Multifaceted role of the Saccharomyces cerevisiae Srs2 helicase in homologous recombination regulation.

Abstract

Homologous recombination (HR) is a major pathway for the elimination of DNA DSBs (double-strand breaks) induced by high-energy radiation and chemicals, or that arise due to endogenous damage and stalled DNA replication forks. If not processed properly, DSBs can lead to cell death, chromosome aberrations and tumorigenesis. Even though HR is important for genome maintenance, it can also interfere with other DNA repair mechanisms and cause gross chromosome rearrangements. In addition, HR can generate DNA or nucleoprotein intermediates that elicit prolonged cell-cycle arrest and sometimes cell death. Genetic analyses in the yeast Saccharomyces cerevisiae have revealed a central role of the Srs2 helicase in preventing untimely HR events and in inhibiting the formation of potentially deleterious DNA structures or nucleoprotein complexes upon DNA replication stress. Paradoxically, efficient repair of DNA DSBs by HR is dependent on Srs2. In this paper, we review recent molecular studies aimed at deciphering the multifaceted role of Srs2 in HR and other cellular processes. These studies have provided critical insights into how HR is regulated in order to preserve genomic integrity and promote cell survival.

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The Nucleus and Gene Expression1447
Multifaceted role of the Saccharomyces cerevisiae
Srs2 helicase in homologous recombination
regulation
M.A. Macris1and P. Sung
Department of Molecular Biochemistry and Biophysics, Yale University, 333 Cedar St., New Haven, CT 06520, U.S.A.
Abstract
Homologous recombination (HR) is a major pathway for the elimination of DNA DSBs (double-strand breaks)
induced by high-energy radiation and chemicals, or that arise due to endogenous damage and stalled
DNA replication forks. If not processed properly, DSBs can lead to cell death, chromosome aberrations and
tumorigenesis. Even though HR is important for genome maintenance, it can also interfere with other
DNA repair mechanisms and cause gross chromosome rearrangements. In addition, HR can generate DNA
or nucleoprotein intermediates that elicit prolonged cell-cycle arrest and sometimes cell death. Genetic
analysesintheyeastSaccharomycescerevisiaehaverevealedacentralroleoftheSrs2helicaseinpreventing
untimelyHReventsandininhibitingtheformationofpotentiallydeleteriousDNAstructuresornucleoprotein
complexes upon DNA replication stress. Paradoxically, efficient repair of DNA DSBs by HR is dependent on
Srs2. In this paper, we review recent molecular studies aimed at deciphering the multifaceted role of Srs2
in HR and other cellular processes. These studies have provided critical insights into how HR is regulated in
order to preserve genomic integrity and promote cell survival.
Formation and resolution of HR
(homologous recombination)
intermediates
DNA DSBs (double-strand breaks) caused by exposure
to DNA-damaging agents or replication fork demise are a
potentinducerofHR[1,2].Onceformed,thetwoendsofthe
DSB are processed nucleolytically to yield a pair of 3?-single-
strandedtails,whichserveasthesubstratefortherecruitment
of HR factors. The assembled HR machinery then conducts
a search for an homologous DNA sequence, most often pro-
vided by the sister chromatid, followed by the invasion of
thelattertoformaDNAjointcalledaD-loop(Figure1).The
primer–template junction in the D-loop then primes DNA
synthesis,whichisnecessaryforreplacingtheDNAsequence
eliminated during the initial nucleolytic processing of the
DSB. In mitotic cells, the resolution of the D-loop structure
most often proceeds via the SDSA (synthesis-dependent
single-strand annealing) pathway, in which the invading
strand containing newly synthesized DNA is dissociated
from the D-loop, followed by annealing of the displaced
DNA strand with the other ssDNA (single-stranded DNA)
tail derived from the initial DNA end-processing reaction.
The repair process is then completed by DNA gap-filling,
Keywords:D-loop,double-strandbreak,homologousrecombination,Saccharomycescerevisiae,
Srs2 helicase, Rad51 recombinase.
Abbreviations used: BRCA1, breast-cancer susceptibility gene 1; DSB, double-strand break; HR,
homologous recombination; PCNA, proliferating-cell nuclear antigen; PRR, post-replicative repair;
RPA, replication protein A; SDSA, synthesis-dependent single-strand annealing; SRS2, suppressor
of RAD six screen mutant 2; ssDNA, single-stranded DNA.
1To whom correspondence should be addressed (email margaret.macris@yale.edu).
DNAsynthesisandligation.IntheSDSApathway,genecon-
version occurs, but no crossover products are generated
(Figure 1). Occasionally in mitotic cells and frequently in
meiotic cells, resolution of the D-loop structure proceeds via
pathways that have the propensity to yield chromatid arm
crossovers [1,3]. The meiotic crossovers are thought to tie the
chromosomal homologues together to ensure their proper
disjunction in meiosis I [3].
The RAD52 epistasis gene group
Genes needed for HR and the recombinational repair of
DSBs were first isolated in the budding yeast Saccharomyces
cerevisiae. These genes, RAD50, RAD51, RAD52, RAD54,
RAD55,RAD57,RAD59,MRE11andXRS2,arecollectively
classified as the RAD52 epistasis group. More recent studies
have revealed a remarkable degree of structural and func-
tional conservation of this gene group among eukaryotes.
Interestingly,theHRmachineryinhighereukaryotesposses-
ses components, such as the tumour suppressors BRCA1
(breast-cancer susceptibility gene 1) and BRCA2, that are
absent in S. cerevisiae [4].
Rad51 recombinase and presynaptic
filament assembly
The RAD51 gene, a key member of the RAD52 epistasis
group, encodes the orthologue of the bacterial recombinase
enzyme RecA [1,5]. Like RecA, Rad51 catalyses the homo-
logous DNA pairing reaction that leads to the formation of
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1448Biochemical Society Transactions (2005) Volume 33, part 6
Figure 1
A DSB is nucleolytically processed to yield 3?-single-stranded tails. One
of the ssDNA tails invades the DNA homologue to form a D-loop and
prime DNA synthesis. The invading DNA strand then dissociates from the
donor DNA molecule and anneals with the other ssDNA tail. Gap-filling
DNA synthesis and DNA ligation complete the process.
The SDSA pathway of HR
DNA joints between recombining DNA molecules ([6]; Fig-
ure 1). Biochemical studies have indicated that homologous
DNA pairing mediated by Rad51 occurs within the confines
of a Rad51-ssDNA nucleoprotein filament, called the pre-
synaptic filament (Figure 2). Significantly, nucleation of
Rad51 on to ssDNA is slow and presynaptic filament as-
sembly is therefore rate-limiting and prone to interference by
the heterotrimeric ssDNA-binding factor RPA (replication
protein A) [5]. In S. cerevisiae, the inhibitory effect of RPA
in HR reactions can be alleviated via the action of a recombi-
nation mediator, Rad52 or the Rad55–Rad57 heterodimer,
members of the RAD52 epistasis group [7–10]. Both Rad52
and the Rad55–Rad57 complex bind ssDNA and physically
interact with Rad51, providing a mechanism to deliver
Rad51 protein to the ssDNA template in order to accelerate
presynapticfilamentassembly[11].Recentbiochemicalstud-
ies have provided evidence for a recombination mediator
activity in the BRCA2 protein [12,13].
Figure 2
(A) In the presence of ATP, Rad51 nucleates on to ssDNA to form
a right-handed helical protein filament. (B) Electron micrograph of a
Rad51-ssDNA nucleoprotein filament.
Rad51 forms a nucleoprotein filament on ssDNA
Attenuation of untimely HR by SRS2
(suppressor of RAD six screen mutant 2)
Even though HR is indispensable for eliminating genotoxic
DNA lesions and maintaining genome integrity, it can inter-
fere with other DNA repair pathways, such as PRR (post-
replicative repair) [14,15]. The PRR pathway comprises
error-free and error-prone sub-pathways that allow bypass
of replication-blocking lesions induced by DNA damage
[14]. Furthermore, DNA or nucleoprotein intermediates
generated by the HR machinery can trigger strong cell-cycle
arrestandcausecelldeathincertaingeneticbackgrounds[14].
For these reasons, cells have evolved mechanisms to prevent
untimely HR events. One such mechanism is dependent on
the SRS2 gene.
A mutant variant of SRS2 was first recognized as a sup-
pressoroftheDNArepairdefectsengenderedbyinactivating
RAD6 or RAD18, whose products form a heterodimer
with DNA-binding and ubiqutin-conjugating activity that
initiates the PRR pathway [16]. Yeast strains bearing srs2
mutations also exhibit a hyper-recombination phenotype
and synthetic lethality with a variety of other mutations
that affect DNA metabolism. In addition, these strains fail
to recover from DNA-damage checkpoint-mediated G2/M
arrest (reviewed in [1]). Interestingly, the suppression of
rad6 and rad18 mutant defects by the srs2 mutation requires
that HR is functional [17]. This observation and the hyper-
recombination phenotype of srs2 mutant cells lend support
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The Nucleus and Gene Expression1449
to the notions that SRS2 negatively regulates HR, and that
enhanced resistance of srs2 rad6 and srs2 rad18 double
mutants to DNA damage as compared with the rad6 or
rad18 single mutant originates from elevated HR capacity
owing to the loss of SRS2. By attenuating HR, it is thought
that SRS2 helps ensure the channelling of DNA lesions
encountered by the DNA replication machinery into the
Rad6/Rad18-mediated PRR pathway. As mentioned above,
the srs2 mutation is lethal when combined with other mu-
tations, such as sgs1, that affect chromosome stability. Sgs1 is
another DNA helicase which is thought to function at later
recombination steps [18]. The synthetic lethality of the srs2
sgs1 double mutant (and of various combinations involving
srs2) can be overcome by eliminating HR [19,20], implicating
inappropriate HR events as the cause of double mutant cell
inviability.
Disruption of the Rad51 presynaptic
filament by Srs2
The Srs2 protein is related to the bacterial UvrD, Rep, and
PcrA proteins, all members of the SF1 family of nucleic
acid helicases [21]. Consistent with this, the Srs2 protein
has ssDNA-dependent ATPase and DNA helicase activities
[22,23]. Using yeast two-hybrid and biochemical assays, Srs2
was shown to physically interact with the Rad51 recombi-
nase [24]. To examine the effect of Srs2 on HR, purified
Srs2 protein was added to in vitro recombination reactions
mediated by Rad51. In concordance with the genetic data,
Srs2 strongly suppressed DNA joint formation in the
in vitro systems [24,25]. Interestingly, RPA had a synergistic
effect with Srs2 in attenuating the ability of Rad51 to form
DNA recombinants [24]. Inhibition of recombination reac-
tions by Srs2 could not be solely attributed to dissociation of
recombinant DNA intermediates by its DNA helicase activ-
ity. Instead, biochemical analyses and electron microscopy
showed that Srs2 efficiently disrupts the presynaptic filament
[24,25], releasing ssDNA that is immediately sequestered
by RPA to prevent re-nucleation of Rad51 (Figure 3). The
inhibitory effect of Srs2 on the Rad51-mediated recombi-
nation reactions is partly overcome by the inclusion of the
recombination mediator, Rad52 [24], which facilitates Rad51
formation on an RPA-coated ssDNA template. This finding
suggests that Srs2 and Rad52 antagonize each other in regu-
lating HR. By replacing Srs2 with mutant variants that lack
ATPase activity in the in vitro recombination reactions and
in assays designed to test the dissociation of the Rad51 pre-
synaptic filament, a requirement for ATP hydrolysis in the
anti-recombinase activity of Srs2 was revealed [26]. In
agreement with the biochemical results, the ATP hydrolysis
defective srs2 mutants exhibit a hyper-recombination pheno-
type and synthetic lethality with the sgs1 mutation [26].
The ability of Srs2 protein to displace Rad51 from ssDNA
elucidatesthemolecularbasisofitsanti-recombinaseactivity.
As mentioned above, srs2 mutant cells fail to recover from
DNA-damagecheckpoint-imposedG2/Marrest,andthesrs2
mutation is lethal in combination with the sgs1 mutation
[20,27,28], phenotypes that can be overcome by deleting
Figure 3
The 3?–5?motor activity of Srs2 can dislodge Rad51 from ssDNA. The free
ssDNA is sequestered by RPA to prevent re-nucleation of Rad51.
Mechanism of Srs2 action
RAD51 [28]. The defects in DNA-damage checkpoint re-
covery in the srs2 mutant and the non-viability of the srs2
sgs1 double mutant could very well stem from an inability to
prevent Rad51 from inappropriately engaging ssDNA, such
as ssDNA resulting from stalling of the DNA replication
machinery.
Replication connection
PCNA (proliferating-cell nuclear antigen), a DNA poly-
meraseprocessivityfactor,ispost-translationallymodifiedby
sumoylation during S phase [29,30]. This post-translational
modification appears to be important for HR regulation
during DNA replication [31]. Mutant yeast strains (e.g. siz1)
in which there is a deficiency in PCNA sumoylation can
suppress the DNA-damage sensitivity of rad6 and rad18
mutants, similar to mutations in SRS2 [30]. In addition,
suppression of the damage sensitivity of rad6 and rad18
mutants by mutations that prevent PCNA sumoylation is
dependent on HR, suggesting that, like Srs2, sumoylated
PCNA can inhibit HR [31]. Yeast two-hybrid and bio-
chemical assays demonstrated that Srs2 physically interacts
with the sumoylated form of PCNA [29,32]. Therefore
PCNA sumoylation serves to recruit Srs2 as a guarding
mechanism to prevent Rad51 filament formation, thereby
avoiding unwanted recombination during DNA replication.
Other cellular functions of SRS2
Interestingly, even though Srs2 suppresses spontaneous re-
combination events, it also plays an important role in DSB
repair by HR [33–35]. In the study published by Kupiec and
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1450Biochemical Society Transactions (2005) Volume 33, part 6
co-workers[34],therepairofasite-specificDSBmadebythe
HO endonuclease was greatly diminished in the srs2 mutant
background. In genetic experiments designed to identify
factors that influence pathway choice in mitotic HR, Srs2
was found to promote SDSA specifically, thereby preventing
the channelling of recombination substrates into pathways
that produce cross-overs [33]. In its SDSA role, Srs2 could
remove Rad51 from one of the ssDNA tails derived from the
initial nucleolytic processing of a DSB. This would preclude
second end invasion to generate a double Holliday structure,
so as to minimize the likelihood of cross-over formation.
Alternatively,Srs2couldbeinvolvedinDNArepairsynthesis
during SDSA or in dissociating the invading strand from
the D-loop structure to allow for DNA strand annealing
in the repair reaction. Aside from its DSB repair function,
Srs2 has also been shown to have a role in DNA-damage
checkpoint activation during S phase [27] and to act with
DNA polymerase δ to suppress expansion of DNA triplet
repeats [36]. In the latter function, it is thought that Srs2
unwinds DNA stem–loop structures formed by the triplet
repeats [37].
Epilogue
Genetic and biochemical studies have provided insights
into the multifaceted role of Srs2 in HR regulation, DSB
repair by HR, DNA-damage checkpoint responses and also
DNAtripletrepeatmaintenance.ThedemonstrationofSrs2’s
ability to disrupt the Rad51 presynaptic filament provides a
remarkable example wherein a DNA motor protein can elicit
its biological activity, not by unwinding nucleic acids, but
by protein displacement from ssDNA [24,25]. Several DNA
helicase enzymes have been implicated in the regulation of
HR in human cells. For example, aberrant recombination
events are believed to contribute to the genomic instability in
cells derived from patients with the cancer-prone syndromes,
Bloom’ssyndromeandWerner’ssyndrome,whicharecaused
by mutations in the RecQ helicases BLM and WRN res-
pectively. Since HR is critical for tumour suppression in
mammalian cells, mechanistic studies of factors that are
involved in regulating the HR process will be important for
providing insights into the mechanism of cancer avoidance.
Research in our laboratory is supported by research grants from the
NIH(NationalInstitutesofHealth).WearegratefultoMichaelSehorn
for providing the electron micrograph.
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