Sie sind auf Seite 1von 8

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
FIRC Institute of Molecular Oncology Foundation and DSBB-University
of Milan, Via Adamello 16, 20139, Milan, Italy
Genetic Dynamics Research Unit Laboratory, RIKEN Research
Institute, Wako, Saitama, 351-0198, Japan
Corresponding author: Foiani, Marco (

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/

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

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.

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.

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].

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

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].

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

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.

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.

References and recommended reading

Papers of particular interest, published within the annual period of
review, have been highlighted as:
 of special interest
 of outstanding interest

Myung K, Kolodner RD: Suppression of genome instability by

redundant S-phase checkpoint pathways in Saccharomyces
cerevisiae. Proc Natl Acad Sci USA 2002, 99:4500-4507.


Deshpande AM, Newlon CS: DNA replication fork pause sites

dependent on transcription. Science 1996, 272:1030-1033.


Takeuchi Y, Horiuchi T, Kobayashi T: Transcription-dependent

recombination and the role of fork collision in yeast rDNA.
Genes Dev 2003, 17:1497-1506.


Dalgaard JZ, Klar AJS: swi1 and swi3 perform imprinting,

pausing, and termination of DNA replication in S. pombe.
Cell 2000, 102:745-751.


Cha RS, Kleckner N: ATR homolog Mec1 promotes fork

progression, thus averting breaks in replication slow zones.
Science 2002, 297:602-606.


Lemoine FJ, Degtyareva NP, Lobachev K, Petes TD:

Chromosomal translocations in yeast induced by low levels of
DNA polymerase a model for chromosome fragile sites.
Cell 2005, 120:587-598.
This study shows that optimal amounts of replication proteins are crucial
for preserving genomic integrity. Budding yeast cells replicating with a
limited amount of DNA polymerases accumulate site-specific genomic
aberrations and hotspots for DSBs.

This study shows that replication fork blockage caused by replication fork
barriers placed in a typical genomic locus in fission yeast is associated
with increased recombination events. The study suggests that recombination usually occurs without breakage of the fork and suggests a role for
Rqh1 in preventing deletion events caused by collapse of the blocked
12. Lambert S, Watson A, Sheedy DM, Martin B, Carr AM: Gross
 chromosomal rearrangements and elevated recombination at
an inducible site-specific replication fork barrier. Cell 2005,
By using a system in which fork progression is inhibited by proteinDNA
complexes at a specific genomic location in S. pombe, this study
describes the consequences of fork collapse. In the system used,
checkpoint activation does not occur and instead recombination events
are induced, allowing cell survival but inducing site-specific chromosomal
13. Kuzminov A: Collapse and repair of replication forks in
Escherichia coli. Mol Microbiol 1995, 16:373-384.
14. McGlynn P, Lloyd RG: Recombinational repair and restart of
damaged replication forks. Nat Rev Mol Cell Biol 2002,
15. Sogo JM, Lopes M, Foiani M: Fork reversal and ssDNA
accumulation at stalled replication forks owing to checkpoint
defects. Science 2002, 297:599-602.
16. Tercero JA, Longhese MP, Diffley JF: A central role for DNA
replication forks in checkpoint activation and response.
Mol Cell 2003, 11:1323-1336.
17. Zou L, Elledge SJ: Sensing DNA damage through ATRIP
recognition of RPA-ssDNA complexes. Science 2003,
18. Byun TS, Pacek M, Yee M-c, Walter JC, Cimprich KA: Functional
 uncoupling of MCM helicase and DNA polymerase activities
activates the ATR-dependent checkpoint. Genes Dev 2005,
This study uses a cell free extract system derived from Xenopus eggs to
examine the mechanism by which checkpoint activation occurs in
response to different types of intra-S damage. This study clearly
indicates that functional uncoupling between the MCM helicase and
polymerase activities at the replication fork is a common mechanism
to generate the checkpoint signal following different types of DNA
19. Cotta-Ramusino C, Fachinetti D, Lucca C, Doksani Y, Lopes M,
 Sogo J, Foiani M: Exo1 processes stalled replication forks and
counteracts fork reversal in checkpoint-defective cells.
Mol Cell 2005, 17:153-159.
By using the 2D gel technique combined with electron microscopy, this
study shows that the Exo1 exonuclease processes stalled replication
forks and counteracts reversed fork accumulation by generating ss-DNA
intermediates. The Exo1- mediated resection of nascent chains is important for both fork stability and replication restart.
20. Pellicioli A, Lee SE, Lucca C, Foiani M, Haber JE: Regulation of
Saccharomyces Rad53 checkpoint kinase during adaptation
from DNA-damage-induced G2/M arrest. Mol Cell 2001,

Cobb JA, Bjergbaek L, Shimada K, Frei C, Gasser SM:

DNA polymerase stabilization at stalled replication forks
requires Mec1 and the RecQ helicase Sgs1. EMBO J 2003,

21. Shimada K, Pasero P, Gasser SM: ORC and the intra-S-phase

checkpoint: a threshold regulates Rad53p activation in S
phase. Genes Dev 2002, 16:3236-3252.


Lucca C, Vanoli F, Cotta-Ramusino C, Pellicioli A, Liberi G,

Haber J, Foiani M: Checkpoint-mediated control of replisomefork association and signalling in response to replication
pausing. Oncogene 2004, 23:1206-1213.

22. Vaze MB, Pellicioli A, Lee SE, Ira G, Liberi G, Arbel-Eden A,

Foiani M, Haber JE: Recovery from checkpoint-mediated arrest
after repair of a double-strand break requires Srs2 helicase.
Mol Cell 2002, 10:373-385.


Torres JZ, Schnakenberg SL, Zakian VA: Saccharomyces

cerevisiae Rrm3p DNA helicase promotes genome integrity by
preventing replication fork stalling: viability of rrm3 cells
requires the intra-S-phase checkpoint and fork restart
activities. Mol Cell Biol 2004, 24:3198-3212.

23. Katou Y, Kanoh Y, Bando M, Noguchi H, Tanaka H, Ashikari T,

Sugimoto K, Shirahige K: S-phase checkpoint proteins Tof1 and
Mrc1 form a stable replication-pausing complex. Nature 2003,

10. Schmidt KH, Kolodner RD: Requirement of Rrm3 helicase for

repair of spontaneous DNA lesions in cells lacking Srs2 or
Sgs1 helicase. Mol Cell Biol 2004, 24:3198-3212.
11. Ahn JS, Osman F, Whitby MC: Replication fork blockage by

RTS1 at an ectopic site promotes recombination in fission
yeast. EMBO J 2005, 24:2011-2023.

24. Nedelcheva MN, Roguev A, Dolapchiev LB, Shevchenko A,

Taskov HB, Stewart AF, Stoynov SS: Uncoupling of unwinding
from DNA synthesis implies regulation of MCM helicase by
Tof1/Mrc1/Csm3 checkpoint complex. J Mol Biol 2005,
By analyzing the DNA topology at the fork as well as genetic and physical
interactions between replisome proteins and the checkpoint proteins Mrc1
Current Opinion in Cell Biology 2005, 17:568575

574 Cell division, growth and death

and Tof1, this study indicates that Mrc1 and Tof1 coordinate DNA synthesis
and replication fork stalling with MCM-mediated unwinding.
25. Liberi G, Maffioletti G, Lucca C, Chiolo I, Baryshnikova A,
 Cotta-Ramusino C, Lopes M, Pellicioli A, Haber JE, Foiani M:
Rad51-dependent DNA structures accumulate at damaged
replication forks in sgs1 mutants defective in the yeast
ortholog of BLM RecQ helicase. Genes Dev 2005, 19:339-350.
This study analyzes the replication intermediates formed during replication of damaged templates in budding yeast and shows that recombination-dependent cruciform structures originating from SCJs accumulate at
damaged forks in sgs1 mutants. Srs2 counteracts their formation, while
the Sgs1Top3 complex is crucial for their resolution.
26. Koszul R, Caburet S, Dujon B, Fischer G: Eucaryotic genome

evolution through the spontaneous duplication of large
chromosomal segments. EMBO J 2004, 23:234-243.
This study uses a growth recovery assay in S. cerevisiae to monitor gene
duplication events induced by deletion of one duplicate from a highly
similar gene pair. The study shows that duplications of large DNA segments encompassing numerous genes occur spontaneously and are
most likely the results of replication accidents. Such events are also
thought to have played a major role in eukaryotic genome evolution.
27. Lopes M, Cotta-Ramusino C, Liberi G, Foiani M: Branch
migrating sister chromatid junctions form at replication
origins through Rad51/Rad52-independent mechanisms.
Mol Cell 2003, 12:1499-1510.
28. Shirahige K, Hori Y, Shiraishi K, Yamashita M, Takahashi K,
Obuse C, Tsurimoto T, Yoshikawa H: Regulation of DNAreplication origins during cell-cycle progression. Nature 1998,
29. Santocanale C, Sharma K, Diffley JF: Activation of dormant
origins of DNA replication in budding yeast. Genes Dev 1999,
30. Santocanale C, Diffley JF: A Mec1- and Rad53-dependent
checkpoint controls late-firing origins of DNA replication.
Nature 1998, 395:615-618.
31. Paulovich AG, Hartwell LH: A checkpoint regulates the rate of
progression through S phase in S. cerevisiae in response to
DNA damage. Cell 1995, 82:841-847.
32. Tercero JA, Diffley JF: Regulation of DNA replication fork
progression through damaged DNA by the Mec1/Rad53
checkpoint. Nature 2001, 412:553-557.
33. Kai M, Boddy MN, Russell P, Wang TS: Replication checkpoint
 kinase Cds1 regulates Mus81 to preserve genome integrity
during replication stress. Genes Dev 2005, 19:919-932.
By using chromatin immunoprecipitation and genetic analysis of the
mutator phenotype of a replication mutant in fission yeast, this study
analyzes the mechanism that regulates Mus81 function in response to
different types of replication perturbation. Cds1-dependent phosphorylation of Mus81 is induced in HU-arrested cells and promotes Mus81
dissociation from chromatin, thereby inhibiting recombination repair from
occurring at stalled forks.
34. Boddy MN, Shanahan P, McDonald WH, Lopez-Girona A,
Noguchi E, Yates IJ, Russell P: Replication checkpoint kinase
Cds1 regulates recombinational repair protein Rad60.
Mol Cell Biol 2003, 23:5939-5946.
35. Pellicioli A, Lucca C, Liberi G, Marini F, Lopes M, Plevani P,
Romano A, Di Fiore PP, Foiani M: Activation of Rad53 kinase in
response to DNA damage and its effect in modulating
phosphorylation of the lagging strand DNA polymerase.
EMBO J 1999, 18:6561-6572.
36. Brush GS, Morrow DM, Hieter P, Kelly TJ: The ATM homologue
MEC1 is required for phosphorylation of replication protein A
in yeast. Proc Natl Acad Sci USA 1996, 93:15075-15080.
37. Meister P, Taddei A, Vernis L, Poidevin M, Gasser SM, Baldacci G:
Temporal separation of replication and recombination
requires the intra-S checkpoint. J Cell Biol 2005, 168:537-544.
38. Lisby M, Barlow JH, Burgess RC, Rothstein R: Choreography of
 the DNA damage response: spatiotemporal relationships
among checkpoint and repair proteins. Cell 2004, 118:699-713.
This study provides an integrated and comprehensive description of the
order in which different repair and checkpoint proteins assemble in
subnuclear foci in response to DSBs or replication fork stalling in budding
Current Opinion in Cell Biology 2005, 17:568575

yeast. Mre11, Rfa1 checkpoints and recombination proteins are

assembled sequentially at DSB sites, but Mre11 and recombination
proteins are not recruited at stalled forks.
39. Wang X, Ira G, Tercero JA, Holmes AM, Diffley JF, Haber JE: Role

of DNA replication proteins in double-strand break-induced
recombination in Saccharomyces cerevisiae. Mol Cell Biol
2004, 24:6891-6899.
This study analyzes the genetic requirements for replication initiation
and elongation factors in the DSB-induced gene conversion process in
budding yeast. Unlike S-phase replication, DNA synthesis during gene
conversion requires only leading strand polymerization.
40. Labib K, Tercero JA, Diffley JF: Uninterrupted MCM2-7 function
required for DNA replication fork progression. Science 2000,
41. Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S:
RAD6-dependent DNA repair is linked to modification of PCNA
by ubiquitin and SUMO. Nature 2002, 419:135-141.
42. Stelter P, Ulrich HD: Control of spontaneous and damageinduced mutagenesis by SUMO and ubiquitin conjugation.
Nature 2003, 425:188-191.
43. Watanabe K, Tateishi S, Kawasuji M, Tsurimoto T, Inoue H,
Yamaizumi M: Rad18 guides poleta to replication stalling sites
through physical interaction and PCNA monoubiquitination.
EMBO J 2004, 23:3886-3896.
44. Kannouche PL, Wing J, Lehmann AR: Interaction of human DNA
polymerase eta with monoubiquitinated PCNA: a possible
mechanism for the polymerase switch in response to DNA
damage. Mol Cell 2004, 14:491-500.
45. Branzei D, Seki M, Enomoto T: Rad18/Rad5/Mms2-mediated
polyubiquitination of PCNA is implicated in replication
completion during replication stress. Genes Cells 2004,
46. Pfander B, Moldovan GL, Sacher M, Hoege C, Jentsch S:
SUMO-modified PCNA recruits Srs2 to prevent recombination
during S phase. Nature 2005.
47. Papouli E, Chen S, Davies AA, Huttner D, Krejci L, Sung P,
Ulrich HD: Crosstalk between SUMO and Ubiquitin on PCNA Is
mediated by recruitment of the helicase Srs2p. Mol Cell 2005,
48. Haracska L, Torres-Ramos CA, Johnson RE, Prakash S,
Prakash L: Opposing effects of ubiquitin conjugation and
SUMO modification of PCNA on replicational bypass of DNA
lesions in Saccharomyces cerevisiae. Mol Cell Biol 2004,
49. Veaute X, Jeusset J, Soustelle C, Kowalczykowski SC, Le Cam E,
Fabre F: The Srs2 helicase prevents recombination by
disrupting Rad51 nucleoprotein filaments. Nature 2003,
50. Krejci L, Van Komen S, Li Y, Villemain J, Reddy MS, Klein H,
Ellenberger T, Sung P: DNA helicase Srs2 disrupts the Rad51
presynaptic filament. Nature 2003, 423:305-309.
51. Kai M, Wang TS: Checkpoint activation regulates mutagenic
translesion synthesis. Genes Dev 2003, 17:64-76.
52. Sugasawa K, Okuda Y, Saijo M, Nishi R, Matsuda N, Chu G, Mori T,
Iwai S, Tanaka K, Hanaoka F: UV-induced ubiquitylation of XPC
protein mediated by UV-DDB-ubiquitin ligase complex.
Cell 2005, 121:387-400.
53. Andreassen PR, DAndrea AD, Taniguchi T: ATR couples
FANCD2 monoubiquitination to the DNA-damage response.
Genes Dev 2004, 18:1958-1963.
54. Zhao X, Blobel G: A SUMO ligase is part of a nuclear

multiprotein complex that affects DNA repair and
chromosomal organization. Proc Natl Acad Sci USA 2005,
This study identifies Mms21/Nse2 as a new SUMO ligase in budding
yeast, and shows that Mms21 is responsible for the sumoylation of
yKu70, Smc5 and Smc6 proteins.
55. Ira G, Malkova A, Liberi G, Foiani M, Haber JE: Srs2 and
Sgs1Top3 suppress crossovers during double-strand break
repair in yeast. Cell 2003, 115:401-411.

The DNA damage response during DNA replication Branzei and Foiani 575

56. Wu L, Davies SL, Levitt NC, Hickson ID: Potential role for the
BLM helicase in recombinational repair via a conserved
interaction with RAD51. J Biol Chem 2001, 276:19375-19381.

58. Mullen JR, Nassaseth FS, Lan YQ, Slagle CE, Brill SJ: Yeast Rmi1/
Nce4 controls genome stability as a subunit of the Sgs1-Top3
complex. Mol Cell Biol 2005, 25:4476-4487.

57. Chang M, Bellaoui M, Zhang C, Desai R, Morozov P,

Delgado-Cruzata L, Rothstein R, Freyer GA, Boone C, Brown GW:
RMI1/NCE4, a suppressor of genome instability, encodes a
member of the RecQ helicase/Topo III complex. EMBO J 2005,

59. Yin J, Sobeck A, Xu C, Meetei AR, Hoatlin M, Li L, Wang W:

BLAP75, an essential component of Blooms syndrome
protein complexes that maintain genome integrity. EMBO J
2005, 24:1465-1476.

Current Opinion in Cell Biology 2005, 17:568575