dna repair 6 ( 2 0 0 7 ) 994–1003
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/dnarepair
Interplay of replication checkpoints and repair
proteins at stalled replication forks
Dana Branzeia,b,∗, Marco Foiania,b,∗
aFIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy
bDipartimento di Scienze Biomolecolari e Biotecnologie, Universit` a degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy
a r t i c l e i n f o
Published on line 26 March 2007
DNA repair and recombination
a b s t r a c t
DNA replication is an essential process that occurs in all growing cells and needs to be
tightly regulated in order to preserve genetic integrity. Eukaryotic cells have developed mul-
tiple mechanisms to ensure the fidelity of replication and to coordinate the progression
of replication forks. Replication is often impeded by DNA damage or replication blocks,
and the resulting stalled replication forks are sensed and protected by specialized surveil-
lance mechanisms called checkpoints. The replication checkpoint plays an essential role in
preventing the breakdown of stalled replication forks and the accumulation of DNA struc-
tures that enhance recombination and chromosomal rearrangements that ultimately lead
to genomic instability and cancer development. In addition, the replication checkpoint is
thought to assist and coordinate replication fork restart processes by controlling DNA repair
pathways, regulating chromatin structure, promoting the recruitment of proteins to sites
of damage, and controlling cell cycle progression. In this review we focus mainly on the
results obtained in budding yeast to discuss on the multiple roles of checkpoints in main-
taining fork integrity and on the enzymatic activities that cooperate with the checkpoint
pathway to promote fork resumption and repair of DNA lesions thereby contributing to
© 2007 Elsevier B.V. All rights reserved.
1.Replication initiation and elongation
The cell division cycle of eukaryotes consists of four differ-
ent phases, G1, S, G2, and M, in which cells are preparing
or actively at work in replicating (S phase) or segregating (M
phase) their chromosomes. The replication of DNA is a com-
plex process, as it needs to occur very accurately, rapidly, and
only once per cell cycle in order to prevent genome abnormali-
ria, which replicate their small genome from a single origin of
ple origins. Budding yeast has well-defined, sequence-specific
∗Corresponding author at: FIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy. Tel.: +39 02 5743 03221;
fax: +39 02 5743 03231.
E-mail addresses: firstname.lastname@example.org (D. Branzei), email@example.com (M. Foiani).
1568-7864/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
replication origins, and genome-wide replication profiles and
fork progression dynamics have been already characterized
[1,2]. S. cerevisiae has about 400 origins of replication, dis-
tributed about every 40–150kb, which are responsible for the
replication of a genome of about 14Mb. The initiation of repli-
cation is one of the most tightly regulated cellular processes,
during the G1 phase of the cell cycle . In addition, origins
are characterized by flexibility and temporal regulation. That
is, firing efficiencies vary from origin to origin and their fir-
ing time of “early” or “late” is dependent on the context rather
gin timing is controlled is not yet well understood, but there
dna repair 6 ( 2 0 0 7 ) 994–1003
is evidence suggesting that different S-phase specific cyclin-
dependent kinase activities target early and late-firing origins
[6,7]. Controlling the timing of origin firing and origin density
is important for replication accuracy and it was shown that
when origin firing is perturbed, replication from fewer origins
leads to chromosome loss and rearrangement . The mecha-
of DNA replication is perturbed, have an extremely high insta-
bility of chromosome XII that contains the highly repetitive
ribosomal RNA gene cluster (rDNA array) .
Origin firing is followed by DNA synthesis, and the factors
required for DNA replication are loaded soon after the origins
are licensed. The binding of the DNA replication machinery to
the origins of replication requires the preloading of the MCM
proteins, likely comprising the unwinding activity during both
initiation and elongation steps of DNA replication, and their
associated factors Cdc45 and GINS, by the ORC/Cdc6/Cdt1 ini-
tiation apparatus. The DNA polymerases ? and ? involved in
replicating both leading and lagging strands, together with
their accessory proteins, such as replication factor C (RFC)
and proliferating cell nuclear antigen (PCNA), are thought to
form a large protein complex called the “replisome” [10,11].
Origin firing generates sister replication forks that proceed
bidirectionally. Originally it was thought that the two repli-
somes travel with the forks and thus behave independently.
However, in order to explain the coordinated termination of
DNA replication, it was proposed that the two replisomes
at sister forks are stationary and remain attached to each
other during DNA replication, and that the newly synthe-
sized DNA strands are extruded as replication proceeds .
This hypothesis was confirmed recently in budding yeast,
by using time-lapse microscopy to study the dynamics of
DNA replication of individual chromosomal loci marked with
bacteria-derived operators . This approach also allowed
visualization of replication factories in budding yeast, and it
appears that an average of 10 replicons are processed in each
replication factory, and likely that adjacent replication forks
are associated with each other in one replication factory .
The formation of replication factories has the same genetic
requirements as replisome assembling; that is, they are
dependent on Cdc6, S phase cyclin CDKs, and origin unwind-
ing. This suggests that the replication factories are composed
of replisomes at advancing forks, rather than of replisome
components loaded prior to DNA replication. The benefits of
having colocalized forks in replication factories still remain to
important consequences for the DNA topology during repli-
cation as well as for coordinating the DNA synthesis rates of
of chromosomes [13,14].
In addition to proteins that mediate DNA synthesis, sev-
eral other regulatory proteins, such as Mrc1, Tof1, and Csm3,
interact with the MCMs and components of the replisome,
and are thought to contribute to the structural and functional
coupling between DNA unwinding and replication [15,16]. In
budding yeast, the Mrc1, Tof1, and Csm3 proteins form a com-
plex that co-localizes with both normal and stalled forks,
and in Mrc1 and Tof1 deficient cells, the MCM complex and
Cdc45 simultaneously become uncoupled from DNA synthe-
sis during hydroxyurea (HU) arrest . As mrc1, tof1, and
csm3 mutants were shown to be synhetic lethal with alleles
affecting the function of the polymerase ?/primase , an
intriguing possibility is that the Mrc1/Tof1/Csm3 complex pre-
vents the lethality of cells in which extensive single-stranded
DNA (ssDNA) regions are generated as a result of uncoupling
between the leading and lagging strand DNA synthesis. Inter-
estingly, Mrc1, Tof1, and Csm3 also affect the checkpoint, and
are required for full activation of Rad53 either in response to
limited levels of dNTPs, or to DNA damage . However, it
is not yet understood whether the presumed lack of synchro-
and csm3 mutants  is due to their checkpoint defects or
rather to the requirement for the Mrc1/Tof1/Csm3 to physi-
cally couple the DNA synthesis and the replicative helicase
complexes at the replication fork. However, we must note
that extended unwinding in front of the fork upon encoun-
tering blocks to DNA synthesis would generate large ssDNA
regions on both sides of the same fork, and yet such structures
were not detected by electron microscopy (EM) coupled with
psoralen crosslinking, in either wild-type or rad53 replication
checkpoint mutants .
2.Checkpoint activation during replication
Stalled replication forks may expose significant amounts of
ssDNA, generated by the uncoupling of either the MCM heli-
case and the replicating apparatus or the leading and lagging
strand DNA synthesis [19,20] (see Fig. 1). When these ssDNA
regions are coated by RPA, they trigger the recruitment of the
Mec1 kinase in yeast or ATR in human cells (reviewed in ),
which together with other checkpoint factors, mediates Rad53
phosphorylation at multiple sites . The Mec1-mediated
Rad53 phosphorylation and activation is crucial for trigger-
ing the full replication checkpoint response both in response
to DNA damage or when DNA synthesis is inhibited by HU.
The activated checkpoint amplifies then the original signal
to different cellular processes that include cell cycle delay,
activation of DNA repair, prevention of late origins from fir-
ing, and stabilization of the replisome and the stalled forks
 (Fig. 1). In particular, an increasing amount of evidence
suggests that the role of the replication checkpoint in stabi-
lizing the stalled fork is crucial for retaining cell viability after
removal of replication stress [22,23].
Stalled fork stabilization and
One important function of the replication checkpoint in
response to replication blocks is to maintain the integrity of
of progressing replication forks after the stress has been
removed [24–26] (Fig. 1). In replication checkpoint mutants,
the replisome dissociates [27,28] and much longer stretches
of ssDNA regions representing gapped and hemireplicated
molecules accumulate as compared to wild-type cells [24,29].
The formation of gapped DNA molecules is perhaps largely
due to Exo1-mediated processing of the nascent chains
dna repair 6 ( 2 0 0 7 ) 994–1003
Fig. 1 – A diagram of the multiple roles of the replication checkpoint during DNA replication. The replication checkpoints
regulate replication initiation, promote replication fork progression through slow zones and fragile sites, and are required to
maintain the stability of stalled forks and coordinate cell cycle delay and repair of DNA lesions with resumption of DNA
deprived of replisome . In addition to having much
longer stretches of ssDNA at stalled forks, rad53 replication
checkpoint mutants accumulate four-branched molecules
resembling reversed forks [24,25] (Fig. 2). Budding yeast rad53
exo1 cells show a much larger increase in reversed forks than
rad53, indicating that Exo1 counteracts fork reversal in vivo,
ing the sister chromatid junctions of the four-way structure
[19,30] (Fig. 2). These DNA structures resembling reversed
forks are likely to provoke unscheduled recombination and
lead to genome instability [19,24,31,32], and indeed, in sup-
port of this view, S-phase checkpoint mutants were shown
to have extremely high elevated rates of gross chromosomal
rearrangements (GCRs) .
promoting replication and stability of fragile
Role of replication checkpoints in
An examination of the dynamics of fork progression of chro-
mosome VI in budding yeast showed that fork progression
rates are similar in wild-type and checkpoint mutants ,
follows a similar stochastic pattern to that of wild-type cells.
These results are consistent with other findings that sug-
gest that replication fork stalling per se occurs stochastically
and independently of the replication checkpoint . How-
ever, it must be noted that even in budding yeast it is still
unknown whether this observation holds true for all the chro-
mosomes, and clearly there are locations in the yeast genome
that are prone to breakage or instability in the absence of
a functional replication checkpoint in an otherwise unper-
turbed S phase [32,34]. The most likely explanation for this is
fork stalling with an unusually high frequency that will ren-
der them particularly sensitive to replication stress or to the
lack of replication checkpoint function, which protect stalling
in yeast have suggested a model in which genome instabil-
ity initiates at specific chromosomal sites, resembling fragile
sites that induce stalling of replication forks . Disruption
of DNA replication or defects in the DNA replication check-
point greatly increases this chromosome instability, leading
to broken forks that undergo recombination and give rise to
Not only do replication checkpoint proteins maintain the
stability of stalled forks, but they are also required to mediate
efficient replication from a number of early replication ori-
gins . In a recent study aimed at examining genome-wide
replication profiles in budding yeast, Bielinsky and collabo-
rators made use of a replication origin microarray approach
and found that 17 early-firing origins are not replicated effi-
in initiation but rather to problems in replication elongation,
as the origins fire but the replication forks accumulate small
intermediates in proximity to the origins. One of these com-
promised sites maps close to the early-firing origin ARS310,
which was identified by another study as a hot-spot for DSB
formation and recombination especially when cells replicate
with limited amounts of polymerases . Furthermore, these
regions are prone to breakage even in the absence of HU ,
and thus can be regarded as the equivalents of fragile sites
in humans . Likely, failure of the replication checkpoint
to protect such regions during replication leads to increased
recombination and chromosomal rearrangements, and may
be relevant to chromosome instability of mammalian fragile
sites and of chromosomes in cancer cells  (see Fig. 1).
Altered S-phase dynamics of replication
The replication checkpoint kinases Mec1 and Rad53 regulate
late and dormant origin firing during normal replication or in
conditions that induce fork stalling or collapse [19,37,38]. Con-
sistent with this concept, a recent study demonstrated that, in
dna repair 6 ( 2 0 0 7 ) 994–1003
Fig. 2 – Intra-S pathways that contribute to resumption of replication. A replication fork in which uncoupling between the
leading and lagging strand has occurred due to a block in the leading strand synthesis is illustrated at the top of the figure.
Replication resumption can occur through bypass of the DNA damage either by template switch (A) or translesion synthesis
(B). Alternatively, replication resumption is envisaged to occur by repriming downstream the lesion (C). This event will
generate a gap, which could be filled in either by template switch mechanisms (D) or translesion synthesis (E). Template
switch mechanisms (D) will lead to hemicatenated molecules that were shown to require the activity of Sgs1/Top3 and
Ubc9/Mms21-mediated sumoylation in order to be resolved. Regression of the stalled replication fork, when unprotected by
the replication checkpoint, can generate four-way junctions called reversed forks (F) that are recombinogenic and can be
further processed by Exo1 leading to gaps and hemireplicated molecules. The pathological structures derived from reversed
forks that can be generated during replication are shown in shaded rectangles.
firing genome-wide . These results were recently extended
in a number of ways to reveal differences between mec1 and
rad53 as well as between checkpoint mutants and wild-type
cells . A large fraction (88%) of the early-firing origins acti-
vated in wild-type cells are also activated in the replication
checkpoint mutants mec1 and rad53, and in agreement with
previous results, both of these replication checkpoint mutants
Fig. 3 – The role of checkpoints in preventing DSB accumulation during replication and in promoting DSB repair.
dna repair 6 ( 2 0 0 7 ) 994–1003
also activate late-firing origins. However, intriguingly, the two
mutants, rad53 and mec1 cells, show an overlap of only 46%
for both early- and late-firing origins, and rad53 cells activate
many more late-firing origins than mec1 cells. Furthermore,
these two replication checkpoint mutants display only 41%
bling fragile sites . Collectively, these results suggest that
Mec1 and Rad53 control origin activation and fork progression
and stability through both common and independent mecha-
nisms (see Fig. 1). The fact that a large fraction of origins are
exclusively regulated by Mec1 or Rad53 could be explained by
the fact that, in addition to Rad53, Mec1 has other targets such
as Chk1 and Pds1 (see ), and Rad53 has functions inde-
pendent of Mec1 in replication, such as those mediated by
its ability to regulate Cdc7/Dbf4 kinase activity and to control
histone protein levels and chromatin structure [39,40].
Replication fork restart and repair
Previous studies have shown that replication checkpoint func-
tion is required not only to stabilize the forks, but also to
promote resumption of replication after transient fork paus-
ing. This assumption was first based on the observation that
replication checkpoints mutants are unable to resume replica-
tion after transient exposure to DNA damaging agents [23,26]
and additional physical evidence was recently obtained that
brings support to this model . EM and two-dimensional
(2D) gel electrophoresis of replicating DNA from UV-irradiated
budding yeast cells revealed that rad53 cells contained a
high fraction of large gapped forks . Indeed, the idea that
been previously proposed by other studies coming from both
budding and fission yeast [41,42]. The questions as to what are
repair and replication through damaged DNA still await eluci-
dation but we will try to describe below the mechanisms that
contribute to repair of damaged DNA during replication and
the function of several proteins whose enzymatic activities
are thought to play an important role in promoting replication
replication forks in bacteria, but less so in eukaryotic cells in
which converging forks from adjacent origins may account
for replication of those regions in which the original replica-
plays a major role in promoting replication of telomeric and
subtelomeric regions, and in processing and in the repair of
double-strand breaks (DSBs) generated during replication.
In certain cases, collision of the fork with the DNA
lesion might result in collapse of the fork and DSB for-
mation. DSB repair by gene conversion or break induced
replication is thought to be a late S or G2 event, and is
under the control of cyclin dependent kinases, Cdks .
Furthermore, a role of damage checkpoints in promoting
recombination repair of DSBs has been proposed  (Fig. 3).
DSB repair involves the assembly of a replication fork by
recombination-mediated processes and requires the function
of many proteins involved in replication elongation . It
is not yet clear which polymerases are implicated in the
DNA synthesis step of homologous recombination (HR) medi-
ated DSB repair, but recent results suggest that translesion
synthesis (TLS) polymerases might be involved. The TLS poly-
merases in yeast involve the activity of Pol?, encoded by
RAD30, and Pol?, whose two subunits are encoded by REV3
and REV7, together with Rev1. Pol? from chicken or human
cells extends 3?strands formed after strand-exchange during
homologous recombination [46,47]. In budding yeast Mec1-
dependent phosphorylation was shown to promote Pol?-Revl
association with DSBs , and a physical interaction between
the damage checkpoint and pol?, has been reported . Thus,
an interesting possibility is that checkpoints might promote
the function of TLS polymerases in HR of DSB repair.
While replication checkpoints
postreplicative DSB repair, they also appear to restrict
recombination from occurring during replication at stalled
forks (Fig. 3). Accordingly, Rad52 recombination foci are not
seen in budding yeast treated with HU, unless the replication
checkpoint is not functional , a situation that gives rise
to collapsed forks, DSBs, and accumulation of recombino-
genic DNA structures [24,25] (Fig. 3). The direct effectors
and mechanisms of this regulation are largely unknown,
although a number of proteins implicated in recombinational
repair, including Srs2, Mus81, Slx4, Esc4, Mre11, Xrs2, Rad51,
Rad55, Rad57, and RPA, were shown to be phosphorylated
in a checkpoint-dependent manner [51–59]. More mecha-
nistic insights are coming from fission yeast, where upon
HU treatment the replication checkpoint Cds1 mediates the
phosphorylation of the recombination proteins Mus81 and
Rad60 and this modification is associated with delocalization
of Rad60 from the nucleus and a reduction in the chromatin
binding ability of the Mus81-Eme1 endonuclease complex
[60,61]. In budding yeast, of particular interest is Srs2, a
3?-to-5?DNA helicase that downregulates HR by dismantling
Rad51 filaments [62,63]. Srs2 was shown to be recruited to
stalled replication forks by sumoylated PCNA [64,65], and
it is phosphorylated in response to replication stress .
However, the role of the checkpoint-mediated Srs2 phos-
phorylation in counteracting recombination events during
replication has not yet been explored.
In addition to recombination, replication forks encounter-
ing damage-induced replication blocks have several choices
of restart or resumption of DNA replication depending on the
nature of the lesion and the DNA structures formed at the
fork as the result of the collision with the lesion. Replica-
and damage-bypass mechanisms, or restart downstream the
lesion leaving gaps that could be filled in by TLS polymerases,
or by template switch mechanisms that use the newly syn-
thesized DNA strand as a template for DNA synthesis across
the gap (Fig. 2). These processes are also known as damage
tolerance processes or the postreplication repair (PRR) path-
way, and in budding yeast damage tolerance depends on the
The high sensitivity of rad6 and rad18 mutants to various
DNA damaging agents attests to their important contribu-
tion in promoting repair or replication fork restart following
intra-S damage. Rad6 and Rad18 interact with each other and
dna repair 6 ( 2 0 0 7 ) 994–1003
this complex is likely recruited to sites of DNA damage by
the single-stranded DNA-binding activity of Rad18 . Rad6
and Rad18 have ubiquitin conjugating and ligating activities,
respectively, and their role in DNA damage tolerance mecha-
nisms is perhaps mediated by ubiquitination of proteins with
functions in DNA replication, either to promote their degra-
dation or as a signaling mechanism to their function [66,67].
Rad6 is not the only ubiquitin conjugating activity of the dam-
age tolerance mechanism. The PRR genes that are involved in
the error-free mechanisms of damage tolerance include RAD5,
MMS2, and POL30 (encoding PCNA). RAD5 encodes a protein
with a putative helicase domain and a RING finger domain
characteristic of ubiquitin ligases, and mediates the interac-
tion between Ubc13-Mms2 and Rad6-Rad18 complexes .
Mms2, encodes a variant ubiquitin-conjugating enzyme that,
in conjunction with the Ubc13 protein, forms a complex capa-
ble of assembling polyubiquitin chains linked through the K63
residue of ubiquitin . The polyubiquitin chains via K63 are
thought to play a specific signaling role in DNA damage tol-
erance as the UbK63R mutation results in repair defects that
are epistatic with rad6 and yet show no obvious impairment
in protein degradation .
One important target of Rad6 and Rad18 is PCNA (Pol30),
a protein with multiple functions in DNA replication and
repair. PCNA ubiquitination was shown to influence both
the translesion synthesis and the error-free branch of the
damage tolerance mechanism [70,71]. Rad6/Rad18 mediated
monoubiquitination of PCNA promotes translesion synthe-
sis and damage-induced mutagenesis by TLS polymerases
. In contrast, Rad5/Mms2/Ubc13-mediated polyubiquiti-
nation of PCNA promotes the error-free damage avoidance
pathway [64,65,70] and replication completion in response
to replication perturbations induced by mutations in repli-
cation factors . Several models have been proposed to
account for the various genetic interactions between mem-
bers of the RAD6/RAD18 epistasis group [68,73]. In addition
to the traditional members of PRR, whose damage sensitivity
and epistatic phenotypes allows their distribution in certain
branches of PRR, different proteins with functions in replica-
tion and the DNA damage checkpoint have been proposed
to promote either TLS or the error-free damage avoidance
pathway of PRR. The current view is that there are two main
pathways of translesion synthesis, one mediated by Pol? and
the other by Pol?, and one error-free PRR pathway of damage
avoidance defined by Rad5, Mms2, Ubc13 and Pol30 multiu-
biquitination. Clearly, however, the situation is much more
complex, as both Pol30 and Rad5 are implicated also in TLS
that likely knocks-out a function of Pol30 specific to error free
processes of damage tolerance, suggested that PCNA func-
tions in a branch separate from RAD5 .
Originally it was thought that TLS occurs via a DNA poly-
merase switch at the time that the replication fork encounters
the lesion, whereby the replicative polymerase is substituted
by another one capable of bypass, allowing the replication
fork to progress through the damage. While this is still the
plausible model, recent studies suggest that many damage
bypass events might occur behind the replicating fork (Fig. 2).
In support of this view, EM analysis of replication intermedi-
ates from UV-irradiated excision deficient cells showed that
internal gaps can be detected on both sides of the fork, and
that cells deficient in translesion synthesis accumulate more
internal gaps than their wild-type counterparts. This suggests
that a large proportion of the TLS events contribute to restor-
ing the integrity of replicated duplexes postreplicatively .
Little is known about how polymerases swap at the fork and
TLS-mediated gap filling events behind the fork are regulated,
but several studies in fission and budding yeast suggest an
involvement of the damage checkpoint pathway and of the
Cdc7-Dbf4 kinase in promoting damage-induced mutagenesis
in response to replication problems [41,42,49,75]. It will be of
interest to understand whether besides the physical interac-
tion already reported between Pol? and the 9-1-1 DNA damage
ity in TLS and whether Cdc7 targets for phosphorylation TLS
polymerases or other replication factors that might ultimately
influence the ability of cells to promote DNA damage bypass.
ance pathway are even less understood. While the genetic
requirements for template switch have been largely worked
the “switch” to the newly synthesized DNA strand remain
largely unknown. The involvement of Rad6/Rad18 and of
Rad5/Mms2 in gap filling was known from studies measuring
the molecular size of DNA synthesized after UV irradia-
tion in excision repair deficient strains, in different mutant
backgrounds [76,77]. In these experiments, the conversion of
nascent DNA from small to large size is very much delayed in
these activities in gap-filling repair. In addition, rad52 strains
also show a delay in this conversion, albeit smaller than that
seen in rad6 strains, suggesting a role also for recombination
proteins in gap-filling . Whether this role is carried out in
cooperation with Rad6 is not known. A recent study measured
the contribution of template strand switching events medi-
mediated by TLS polymerases, in assisting the completion
of replication of plasmids having single thymine–thymine
pyrimidine (6-4) pyrimidinone photoadducts in each strand in
rad1 excision defective strains . The results indicate that
more than 90% of the successful events involve the template
switch mechanism, with 60–70% of these events depending on
the RAD18/RAD5, and the remaining events on RAD52 .
Thus, these results suggest that Rad18/Rad5-mediated
template switch plays a prominent role in the restart of the
stalled replication forks, and this contribution is also attested
by the high increase in GCR rates of rad18/rad5 mutants .
The GCR increase in rad5/rad18 mutants is dependent on HR
activities, suggesting that, in the absence of Rad18/Rad5, the
ssDNA gaps can be filled in by recombination mechanisms.
Genetic evidence brings support for this model and suggests
that Srs2 together with sumoylated PCNA might provide the
activity that promotes Rad18/Rad5-mediated template switch
and inhibits certain recombination events [64,65,80,81]. This
presumptive role of Srs2 in stabilizing the replication forks
stalled by DNA damage is somehow reminiscent of that of the
replication checkpoint, although 2D gel analysis of replica-
tion intermediates showed no accumulation of pathological
or recombinogenic structures at damaged forks in srs2 or
mutants affecting PCNA sumoylation [82,83]. Since Srs2 is a
dna repair 6 ( 2 0 0 7 ) 994–1003
checkpoint target , it will be of interest to address whether
Srs2 checkpoint mediated phosphorylation plays any role in
regulating the pathway that promotes restart of stalled repli-
In addition to Srs2, RecQ helicases have also been sug-
gested to regulate recombination events occurring during
replication and to promote efficient resumption of replication
forks. Furthermore, Sgs1 was shown to act as a negative regu-
lator of spontaneous  as well as of DSB-induced crossovers
. In budding yeast, the Sgs1-Top3 complex was proposed
junctions during template switch or during replication ter-
mination when replication forks converge [82,86,87] (Fig. 2).
This model is consistent with in vitro studies that show
that the Sgs1 orthologue in humans, BLM, could merge a
double Holliday junction (dHJ) and create an intermediate
that can be subsequently resolved by dissolution through
the specific single-strand decatenating activity of Top3 .
Recently, it was shown that the ability of Sgs1 to resolve
ated mechanism, is regulated by Ubc9 and Mms21 dependent
sumoylation, but is independent of PCNA sumoylation .
Since Sgs1 is itself sumoylated, but in an Mms21-independent
manner, these results suggest that there must be other tar-
gets whose sumoylation is crucial for Sgs1 ability to resolve
hemicatenanes, or which, upon their sumoylation, need to
cooperate with Sgs1 in this process . This role of Sgs1
and sumoylation in preventing the accumulation of cruci-
form structures during replication and possibly in promoting
the efficient resolution of the hemicatenane-like structures
formed during replication termination [83,89], is likely to have
important consequences for genome stability. It is expected
that such intermediates will need to undergo a recombino-
genic strand break during anaphase to separate the connected
molecules and permit chromosome segregation. As this role is
different from that of replication checkpoints, it is envisioned
that sumoylation is a novel regulatory pathway that, together
with the replication checkpoint, functions to promote genome
stability during replication.
7. Concluding remarks
Increasing evidence suggests that many of the chromosomal
abnormalities present in cancer cells are caused by faulty
DNA replication. Although tightly regulated, DNA replication
is still a perilous event as various types of exogenous dam-
age and endogenous events interfere with the progression,
stability, and restart of replication forks. In addition, the cells
need to coordinate the repair of different types of lesions with
progression of replication, and all of these events take place
in the chromatin context, a highly condensed structure that
needs additional levels of regulation to allow DNA accessibil-
ity and its subsequent replication and repair. A coordinated
interplay between the replisome and factors involved in reor-
ganizing nucleosomes and promoting establishment of sister
chromatid cohesion has already been shown to be required for
pinpointed a role for chromatin modifications in checkpoint
activation or in the amplification of the checkpoint signal, as
well as a role for checkpoints in maintaining chromatin struc-
ture and in regulating chromatin assembly to promote fork
stabilization or restart in response to DNA lesions or replica-
tion blocks. New ideas are emerging about the existence of
topological boundaries that might facilitate replication, cate-
nation as well as repair of DNA. The ongoing research on DNA
replication and its regulation will need to take more in consid-
eration all of these aspects, and clearly the interplay of factors
at stalled replication forks will likely turn to be much more
complex than presently described.
The authors apologize for the many interesting articles that
they were not able to discuss or acknowledge due to space
limitations. This work was supported by grants form the
Associazione Italiana per la Ricerca sul Cancro, European
Community, Telethon-Italy, Italian Ministry of Education, and
D. Branzei. D. Branzei is supported by the Buzzati-Traverso
 M. Raveendranathan, S. Chattopadhyay, Y.T. Bolon, J.
Haworth, D.J. Clarke, A.K. Bielinsky, Genome-wide
replication profiles of S-phase checkpoint mutants reveal
fragile sites in yeast, EMBO J. 25 (2006) 3627–3639.
 M.K. Raghuraman, E.A. Winzeler, D. Collingwood, S. Hunt, L.
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