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Biol. Cell (2009) 101, 617627 (Printed in Great Britain) doi:10.

The S-phase checkpoint: targeting
the replication fork
M onica Segurado and Jos e Antonio Tercero
Centro de Biologa Molecular Severo Ochoa (CSIC/UAM), Cantoblanco, 28049 Madrid, Spain
The S-phase checkpoint is a surveillance mechanism, mediated by the protein kinases Mec1 and Rad53 in the
budding yeast Saccharomyces cerevisiae (ATR and Chk2 in human cells, respectively) that responds to DNA
damage and replication perturbations by co-ordinating a global cellular response necessary to maintain genome
integrity. A key aspect of this response is the stabilization of DNA replication forks, which is critical for cell survival.
A defective checkpoint causes irreversible replication-fork collapse and leads to genomic instability, a hallmark
of cancer cells. Although the precise mechanisms by which Mec1/Rad53 maintain functional replication forks are
currently unclear, our knowledge about this checkpoint function has signicantly increased during the last years.
Focusing mainly on the advances obtained in S. cerevisiae, the present review will summarize our understanding of
how the S-phase checkpoint preserves the integrity of DNA replication forks and discuss the most recent ndings
on this topic.
Precise and complete DNA replication in every cell
cycle and repair of DNA lesions are critical for
the maintenance of genetic stability (Aguilera and
Gomez-Gonzalez, 2008; Branzei and Foiani, 2008).
Failures in these processes reduce cell survival and
lead to cancer and other diseases in higher metazoans
(Hoeijmakers, 2001; Friedberg, 2003). Problems
arising during chromosome replication are inherent
to the complexity of the process and a major
source of genomic instability. They are aggravated
and frequently caused by exogenous environmental
agents and reactive metabolic products that con-
stantly and inevitably damage the DNA, raising
potential obstacles to the progression of replication
forks. In addition, particular regions in the genome
constitute a challenge to replication-fork movement
and are associated to a high incidence of chromosomal
rearrangements. In all these cases, replication forks
must maintain their integrity in order to be able to
To whom correspondence should be addressed (jatercero@cbm.uam.es).
Key words: budding yeast (Saccharomyces cerevisiae), chromosome
replication, checkpoint, genome stability, replication-fork integrity.
Abbreviations used: ChIP, chromatin immunoprecipitation; HU, hydroxyurea;
MCM, minichromosome maintenance complex; MMS, methyl
methanesulfonate; ORC, origin recognition complex; Pol, DNA polymerase ;
Pol, DNA polymerase ; RFC, replication factor C; RPA, replication protein A;
ssDNA, single-stranded DNA.
nish chromosome replication accurately when con-
ditions that halt them are eliminated (Paulsen and
Cimprich, 2007; Tourriere and Pasero, 2007; Friedel
et al., 2009).
To cope with situations where the genome is at
risk owing to DNA damage or replicative stress,
eukaryotic cells activate surveillance mechanisms,
called checkpoints, which detect the problems and
co-ordinate a global response to maintain genome
integrity (Zhou and Elledge, 2000; Harrison and
Haber, 2006). When replication forks stall because of
DNAlesions or replication perturbations, the S-phase
or replication checkpoint pathway (Paulovich and
Hartwell, 1995; Boddy and Russell, 2001; Nyberg
et al., 2002; Osborn et al., 2002) becomes a main
actor to maintain functional replication forks, mak-
ing successful chromosome replication possible. In
the present review we will discuss the crucial role
of the S-phase checkpoint in preserving replication-
fork integrity, with special focus on the work done in
the eukaryotic model Saccharomyces cerevisiae, where
many of the advances on this topic have been
The S-phase checkpoint response
The S-phase checkpoint is an evolutionarily conserved
intranuclear signal-transduction pathway (Paulovich
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M. Segurado and J.A. Tercero
and Hartwell, 1995; Zhou and Elledge, 2000) the
central players of which, in the budding yeast
S. cerevisiae, are two kinases, namely Mec1 and Rad53.
They are the homologues of Rad3 and Cds1 respect-
ively in the ssion yeast Schizosaccharomyces pombe, or
ATR and Chk2 in mammalian cells, which are de-
cient in many cancer cells. An additional checkpoint
kinase, Chk1, has an apparently minor function in
budding yeast during S-phase, unlike the metazoan
Chk1, which could be considered as the analogue
of Rad53. However, a new role for this protein in
S. cerevisiae has been recently uncovered and will be
discussed below.
Under conditions that threaten DNA replication,
such as DNA damage or nucleotide depletion, the
S-phase checkpoint pathway gets activated (reviewed
in Branzei and Foiani, 2007; Paulsen and Cimprich,
2007; Tourriere and Pasero, 2007). The activa-
tion requires the establishment of DNA replica-
tion forks (Lupardus et al., 2002; Stokes et al.,
2002; Tercero et al., 2003) and the generation of
ssDNA (single-stranded DNA). The accumulation
of ssDNA regions at stalled forks occurs probably be-
cause the MCM(minichromosome maintenance com-
plex) helicase continues DNA unwinding, although
uncoupled from DNA synthesis (Sogo et al., 2002;
Byun et al., 2005; Nedelcheva et al., 2005). The
ssDNA binds RPA (replication protein A), which
triggers the activation of the checkpoint response
(You et al., 2002; Zou et al., 2003). This process is
initiated by the recruitment of the Mec1/ATR sensor
to RPA-coated ssDNA at stalled forks by its regu-
latory subunit, Ddc2 (ATRIP in human cells). Mec1
then phosphorylates Mrc1 (the homologue of human
Claspin), a mediator that transduces the signal from
Mec1 to the effector kinase Rad53 (Alcasabas et al.,
2001), which becomes phosphorylated and activated.
The checkpoint response during S-phase is depicted
schematically in Figure 1.
The S-phase checkpoint response co-ordinates
DNAreplication, DNArepair and cell-cycle progres-
sion and regulates processes such as ring of replica-
tion origins (Santocanale and Difey, 1998; Shirahige
et al., 1998; Santocanale et al., 1999), stabilization of
DNA replication forks in response to DNA damage
or replicative stress (Lopes et al., 2001; Tercero and
Difey, 2001), resumption of stalled DNA replica-
tion forks (Desany et al., 1998; Szyjka et al., 2008),
transcriptional induction of DNA damage response
genes (Allen et al., 1994), choice of the repair path-
way (Kai et al., 2007) and inhibition of mitosis until
replication is completed (Allen et al., 1994).
The S-phase checkpoint is required for
cellular viability in DNA damage or
replicative-stress conditions
Genome integrity is profoundly affected when the
checkpoint is defective. For example, work in
S. cerevisiae showed that spontaneous and induced
chromosomal rearrangements are signicantly in-
creased in cells mutated for checkpoint components
(Myung et al., 2001; Kolodner et al., 2002; Myung
and Kolodner, 2002). In vertebrates, loss of ATR
causes chromosome breaks, cell-cycle arrest and em-
bryonic lethality, and the absence of Chk1 originates
DNA double-strand breaks and non-viable embryos
(Brown and Baltimore, 2000, 2003; Liu et al., 2000;
Takai et al., 2000).
In S. cerevisiae, mec1 and rad53 checkpoint mutants
are extremely sensitive to agents that cause replicat-
ive stress, such as HU (hydroxyurea), that depletes
the dNTP pool by inhibiting the ribonucleotide re-
ductase. These mutants are also highly sensitive to
different forms of DNAdamage, such as DNAalkyla-
tion originated by MMS (methyl methanesulfonate),
ionizing radiation or UVlight. As cell survival is very
compromised under those conditions in the absence of
a functional S-phase checkpoint, a fundamental ques-
tion to address was which of the process(es) regulated
by the checkpoint is/are crucial for the maintenance
of cellular viability.
MEC1 and RAD53 are essential genes, but the
lethality can be avoided by increasing the levels of
dNTP (Desany et al., 1998; Zhao et al., 1998). How-
ever, the checkpoint functions cannot be rescued by
simply regulating dNTP levels (Desany et al., 1998).
In mec1 and rad53 checkpoint mutants, mitosis can-
not be prevented when cells are exposed to HU or
MMS, despite incomplete DNA replication, and it
could be the major cause of cell death. However,
restraining mitosis using microtubule inhibitors is
not enough to rescue the lethality of mec1 or rad53
mutant cells treated with HU or MMS (Desany et al.,
1998; Tercero and Difey, 2001). Another import-
ant function of the checkpoint is the induction of
genes involved in the DNA damage response (Allen
et al., 1994), but it does not seem essential for cell
The Authors Journal compilation
2009 Portland Press Ltd
S-phase checkpoint and replication-fork integrity Review
Figure 1 A schematic illustration of the S-phase checkpoint response
When replication forks hit DNA lesions or stall because of dNTP deprivation, the helicase and the polymerases may uncouple,
exposing regions of ssDNAthat cause the activation of the checkpoint response. RPAbinds to ssDNAand triggers the recruitment
of S. cerevisiae Mec1 to the stalled fork by its regulatory subunit Ddc2. Mec1 phosphorylates the mediator Mrc1 and the signal is
transduced to the downstreameffector kinase Rad53, which is phosphorylated and activated. Rad53 maintains stable, functional
DNA replication forks, inhibits ring of late origins, activates gene expression and prevents entry into mitosis and unscheduled
recombination. Abbreviations: HR, homologous recombination; PCNA, proliferating cell nuclear antigen.
viability either, as the inhibition of protein synthesis
by cycloheximide during S-phase does not render cells
sensitive to HU, nor does it impede fork resumption
after HU block (Tercero et al., 2003).
The Mec1/Rad53 checkpoint regulates at least two
aspects of DNA replication: replication fork stabil-
ization and late origin ring. Using a hypomorphic
mec1 mutant, mec1-100 (Paciotti et al., 2001), which
has a delayed and reduced level of Rad53 activation
in response to HU or MMS, it was shown that fork
stabilization and late origin ring are genetically sep-
arable functions (Tercero et al., 2003). Like the mec1
mutant, mec1-100 mutant cells cannot block the r-
ing of late replication origins in the presence of HU
or MMS. However, unlike the mec1 mutant, mec1-
100 cells are not hypersensitive to MMS or HU and
can stabilize DNA replication forks in the presence
of DNA damage. Thus, although the control of late
origin ring extends S-phase, it has only a minor role
in preserving cellular viability. Therefore, the stabil-
ization of DNA replication forks appears as the most
critical checkpoint function to ensure cell survival
when the DNA is damaged. How the replication
checkpoint prevents fork collapse has been extens-
ively investigated, but, despite considerable progress,
the precise mechanisms by which the checkpoint ex-
ecutes this critical function remain elusive. The emer-
ging picture from recent works suggests that it is a
much more complex task than rst thought.
Stabilization of DNA replication forks by
the S-phase checkpoint
Parallel studies in budding yeast proposed a role for
the S-phase checkpoint proteins in preserving the in-
tegrity of DNA replication forks in response to DNA
damage by MMS or HU blocks (Lopes et al., 2001;
Tercero and Difey, 2001). Direct evidence for a func-
tion of Mec1 and Rad53 in fork stability came from
a functional assay based on dense isotope substitu-
tion (Tercero and Difey, 2001). The progression of
DNA replication forks was monitored along a chro-
mosomal replicon of S. cerevisiae in the presence of
MMS, in wild-type cells and checkpoint mutants.
It was found that MMS diminishes drastically the
replication fork rate, in both wild-type and mutant
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M. Segurado and J.A. Tercero
cells, which is probably the consequence of a physical
impediment of replication-fork progression either by
methylated DNAbases or some intermediates formed
during the processing of damaged DNA (Vazquez
et al., 2008). Unlike wild-type cells, whose replica-
tion forks move slowly through alkylated DNA, but
continue to progress and nish replication, a high
proportion of DNA remains unreplicated in check-
point mutants under the same conditions. These ex-
periments showed that DNA replication forks ter-
minate irreversibly at a high rate in mec1 and rad53
mutants, very likely accounting for the elevated leth-
ality of checkpoint mutant cells after exposure to
Lopes et al. (2001) also demonstrated that the
S-phase checkpoint was required to stabilize DNA
replication forks, in this case under replicative
stress originated by the decrease in dNTP levels
caused by HU. Using a two-dimensional gel-
electrophoretic approach, they clearly showed that,
unlike checkpoint-procient cells, treatment of rad53
cells with HU resulted in a reduced percentage of
DNA replication bubbles, together with an accu-
mulation of unusual DNA structures at forks, visu-
alized as small Y molecules and cone-shaped sig-
nals. These structures were interpreted as broken
bubbles resulting from the collapse of stalled rep-
lication forks and were associated with the incapacity
of these mutant cells to complete chromosome replic-
ation (Lopes et al., 2001). This nding was in agree-
ment with those of a previous study where pulse-eld
gel-electrophoretic experiments indicated that rad53
mutants failed to complete DNA replication after
HU arrest (Desany et al., 1998), suggesting a role
for the checkpoint in fork recovery after replicative
stress. The failure in completing DNA replication in
rad53 mutant cells after the HU block is irreversible,
as abnormal structures persist if the HU is removed,
and adding new Rad53 does not restore the replica-
tion defects (Lopes et al., 2001; Tercero et al., 2003;
Rouse, 2004). Therefore the function of Mec1/Rad53
at replication forks is not only to resume DNA syn-
thesis after fork stalling. Instead, these checkpoint
proteins are required to avoid catastrophic irrevers-
ible events that cause the collapse of DNAreplication
forks and correlate with cell death.
A role for the checkpoint in preventing replica-
tion fork collapse has also been reported in other
organisms. Thus, in the ssion yeast S. pombe, HU
treatment of Cds1-defective cells causes irreversible
accumulation of structures resembling those found in
budding yeast (Noguchi et al., 2003; 2004; Meister
et al., 2005; Froget et al., 2008). Several studies
have also suggested that the S-phase checkpoint is
required to maintain stable functional forks in ver-
tebrates (Zachos et al., 2003, 2005; Petermann et al.,
2006; Trenz et al., 2006). Therefore the requirement
of the S-phase checkpoint for the integrity of DNA
replication forks seems to be conserved in eukaryotes.
Structural insight into checkpoint function in pro-
tecting the stability of DNA replication forks came
fromelectron-microscopic analysis of rad53-defective
cells. These experiments showed that stalled rep-
lication bubbles accumulate long ssDNA regions
indicative of hemireplicated or gapped DNA mo-
lecules (Sogo et al., 2002). These ssDNA regions
probably arose by resection of the nascent chains by
the Exo1 nuclease in the absence of the replisome
(Cotta-Ramusino et al., 2005). Other studies per-
formed a genome-wide analysis in the presence of
HUand also observed ssDNAregions remaining over
time at most known replication origins in rad53 cells,
suggesting fork stalling and breakdown (Feng et al.,
2006). Replication intermediates formed in rad53
mutants in response to replication blocks also accu-
mulated reversed forks (Sogo et al., 2002), resulting
fromthe conversion of sister chromatid junctions into
four-branched structures (Lopes et al., 2003). It was
proposed that the nuclease Exo1 counteracts the accu-
mulation of these reversed replication forks by gener-
ating ssDNA intermediates (Cotta-Ramusino et al.,
In addition to the stabilization of replication forks
in situations of DNA damage or dNTP depriva-
tion, the S-phase checkpoint plays an important role
in maintaining fork integrity through fragile sites
or slow replicating zones of the genome (Casper
et al., 2002; Cha and Kleckner, 2002; Lahiri et al.,
2004; Admire et al., 2006; Arlt et al., 2006;
Raveendranathan et al., 2006). Replication forks
probably stall at these regions with elevated fre-
quency and are prone to breakage. Interestingly, how-
ever, unlike replication forks passing through these
fragile or slow zones, or those stalled by HU or DNA
damage, the stable fork arrest at programmed paused
sites such as replication fork barriers is independ-
ent of the S-phase checkpoint (Calzada et al., 2005;
Lambert et al., 2005; Tourriere et al., 2005).
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S-phase checkpoint and replication-fork integrity Review
Possible targets of the S-phase
checkpoint at DNA replication forks
Efforts from many laboratories over the last years
have tried to identify the target(s) of the checkpoint
at DNAreplication forks. Studies in different systems
indicate that the S-phase checkpoint regulates several
processes that would co-operate to preserve the integ-
rity of DNA replication forks, but the whole view is
not yet complete.
Obvious checkpoint target candidates were the
components of the replication machinery, and sev-
eral studies suggest that the checkpoint stabilizes the
association of the replisome with the fork. However,
it is not completely clear whether those results re-
ect that the replisome is targeted directly by the
Mec1/Rad53 kinases or are a consequence of an in-
direct effect. ChIP (chromatin immunoprecipitation)
experiments in budding yeast showed that, in HU-
arrested cells, the chromosome association of Pol
and Pol (polymerases and ) at replication forks
is dependent on an active S-phase checkpoint (Cobb
et al., 2003, 2005; Lucca et al., 2004). In addition to
the proposed role for Pol in checkpoint activation
(Michael et al., 2000; Byun et al., 2005), checkpoint-
dependent phosphorylation of this polymerase might
be also important to stabilize the replisome (Pellicioli
et al., 1999; Lemoine et al., 2005). Another replica-
tion protein involved in the response to DNAdamage
is RPA (Zou and Elledge, 2003; Binz et al., 2004),
the phosphorylation of which is dependent on Mec1
(Brush et al., 1996), although the relevance of this
modication for fork stabilization is currently un-
known. The Mrc1 transducer is also phosphorylated
in a checkpoint-dependent manner (Alcasabas et al.,
2001). This protein is associated with the replica-
tion fork under normal replication conditions, and,
when cells are treated with HU, forms a pausing com-
plex with Tof1 (Swi1 in S. pombe) that is essential to
avoid the uncoupling of the replication machinery
components (including the polymerases, MCMs and
Cdc45) from the place of DNA replication (Katou
et al., 2003). The MCMhelicase complex has always
been an attractive checkpoint target candidate, since
disassembly of Mcm2Mcm7 (MCM components 2
7) from stalled replication forks causes an irrevers-
ible HU arrest accompanied by loss of cell viability
(Labib et al., 2000). This is not the case for other
fork progression complex components associated with
MCMs, such as Cdc45 (Tercero et al., 2000) and it
might not be the case for DNA polymerases either,
as Pol can be reloaded in Xenopus (South African
clawed frog) in the presence of ATR/ATM (Trenz
et al., 2006). MCMs undergo ATR/ATM-dependent
phosphorylation in metazoans (Ishimi et al., 2003;
Cortez et al., 2004; Yoo et al., 2004; Shi et al.,
2007; Trenz et al., 2008), suggesting themas possible
checkpoint targets. Arecent report also indicates that,
in S. pombe, Mcm4 could be a target of the Cds1 kinase
(Bailis et al., 2008). Moreover, ChIP experiments in
budding yeast suggested that loss of Rad53 leads to
MCM dissociation from the fork (Cobb et al., 2005).
Recently, a phosphoproteomic analysis in human cells
searching for ATR and ATM targets in response to
DNA damage has also identied Mcm2Mcm7 pro-
teins, together with other essential replication
proteins, such as subunits of the ORC (origin recog-
nition complex), RFC (replication factor C) compo-
nents and DNA polymerases (Matsuoka et al., 2007).
The stabilization of stalled replication forks by the
S-phase checkpoint is also thought to happen by pre-
venting unscheduled recombination (Meister et al.,
2005; reviewed in Lambert et al., 2007). Consist-
ent with this idea, Rad52 foci are not formed in S.
cerevisiae cells arrested with HU. They only appear
when the checkpoint is defective (Lisby et al., 2004)
and forks collapse, forming aberrant DNA structures
(Lopes et al., 2001; Sogo et al., 2002), including
double-strand breaks and recombinogenic interme-
diates. The mechanisms by which the S-phase check-
point restrains recombination at forks are not well
known, but several proteins related to this process ap-
pear to undergo checkpoint-dependent phosphoryla-
tion (Lambert et al., 2007) and are potential targets.
In S. pombe, the recombination proteins Mus81 and
Rad60 are regulated negatively by the checkpoint.
In response to DNA damage, the Mus81 nuclease
is phosphorylated by Cds1, causing its dissociation
from chromatin (Kai et al., 2005). This regulation
may prevent Mus81 from cleaving DNA intermedi-
ates originated at stalled or reverse forks, therefore
avoiding recombination events. Rad60 is also modi-
ed by the checkpoint when forks stall, which causes
the nuclear delocalization of the protein (Boddy et al.,
Increasing evidence indicates that the check-
point targets chromatin remodellers and histone-
regulating enzymes, which has an important impact
on the maintenance of functional DNA replication
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M. Segurado and J.A. Tercero
forks. Recently, new insights have been gained into
the mechanism by which the checkpoint regulates
the ATP-dependent chromatin remodelling complex
Ino80. The Ies4 subunit of the Ino80 complex is phos-
phorylated by Mec1/Tel1 in the presence of DNA
damage, and the function of phosphorylated Ies4
seems to be redundant with the checkpoint protein
Tof1 (Morrison et al., 2007). Ino80 regulates efcient
fork progression under low levels of replication stress
and is enriched at stalled replication forks, where
it contributes to the stabilization of the replisome
and facilitates replication fork recovery, probably
mobilizing histones ahead of the fork (Papamichos-
Chronakis and Peterson, 2008; Shimada et al., 2008).
The Hst3 deacetylase has been also implicated in the
S-phase checkpoint response and the maintenance
of genome integrity. Thus Hst3 undergoes Mec1-
dependent phosphorylation in response to genotoxic
stress, which causes an accumulation of the acetylated
histone H3-K56 at chromatin with damaged DNA.
The H3-K56 accumulation could contribute to the
recruitment of proteins required to signal DNA le-
sions or stabilize stalled replication forks (Thaminy
et al., 2007). Interestingly, histone H3 is acetylated
at Lys
by the acetyltransferase Rtt109, which is
necessary for replisome integrity (Han et al., 2007).
Rtt109, together with the cullin Rtt101, is also re-
quired for the recruitment of Rtt107/Esc4 to chro-
matin, at or near damaged or stalled replication forks
(Roberts et al., 2008). However, in this case, Lys
acetylation is not necessary, suggesting the existence
of another unidentied Rtt109 target involved in the
recruitment of Rtt107. Rtt107/Esc4 is a Mec1 tar-
get (Rouse, 2004; Chin et al., 2006; Roberts et al.,
2006), and its function at replication forks could
be the recruitment of DNA repair proteins to pro-
mote the restart of DNAsynthesis after DNAdamage
or replicative stress.
An Exo1-dependent Rad53 pathway for
the stabilization of DNA replication forks
in response to DNA damage
Recent results have situated the nuclease Exo1 at
the centre of the checkpoint response at replication
forks (depicted schematically in Figure 2), as fork col-
lapse in rad53-null mutants exposed to DNA dam-
aging agents is dependent on Exo1 (Segurado and
Difey, 2008). Thus elimination of this protein sup-
Figure 2 Rad53, Mec1 and Chk1 kinases stabilize DNA
replication forks
Rad53 maintains fork integrity through inhibition of Exo1 nuc-
lease activity as well as by Exo1-independent mechanisms.
Mec1 activates Rad53, but also has Rad53-independent func-
tions in the stabilization of replication forks. Chk1 is also in-
volved in fork stabilization in the absence of Rad53. Moreover,
these two protein kinases contribute to prevent mitosis.
Abbreviation: HR, homologous recombination.
presses replication-fork catastrophe in the presence of
MMS and is sufcient for rad53 mutant cells to com-
plete DNA replication and restore viability. It was
shown previously that fork collapse in cells blocked
with HU relies also on Exo1 (Cotta-Ramusino et al.,
2005). However, although the generation of aberrant
replication intermediates in HU-treated rad53 cells
could be suppressed in the absence of this nuclease,
it is not accompanied by an increase in cell viabil-
ity or replication-fork restart (Cotta-Ramusino et al.,
2005; Segurado and Difey, 2008). These results sug-
gest that the Rad53 kinase has Exo1-dependent and
Exo1-independent roles required for the stabiliza-
tion of DNA replication forks in cells blocked with
HU, but when cells are exposed to DNA damage
(MMS, ionizing radiation or UV light), the func-
tion of Rad53 would be executed mainly through an
Exo1-dependent pathway.
The mechanism by which Exo1 affects the integ-
rity of DNA replication forks is presently unknown.
It is possible that when forks stall in the absence of a
functional checkpoint, some pathological structures
are generated on the DNA that could be targeted
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S-phase checkpoint and replication-fork integrity Review
and degraded by Exo1. Alternatively, Exo1 might
process normal DNA replication intermediates that
become inappropriately exposed when Rad53 is de-
fective, yielding abnormal structures (Sogo et al.,
2002; Cotta-Ramusino et al., 2005). It is also pos-
sible that, if the checkpoint is required to maintain
the replisome at forks, as previously explained, loss
of some components in rad53 mutants could expose
forks to degradation by the Exo1 nuclease. The fact
that Exo1 is bound to chromatin in both wild-type
cells and rad53 checkpoint mutants in the presence
of HU (Cotta-Ramusino et al., 2005) excludes the
possibility of an inappropriate association of Exo1
with chromatin in the latter. It is worth consider-
ing that replisome instability in checkpoint mutants
has been only documented in HU-treated cells (Cobb
et al., 2003, 2005; Lucca et al., 2004) and there
is no evidence supporting this notion in the pres-
ence of DNA-damaging agents. It is possible that
the HU treatment reects a particular case where
altered dNTP levels specically affect the stability of
the polymerases at forks, whereas DNA lesions gen-
erated by genotoxic agents like MMS may represent
a physical impediment to fork movement (Vazquez
et al., 2008) with no effect on polymerase association.
In fact, rad53 exo1 double-mutant cells exposed to
MMS maintain functional forks that are capable of
continuous chromosome replication (Segurado and
Difey, 2008), suggesting a stable replisome-fork
One possibility to explain the role of Exo1 during
a perturbed S-phase is that this nuclease is directly
regulated by Rad53, which would explain the Exo1-
specic role at stalled forks. In agreement with this
possibility, in a quantitative phosphoproteomic ap-
proach carried out in budding yeast, Exo1 has been
found to be phosphorylated when cells are treated
with MMS, and this phosphorylation is dependent
on Rad53 (Smolka et al., 2007). It is also known that
Exo1 is responsible for the generation of ssDNA at
uncapped telomeres (Zubko et al., 2004), which is
inhibited in a Mec1/Rad53-dependent manner (Jia
et al., 2004). Similar results have been found in
HU-treated rad53 cells, where a high frequency of
ssDNA, dependent on Exo1, is generated at telomeres
(Feng et al., 2006). In addition, a recent report indic-
ates that Exo1 activity is down-regulated directly by
Rad53 when telomeres are unprotected (Morin et al.,
Mec1, Chk1 and DNA replication-
fork stabilization
mec1-null mutants are considerably more sensitive to
HU and DNA-damaging agents than are rad53-null
mutant cells (Desany et al., 1998; Gardner et al.,
1999), and DNA replication fork breakdown is also
more severe in the former (Tercero and Difey, 2001),
indicating that Mec1 has a direct role in replication-
fork stabilization different fromits function in Rad53
activation (shown schematically in Figure 2). Re-
cently obtained results have given additional sup-
port to this notion. In contrast to the situation in
rad53 mutant cells, deletion of EXO1 is not sufcient
to prevent replication-fork collapse in mec1 mutants
treated with MMS and, consistently, cell lethality is
not suppressed (Segurado and Difey, 2008). Thus,
whereas the primary role of Rad53 at forks in the
presence of DNAdamage is to avoid Exo1-dependent
replication-fork collapse, it seems that Mec1 has
Rad53-independent function(s) at forks, at least if
cells are treated with MMS. This role is not just
the activation of Chk1, as mec1 mutants are more
sensitive to MMS than rad53 chk1 double mutants.
In this regard, Smolka et al. (2007) have identied
Mec1/Tel1-dependent, Rad53-independent possible
targets in response to DNA damage, some of which
could have a function at replication forks. Thus, des-
pite Mec1 having been traditionally considered in
budding yeast as the sensor kinase and Rad53 and
Chk1 as the effector kinases, Mec1 is also very likely
an effector kinase. Nevertheless, the precise nature
of its direct role at replication forks is presently un-
clear, and future work will be necessary to understand
how it co-operates to maintain fork integrity inde-
pendently of Rad53.
A role for S. cerevisiae Chk1 in the maintenance of
replication-fork stability in DNA-damaging condi-
tions has been proposed too (Segurado and Difey,
2008) (Figure 2). This was unexpected, as chk1
mutants are not very sensitive to MMS or HU (Sanc-
hez et al., 1999) and the double mutant rad53 chk1
is neither more sensitive to MMS nor has more
fork breakdown rates than rad53 cells (Segurado and
Difey, 2008). This phenotype has been always at-
tributed to a minor role of Chk1 in the S-phase
checkpoint. However, Exo1-dependent suppression
in rad53 mutant cells requires the presence of the
kinase Chk1, indicating that this protein has also a
function in the stability of replication forks, at least
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M. Segurado and J.A. Tercero
when Rad53 is not functional. This work showed
that, when Rad53 is not present, Chk1 prevents
replication-fork collapse, for which the absence of
Exo1 is necessary. Thus Rad53 and Chk1 may have
redundant roles at replication forks, but the action of
Exo1 can only be counteracted by Rad53. Similarly,
the S. pombe homologues of Rad53 and Chk1, namely
Cds1 and Chk1 respectively, have also redundant roles
at forks, at least in HU-treated cells (Boddy et al.,
1998; Lindsay et al., 1998; Martinho et al., 1998;
Zeng et al., 1998; Brondello et al., 1999). The re-
dundancy between different kinases may provide a
safety mechanism to ensure a checkpoint response
that guarantees cell survival. Future work will be re-
quired to explore this new role of Chk1 in budding
Conclusions and perspectives
The stabilization of DNA replication forks by the
S-phase checkpoint in the presence of DNA dam-
age or replicative stress has been a main focus of
research during the last few years because of its rel-
evance for cell survival. Importantly, avoiding fork
collapse is essential to prevent genome instability,
which can be a major cause of cancer. Moreover, un-
derstanding the processes that maintain stable rep-
lication forks may contribute to improve the ef-
ciency of drugs that disrupt DNA replication and
are utilized in chemotherapy. Although several pos-
sible Mec1 and Rad53 target candidates have been
identied, many questions are still unanswered or
remain unclear and it is necessary to elucidate the
molecular mechanisms responsible for fork stabiliz-
ation. Recent results have indicated that this pro-
cess is much more complex than expected, show-
ing unexplored Rad53-independent roles for Mec1
and Chk1 in the stabilization of replication forks.
Clearly, it would be important to investigate further
the functions of the individual kinases to fully under-
stand how eukaryotic cells overcome perturbations
during chromosome replication. In addition to the
Mec1(ATR)/Rad53 checkpoint, SUMOylation path-
ways control stalled and damaged replication fork sta-
bility by preventing the accumulation of X-shaped
structures at forks (Branzei et al., 2006, 2008). Like-
wise, a new pathway controlling terminal fork in-
tegrity, mediated by Mre11 and Tel1/ATM, has been
recently uncovered (Doksani et al., 2009). Under-
standing the interplay among all these pathways
and their relation with the different DNA-repair
and DNA-damage-tolerance mechanisms to main-
tain chromosome integrity during S-phase will be an
exciting challenge for the coming future.
We apologize for any omission of articles that we
were unable to cite. We thank Dr Crisanto Guti errez
(CBMSO) for critical reading of the manuscript.
This work was supported by the Ministerio de
Ciencia e Innovaci on (MICINN), Spain [grant num-
bers BFU2007-67445 and Consolider CSD2007-
00015 (to J.A.T.s group)]; the Fundaci on Ram on
Areces (to the Centro de Biologa Molecular Severo
Ochoa); and the Consejo Superior de Investigaciones
Cientcas (CSIC) (JAE-Doc contract with M.S.).
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Received 8 April 2009/14 May 2009; accepted 15 May 2009
Published on the Internet 19 August 2009, doi:10.1042/BC20090053
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