, preferentially in the slow, late replicating
zones of the chromosomes [26].

Checkpoint-mediated stabilization of
stalled forks
One of the most important and so far best-studied
mechanisms guarding genomic integrity during S phase
is provided by replication checkpoints. Electron microscopic analysis of the budding yeast rad53 mutant 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
Current Opinion in Cell Biology 2005, 17:568575

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 accumulate 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 [2830]
(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].
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The DNA damage response during DNA replication Branzei and Foiani 571

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 checkpoint-dependent phosphorylation of replication or recombination/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 replisomefork 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 Mus81Eme1
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 immunoprecipitation have failed [8] (K Shirahige, personal communication). 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
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with cells that operate with an altered replication program. In this respect, it is interesting that cells experiencing 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 mechanisms


Unlike E. coli, which rely on a single replication origin and
need to engage the collapsed forks into replicationcoupled recombination processes in order to complete
replication, eukaryotic organisms are endowed with several routes to restart the fork (Figure 3).
Recombination-mediated replication-restart and damagebypass mechanisms are frequently used by eukaryotic
organisms to replicate damaged DNA or to resume repliCurrent Opinion in Cell Biology 2005, 17:568575

572 Cell division, growth and death

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 replication 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 impediments. 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 posttranslational modification mechanisms capable of coordinating 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 errorprone polymerases [4244], 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 [4648]. 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 coordination 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
Current Opinion in Cell Biology 2005, 17:568575

the DNA damage response. In this respect, UV-DDBdependent ubiquitination of XPC upon UV irradiation
appears to be critical for nucleotide excision repair of UVinduced 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 SUMO by a newly identified SUMO ligase,
Mms21/Nse2 [54].
RecQ helicases are also targets for checkpoint and posttranslational 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 indicated
that the RecQTop3 complex functions to resolve recombination 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 templates 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 resolution [25]. With regard to how the RecQ/Top3 complex
might be recruited to such recombination structures, fresh
hints came from the identification of a new member of the
RecQ/Top3 complex. The RMI1 gene encodes a DNAbinding 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, top3 mutant cells in budding yeast with respect to
genomic instability and the intra-S damage checkpoint
response [5759].

Conclusions
The past few years have brought significant contributions
in understanding the molecular basis of replication initiation, 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 coordination between replication progression and recombination induction in the meiotic cycle, still await elucidation.
Recent advances in imaging, microarray analysis, proteomic technologies, mouse modelling and genome-wide
techniques have made 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
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The DNA damage response during DNA replication Branzei and Foiani 573

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.

Acknowledgements
The authors apologize for the many interesting articles that they were not
able to discuss or acknowledge due to space limitations. We thank all
members of Foianis laboratory and Joel A. Huberman for useful
discussions, and Katsuhiko Shirahige for communicating unpublished
results. The work carried out in Foianis laboratory was supported by grants
of the Italian Association for Cancer Research and the European
Community, and D. Branzei was supported by the RIKEN Special
Postdoctoral Research Program in Japan and the European Community
Grant LSHC-CT-2005-512113.

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574 Cell division, growth and death

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Current Opinion in Cell Biology 2005, 17:568575

yeast. Mre11, Rfa1 checkpoints and recombination proteins are


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Current Opinion in Cell Biology 2005, 17:568575