A Genetic Screen for top3 Suppressors in Saccharomyces ... · The sole RecQ helicase in budding yeast, Sgs1, interacts with Top3 both physically and genetically, and the two proteins

Copyright © 2005 by the Genetics Society of AmericaDOI: 10.1534/genetics.104.036764

A Genetic Screen for top3 Suppressors in Saccharomyces cerevisiae IdentifiesSHU1, SHU2, PSY3 and CSM2: Four Genes Involved

in Error-Free DNA Repair

Erika Shor,1 Justin Weinstein and Rodney Rothstein2

Department of Genetics and Development, Columbia University College of Physicians & Surgeons, New York, New York 10032-2704

Manuscript received September 23, 2004Accepted for publication December 2, 2004

ABSTRACTHelicases of the RecQ family and topoisomerase III are evolutionarily conserved proteins important for

maintenance of genome stability. In Saccharomyces cerevisiae, loss of the TOP3 gene, encoding topoisomeraseIII, results in a phenotype of slow growth, DNA damage sensitivity, meiotic defects, and hyperrecombination.The sole RecQ helicase in budding yeast, Sgs1, interacts with Top3 both physically and genetically, andthe two proteins are thought to act in concert in vivo. Much recent genetic and biochemical evidencepoints to the role of RecQ helicases and topoisomerase III in regulating homologous recombination (HR)during DNA replication. Previously, we found that mutations in HR genes partially suppress top3 slowgrowth. Here, we describe the analysis of four additional mutational suppressors of top3 defects: shu1,shu2, psy3, and csm2. These genes belong to one epistasis group and their protein products interact witheach other, strongly suggesting that they function as a complex in vivo. Their mutant phenotype indicatesthat they are important for error-free repair of spontaneous and induced DNA lesions, protecting thegenome from mutation. These mutants exhibit an epistatic relationship with rad52 and show altereddynamics of Rad52-YFP foci, suggesting a role for these proteins in recombinational repair.

ALL living cells possess mechanisms that ensure faith- type at the SUP4-o locus (Wallis et al. 1989). In additionful reproduction of genetic material and protec- to causing increased recombination, mutation of TOP3

tion of the genome from excessive mutation. Mutagenic results in slow growth, hypersensitivity to genotoxicDNA lesions can either arise spontaneously, for in- agents, such as hydroxyurea (HU) and methyl methane-stance, during the process of DNA replication, or be sulfonate (MMS), mitotic chromosome missegregation,induced by exogenous agents, such as chemicals or irra- and inability to undergo meiosis and sporulate (Wallisdiation. One of the pathways used to repair both sponta- et al. 1989; Gangloff et al. 1999; Chakraverty et al.neous and induced DNA double-strand breaks (DSBs) 2001). Mutation of SGS1, the gene encoding the soleor single-strand gaps is homologous recombination (HR), budding yeast RecQ helicase, suppresses top3� slowa process of exchange of genetic material between two growth and the two proteins physically interact withDNA molecules of nearly or perfectly identical sequence each other (Gangloff et al. 1994; Bennett et al. 2000;(Symington 2002). Since it uses a homologous se- Fricke et al. 2001). Mutation of SGS1 in an otherwisequence as template for DNA repair, normal HR is error wild-type background causes a phenotype reminiscentfree, thus playing an important role in protection of of that of the top3� strain, although milder in severitythe genome from mutation. However, recombination (Gangloff et al. 1994; Watt et al. 1995, 1996).has to be regulated, since excessive or inappropriate RecQ helicases and topoisomerase III are evolution-recombination can also contribute to genomic instabil- arily conserved proteins and play important roles inity. A growing amount of evidence suggests that helicases maintenance of genome stability in higher eukaryotes.of the RecQ family in conjunction with type III topo- For instance, mutations in three of five human RecQisomerases play crucial roles in regulating HR (Oakley homologs, BLM, WRN, and RECQL4, cause the cancerand Hickson 2002; Khakhar et al. 2003). predisposition syndromes Bloom, Werner, and Roth-

Topoisomerase III was first identified in budding yeastmund-Thomson (reviewed in Hickson 2003). Werner

as a mutation that causes a hyperrecombination pheno-syndrome and Rothmund-Thomson syndrome patientsalso exhibit symptoms of premature aging. At the cellu-lar level, all of these syndromes are characterized by1Present address: Department of Biomolecular Chemistry, University

of Wisconsin, Madison, WI 53706-1532. genomic instability that is, at least in part, caused by2Corresponding author: Department of Genetics and Development, inappropriate recombination events. The functional in-

Columbia University College of Physicians & Surgeons, 701 W. 168th teraction between RecQ-like helicases and topoisomer-St., New York, NY 10032-2704.E-mail: [email protected] ase III homologs also appears to be conserved in human

Genetics 169: 1275–1289 (March 2005)

1276 E. Shor, J. Weinstein and R. Rothstein

cells. BLM physically interacts with an isoform of topo- genetic interaction with sgs1 and is thus thought to act ina pathway parallel to Sgs1-Top3. Recently, two differentisomerase III, Top3�, and this interaction is necessary

for BLM function (Wu et al. 2000). Mutation of topo- laboratories showed that, in vitro, Srs2 can dislodge theyeast RecA homolog, Rad51, from ssDNA and preventisomerase III is not known to be associated with a human

genetic disorder. However, deletion of TOP3� in the Rad51-mediated recombination reactions (Krejci et al.2003; Veaute et al. 2003).mouse results in embryonic lethality, while deletion of

TOP3� produces mice with a shortened life span (Li Regulation of HR is especially important during theprocess of DNA replication. Impediments to replicationand Wang 1998; Kwan and Wang 2001).

As described above, mutation of human or yeast RecQ fork movement, such as DNA lesions, can result in forkpausing or arrest. Stalled forks contain regions of ssDNA,helicases causes a genomic instability phenotype charac-

terized by increased or inappropriate recombination which can be used as substrate for Rad51 binding andinitiation of HR (Sogo et al. 2002; Lucca et al. 2004).events. Other evidence, both genetic and biochemical,

indicates that RecQ family proteins have important roles While HR is important for resumption of replication incertain contexts, for instance, when the fork collapsesin regulating HR. In Saccharomyces cerevisiae, sgs1� exhib-

its extreme synthetic sickness or lethality with mutation and forms a DSB, in the instance of replication arrestrecombination can destabilize the fork and create un-of the gene encoding another DNA helicase, Srs2 (Gang-

loff et al. 2000; McVey et al. 2001). Mutation of SGS1 timely genomic rearrangement (Cox 2001; Michel etal. 2001; Lucca et al. 2004). In fact, analyses of yeast cellsalso exhibits synthetic lethality with mutation of either

of the two genes encoding the Mms4-Mus81 endonucle- treated with HU, a chemical that inhibits replicationprogression, suggest that HR proteins are excluded fromase (Kaliraman et al. 2001; Mullen et al. 2001). These

synthetic defects are fully suppressed by deletion of HR arrested replication forks (Lisby et al. 2004). Severallines of evidence suggest that RecQ helicases and associ-genes RAD51, RAD52, RAD55, or RAD57 (Gangloff et

al. 2000; Fabre et al. 2002). Additionally, mutation of ated factors provide a mechanism to regulate recombi-nation during the process of DNA replication. In yeastHR genes partially suppresses the slow growth and DNA

damage sensitivity of the top3� strain (Shor et al. 2002). and in human cells, RecQ homologs were shown tocolocalize with sites of DNA synthesis and with otherDiploids lacking both copies of the TOP3 gene have a

severe meiotic defect that can be bypassed by deleting proteins involved in DNA replication (Brosh et al. 2000;Constantinou et al. 2000; Bischof et al. 2001). Also,SPO11, the gene responsible for initiating meiotic re-

combination (Gangloff et al. 1999). In fission yeast, both yeast and human cells carrying mutations in RecQhomologs exhibit various S-phase-specific defects. Forseveral defects of the mutant lacking the RecQ homolog

Rqh1 can be partially rescued by expression of bacterial instance, fission yeast rqh1 cells fail to recover normallyfrom replication arrest and subsequently exhibit in-Holliday junction (HJ) resolvase, RusA (Doe et al. 2000).

These genetic data indicate that in the absence of the creased recombination and defects in chromosome seg-regation (Stewart et al. 1997). In S. cerevisiae, both Sgs1Sgs1-Top3 complex and other proteins that function in

parallel pathways, HR plays a detrimental role, causing protein levels and Top3 mRNA levels were shown to becell cycle regulated, peaking during S phase (Frei andgenomic instability and slow growth or lethality.

Biochemical analyses of purified proteins in vitro also Gasser 2000; Chakraverty et al. 2001). Additionally,Sgs1 has a role in the intra-S checkpoint and is importantsuggest a role for RecQ helicases and associated factors

in regulating HR. In addition to exhibiting “classic” for the stable association of DNA polymerases with repli-cation forks during replication arrest (Frei and Gasserhelicase activity, that of unwinding dsDNA substrates,

several RecQ homologs can unwind DNA structures that 2000; Cobb et al. 2003).In a previous article, we described a genetic screenresemble HR intermediates, such as HJs and D-loops

(Bennett et al. 1998, 1999; Constantinou et al. 2000; to isolate non-sgs1 suppressors of top3� slow growth(Shor et al. 2002). We reported that mutation of theKarow et al. 2000; van Brabant et al. 2000). Topoisom-

erase III is a type I topoisomerase, acting on DNA that HR genes RAD51, RAD52, RAD54, RAD55, or RAD57partially suppresses top3� slow growth and DNA damageis negatively supercoiled and/or contains single-strand

regions (Harmon et al. 1999; Champoux 2001). In sev- sensitivity. In addition to HR mutants, we isolated severalother mutational suppressors of top3� defects. In thiseral studies, the concerted action of RecQ helicase and

topoisomerase III in vitro produces a novel activity not article, we describe our analyses of four such suppres-sors: shu1, shu2, psy3, and csm2. Through the efforts ofassociated with either protein alone. For instance, Esche-

richia coli RecQ and topoisomerase III can catenate and several research groups performing mutant hunts usingthe S. cerevisiae deletion library, some of these mutantsdecatenate covalently closed dsDNA circles (Harmon

et al. 1999). Also, the BLM protein, in concert with human have been previously identified as causing a phenotypeof MMS sensitivity, platinum sensitivity, increased for-topoisomerase III�, can dissolve a double-HJ-containing

DNA substrate to yield exclusively noncrossover prod- ward mutation rates and gross chromosomal rearrange-ments, and meiotic chromosome segregation defectsucts (Wu and Hickson 2003). As mentioned earlier, the

SRS2 gene encodes another DNA helicase that exhibits a (Rabitsch et al. 2001; Hanway et al. 2002; Huang et

1277Shu1, Shu2, Psy3 and Csm2 Function in DNA Repair

�Leu medium. The transformants were then replica platedal. 2003). In genome-wide two-hybrid screens, severalonto �Leu medium containing 0.03% MMS. Library plasmidsinteractions were observed among these proteins (Itowere isolated from colonies able to grow on MMS-containing

et al. 2001). In this article, we expand on some of these medium. The plasmid inserts were sequenced using thedata and also analyze the genetic interactions of these primer CTTCTTTGCGTCCATCC. To confirm that the com-

plementing insert indeed contains the SHU gene of interest,genes with each other, with SGS1-TOP3, and with HR.we deleted the corresponding ORF in the genome and showedWe also analyze the impact of these mutations on recom-that the resulting deletion has the same phenotype and failsbination rates at several loci and on Rad52 focus dynam-to complement the shu mutant. We also sequenced the muta-

ics. Our data show that these four proteins function as tional alteration in each shu mutant.part of a single pathway, possibly a complex, in vivo. Two-hybrid assays: Strain PJ69-4A MATa was transformed

with the Gal4 activation domain (GAD) plasmids and PJ69-Genetic analyses place them downstream of RAD52 in4A MAT� with the Gal4-binding domain (GBD) plasmids con-MMS resistance and mutation avoidance. Analyses oftaining the SHU ORFs (Ito et al. 2001). Each MATa trans-Rad52 foci in the shu1 mutant indicate that recombina-formant was crossed to each MAT� transformant and the

tional repair is affected by mutation of genes in this diploids were selected on SC leucine- and tryptophan-dropoutgroup, especially after high doses of DNA damage. Over- medium (�Leu �Trp; Bendixen et al. 1994). To use HIS3 as

the two-hybrid reporter, each diploid was replica plated fromall, our data indicate that Shu1, Shu2, Psy3, and Csm2�Leu �Trp onto �Leu �Trp �His and onto �Leu �Trppromote efficient recombinational repair and function�His � 3 mm 3-AT. To use the lacZ gene as the reporter, thein the protection of the genome from spontaneous anddiploids were grown in liquid �Leu �Trp medium, the cells

induced DNA damage. were permeabilized with chloroform and incubated witho-nitrophenyl-�-d-galactoside, and units of �-galactosidase ac-tivity were calculated as described (Ausubel et al. 1998).

MATERIALS AND METHODS Sensitivity to DNA-damaging agents: To measure sensitivityto MMS, the appropriate numbers of cells were plated ontoMedia and strains: Yeast extract/peptone/dextrose mediumYPD medium containing various concentrations of MMS as(YPD) and synthetic complete (SC) media lacking specificdescribed in Shor et al. (2002) and viable cell counts wereamino acids or containing 5-fluoroorotic acid (5-FOA), genet-calculated.icin, canavanine, or 3-aminotriazole (3-AT) were prepared as

SUP4-o recombination assay: The basis of the SUP4-o recom-described (Rose et al. 1990) with twice the amount of leucinebination assay has been described previously (Rothstein et al.(60 �g/ml) in all SC-based media. 5-FOA was purchased from1987; Shor et al. 2002). Strains containing the SUP4-o::URA3American Bioanalytical and the other chemicals from Sigmaconstruct were grown overnight in YPD and plated onto(St. Louis). Standard procedures were used for mating, sporu-SC arginine- and uracil-dropout plates containing 1 mg/mllation, and dissection (Sherman et al. 1986). Yeast strains5-FOA, 60 �g/ml canavanine, and 10 �g/ml adenine. Threeused in this study are listed in Table 1. In most experiments,days later, red 5-FOA- and canavanine-resistant colonies weremultiple meiotic segregants of the same genotype were ana-counted and SUP4 recombination rates were calculated onlyzed. When necessary, the figure legends state which diploidthe basis of the method of the median (Lea and Coulsonstrains from Table 1 produced the haploid strains shown in1949). From 9 to 11 cultures were tested for each strain. SUP4the figure.deletion class identification was performed for independentlyReplacements of the SHU1 ORF by the HIS3 gene and ofgenerated recombinants by the PCR-Xho I digestion approachthe SHU2 ORF by the Kluyveromyces lactis URA3 gene wereas described (Shor et al. 2002).done using a PCR-based allele replacement method (Erdeniz

Measuring the CAN1 forward mutation rate: CAN1 strainset al. 1997). psy3::kanMX4, csm2::kanMX4, and rev3::kanMX4were grown overnight in YPD and plated onto SC arginine-constructs were amplified by PCR from the S. cerevisiae genomedropout medium containing 60 �g/ml canavanine. After 3deletion library (Research Genetics, Huntsville, AL) and trans-days, canavanine-resistant colonies were counted and muta-formed into the W303 background (Thomas and Rothsteintion rates calculated on the basis of the method of the median1989). Transformants were selected on YPD medium con-(Lea and Coulson 1949). From five to nine cultures weretaining 200 �g/ml geneticin and the gene deletions weretested for each strain. To measure the MMS-induced rate ofverified by PCR.mutagenesis, colonies were inoculated into YPD and pregrownFusions of yellow fluorescent protein (YFP) at the Shu Cfor 8 hr. MMS was then added to a final concentration oftermini were done using the PCR-based cloning-free chromo-0.01% and the cultures were grown overnight. The rest of thesomal integration strategy as described (Lisby et al. 2001; Reidassay was done as described above for the measurements ofet al. 2002). The second YFP gene was subsequently integratedspontaneous mutation rates.at the C terminus of Shu1-YFP using the same method. Shu1

Microscopy: Strains of the desired genotype carrying theand the two YFPs are each separated by a four-alanine linker.RAD52-YFP fusion and the wild-type ADE2 allele were grownPlasmids: Construction of pWJ1323 expressing Nup49 fusedat 23� in SC medium supplemented with 50 �g/ml adenineto cyan fluorescent protein (Nup49-CFP) was described in M.and harvested for microscopy during midlog phase. The strainWagner, G. Price and R. Rothstein (unpublished results).expressing Shu1-YFP-YFP and Nup49-CFP contained theConstruction of the two-hybrid plasmids expressing Shu pro-ade2-1 allele but was otherwise treated the same as above. Inteins fused to the Gal4 activation or binding domain was de-the case of monitoring Rad52-YFP foci after DNA damage,scribed as part of the genome-wide two-hybrid study (Ito etMMS or HU were added to indicated concentrations and theal. 2001).cultures were further incubated at 23� for the indicated timesGrowth rate determination: The OD600 of cultures in expo-prior to harvesting. Fluorescent images were captured withnential growth phase in YPD was determined every hour fora Zeiss Axioplan equipped with a Hamamatsu Orca camera5 hr. Three isolates were analyzed for every strain.(Hamamatsu Photonics) and processed with Openlab 3.0.4Cloning SHU1 and CSM2 by complementation of MMS sensi-(Improvision) and Adobe Photoshop. Exposure times weretivity: We transformed shu mutants with a genomic DNA library

(Akada et al. 1997) and selected transformants on synthetic 1000 msec for Rad52-YFP, 3000 msec for Shu-YFP-YFP, and


TABLE 1

Strains

Straina Genotype

W1588-4C MATaW1588-4A MAT�

ESD50MAT� sgs1::URA3 shu1::HIS3MATa top3::TRP1

ESD51MATa sgs1::HIS3 top3::TRP1MAT� shu2::URA3 K.lactis

W4105MAT� top3::TRP1 sgs1::HIS3MATa psy3::KANMX

W4116MATa sgs1::URA3 top3::LEU2MAT� csm2::KANMX srs2::HIS3

W4173MATa shu1::HIS3 csm2::KANMXMAT� shu2::URA3K.lactis psy3::KANMX

ESD35MATa RAD52-YFP bar1::LEU2 ADE2MAT� sgs1::URA3 shu1::HIS3

ESD46MATa RAD52-YFP bar1::LEU2 ADE2MAT� top3::TRP1 shu1::HIS3

ESH38 MAT� SHU1-YFP-YFPESD20-7A MATa shu1::HIS3 rad52::HIS5 leu2�BstEII TRP1 lys2�ESD20-22C MATa shu1::HIS3 leu2�BstEII TRP1 lys2�ESD21-17B MAT� shu1::HIS3 leu2�EcoRIESD28-4B MATa csm2::KANMX leu2�BstEII TRP1 lys2�ESD24-10C MAT� csm2::KANMX leu2�EcoRIESD24-6C MAT� csm2::KANMX rad52::HIS5 leu2�EcoRI

ESD38MATa csm2::KANMXMAT� rDNA::ADE2-CAN1

ESD39MATa shu1::HIS3MAT� rDNA::ADE2-CAN1

ESD49MAT� shu1::HIS3 CAN1MATa rev3::KANMX

ESD41MATa csm2::KANMXMAT� CAN1 lys2� URA3K.lactis

ESD44MATa CAN1 lys2�

MAT� shu1::HIS3 rad52::HIS5 leu2�EcoRI

ESD45MATa csm2::KANMXMAT� SUP4-o::URA3

ESD47MAT� top3::TRP1 shu1::HIS3MATa SUP4-o::URA3

ESD48MAT� sgs1::HIS3 SUP4-o::URA3MATa shu1::HIS3

PJ69-4Aab MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4� gal80�

LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ

PJ69-4A�b MAT� trp1-901 leu2-3,112 ura3-52 his3-200 gal4� gal80�

LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZa All strains are RAD5 derivatives of W303 (Thomas and Rothstein 1989) and contain the following markers

(unless specified otherwise): ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1.b The strains used for the two-hybrid assays are described in James et al. (1996).

1000 msec for Nup49-CFP. For the time-lapse experiments, 5 hr. For each field of cells, 11 fluorescent images were ob-tained at 0.4-�m intervals along the z-axis. Only cells that hadMMS was added to the culture to the final concentration of

0.01% immediately before mounting cells on the microscope exhibited growth and/or budding during the span of the timelapse were taken into account.slide. Images were captured every 5 min for the duration of


Figure 1.—Genetic interactions among sgs1, top3, and the shu mutants. All the mutations shown are deletions of the respectivegenes. The haploid strains are meiotic segregants of diploids ESD50, ESD51, W4105, and W4173. For the DNA damage sensitivityassays in B–D, 10-fold serial dilutions of overnight cultures were spotted onto YPD medium containing the indicated drugs. (A)Deletion of SHU1, SHU2, PSY3, or CSM2 suppresses top3� slow growth. Doubling times in rich medium are shown. (B) Deletionof SHU1, SHU2, PSY3, or CSM2 suppresses top3� sensitivity to HU and MMS. (C) Deletion of SHU1, SHU2, PSY3, or CSM2suppresses sgs1� HU sensitivity. (D) SHU1, SHU2, PSY3, and CSM2 are in one epistasis group for MMS resistance. The quadrupledeletion mutant is no more MMS sensitive than any of the single mutants.

RESULTS Genome-wide two-hybrid screens of S. cerevisiae geneproducts suggest that Shu1p and Csm2p are connectedIdentification of shu1, shu2, psy3, and csm2 as suppres-by a series of protein-protein interactions also involvingsors of top3� slow growth: The screen for non-sgs1 sup-the gene products of YDR078C and PSY3 (Ito et al.pressors of top3� slow growth was performed as pre-2001). PSY3 was identified and named as part of a ge-viously described (Shor et al. 2002). All suppressornome-wide search for mutants that confer p latinum sen-mutations not linked to SGS1 were backcrossed intositivity (psy ; Wu et al. 2004). This network of two-hybrida TOP3 background, tested for sensitivities to severalinteractions suggests that the proteins also interact func-genotoxic agents, and checked for complementationtionally in vivo. Thus, we created chromosomal deletionswith other non-sgs1 top3� suppressors, such as HR mu-of each of the four ORFs and tested their genetic interac-tants. A number of the new top3� suppressor mutationstions with top3� and sgs1� mutations. We find that eachhad a similar phenotype and fell into four complementa-deletion mutant suppresses top3� slow growth, DNAtion groups. In addition to suppressing the slow growthdamage sensitivity, and sgs1� HU sensitivity (Figure 1,of the top3� mutant, these mutants partially suppressA–C). Moreover, complementation analyses indicatedtop3� sensitivity to HU and MMS (Figure 1B), the HUand DNA sequencing confirmed that shu2 and shu3sensitivity of the sgs1� strain (Figure 1C), and the lethal-mutants isolated in the screen were in fact alleles ofity/extreme sickness of the sgs1� srs2� and sgs1� mus-YDR078C and PSY3. Thus, we gave YDR078C the name81� double mutants (data not shown). The new mutantsSHU2. Overall, we identified six alleles of shu1, one ofwere named shu (suppresses sgs1 HU sensitivity). In anshu2, one of psy3, and five of csm2 during the screen.otherwise wild-type background, the shu mutations re-

Shu1, Shu2, Psy3, and Csm2 are all relatively smallsulted in moderate sensitivity to MMS (Figure 1D). Thisproteins (under 250 aa). We attempted to glean infor-observation allowed us to clone two of the genes bymation about their functions by searching for possiblecomplementation of the MMS sensitivity with a genomichomologs in other organisms. However, BLAST searchesDNA library (for details see materials and methods).identified no orthologs of any of the four proteins inIn this manner, we identified yhl006c and csm2 as top3bacteria, fission yeast, or higher eukaryotes. We did iden-slow-growth suppressors. YHL006C was named SHU1.tify orthologs of all four proteins in sensu stricto andCSM2 was previously identified and named as part of asensu lato Saccharomyces species, as well as those of Shu2genome-wide screen for mutations that affect chromo-

some segregation in meiosis (csm ; Rabitsch et al. 2001). and Psy3 in several species of the genus Kluyveromyces.


TABLE 2

Two-hybrid Interactions

GADa GAD-Shu1 GAD-Shu2 GAD-Psy3 GAD-Csm2

GBDa — — — — —GBD-Shu1 — — 25 � 7 10 � 1 �GBD-Shu2 — 28 � 8 — 6 � 1 �GBD-Csm2 — — � 19 � 3 —

The Shu proteins interact with each other in the two-hybrid system. The numbers refer to units of�-galactosidase produced as a result of transcriptional activation of the LacZ reporter by the two-hybrid interac-tions. The basal level of LacZ activation results in 0–2 units of �-galactosidase. Another transcriptional reportertested was HIS3. In certain cases, a weak two-hybrid interaction resulted in detectable activation of the HIS3reporter but not of the LacZ reporter. These interactions are indicated as “�.” The GBD-Psy3 construct is notincluded in the table since it does not exhibit any two-hybrid interactions due to a frameshift mutation at aa7 of the Psy3 protein.

a Empty vector.

Since these genomes have not been completely se- These observations suggest that the four proteins func-tion together in vivo, possibly as part of a single pathwayquenced, it is possible that they also contain orthologs

of Shu1 and Csm2. A BLAST search against the fully or complex. To test this idea, we created double, triple,and quadruple deletion mutants and tested their MMSsequenced Candida albicans genome revealed a possible

ortholog of Csm2, with 21% identity and 35% similarity. sensitivities. Indeed, we find that all of the mutant com-binations exhibit the same degree of MMS sensitivity asAdditionally, we have used the sequence analysis tools

available at the Saccharomyces Genome Database (http:// any of the single mutants (Figure 1D; data not shown).Thus, shu1, shu2, psy3, and csm2 are in one epistasisgenome-www.stanford.edu/Saccharomyces) to identify

possible motifs and domains within the Shu proteins. group and likely function in the same pathway. Forbrevity, in the remainder of this article, we refer to themHowever, no significant matches were found.

Characterization of the physical and genetic interac- collectively as the Shu proteins or the SHU genes, eventhough two of the genes are named PSY3 and CSM2.tions among Shu1, Shu2, Psy3, and Csm2: To character-

ize the two-hybrid interactions among Shu1, Shu2, Psy3, Mutation of SHU1 suppresses increased Rad52-YFPfoci in top3 and sgs1 mutants: HR proteins form sponta-and Csm2 in more detail, we obtained the relevant two-

hybrid plasmids (Ito et al. 2001) and measured the two- neous and DNA-damage-induced foci in both yeast andmammalian cells (Haaf et al. 1995; Lisby et al. 2001,hybrid interactions using HIS3 and �-galactosidase as

reporters. The results of the two-hybrid assays are sum- 2004). These foci are sites of repair of multiple DNAlesions, such as DSBs (Lisby et al. 2003). In buddingmarized in Table 2. We also sequenced the relevant

ORFs on the plasmids and tested them for complemen- yeast, spontaneous Rad52-YFP foci are mostly observedin S and G2/M cells, suggesting that these foci are sitestation of the MMS sensitivity of the corresponding dele-

tion mutant. DNA sequencing revealed a �1 frameshift of replication fork repair (Lisby et al. 2001, 2003). Dy-namics of focus formation can be affected by mutationmutation at amino acid 7 (ATT to AT) of the Psy3

protein in the GBD-Psy3 construct. The resulting mu- of genes involved in DNA metabolism (Lisby et al. 2004;D. Alvaro and R. Rothstein, unpublished results; Fig-tant Psy3 polypeptide is 22 aa long and fails to exhibit

any two-hybrid interactions or to complement the MMS ure 2). For instance, budding yeast top3� and sgs1�mutants, both of which exhibit hyperrecombination andsensitivity of psy3�. The GAD-Psy3 construct, while fail-

ing to complement, is functional in the two-hybrid assay increased genomic instability, also show increased inci-dence of spontaneous Rad52-YFP foci (M. Wagner, G.and does not contain errors at the sequence level. The

rest of the constructs fully complement the MMS sensi- Price and R. Rothstein, unpublished results; Figure2A). We tested whether deletion of SHU1 would influ-tivity of the respective shu� strains, do not contain any

sequence errors, and interact with each other in the ence this phenotype. The data, shown in Figure 2, arepresented as the proportion of all G1 (i.e., unbudded)two-hybrid system (Table 2).

As described above, mutation of SHU1, SHU2, PSY3, and S/G2/M (i.e., budded) cells that contain Rad52-YFP foci. The insets in B and C show the cell cycleor CSM2 leads to suppression of top3� slow growth and

DNA damage sensitivity (Figure 1, A and B), suppression distribution of all the strains tested (i.e., the fraction ofthe population in G1 or S/G2/M). We find that deletionof sgs1� HU sensitivity (Figure 1C), and, in an otherwise

wild-type background, a similar level of sensitivity to of SHU1 does partially suppress the increased Rad52-YFP focus formation in the top3� mutant: 71% of allMMS (Figure 1D). Also, as indicated by the two-hybrid

results, these proteins likely interact among themselves. S/G2/M top3� cells contain Rad52-YFP foci, compared


Figure 2.—Effects of shu1� on the formation of Rad52-YFP foci in the top3� and sgs1� mutants. The haploid strains for theseexperiments were generated from diploids ESD35 and ESD46. (A) The top3� strain exhibits an increase in Rad52-YFP focicompared to the wild-type strain. (B) shu1� partially suppresses increased Rad52 foci in the top3 mutant. The graph shows thepercentage of cells in G1 (i.e., unbudded cells) or in S/G2/M (i.e., all budded cells) that contain Rad52-YFP foci in the indicatedstrains. The inset shows the cell cycle distribution of the same four strains. The graph represents data from at least 300 cells ofeach genotype. (C) shu1� partially suppresses increased Rad52-YFP foci in the sgs1� mutant after HU treatment. The graphshows the percentage of HU-treated and untreated cells in G1 or S/G2/M that contain Rad52-YFP foci. The two insets show thecell cycle distribution of untreated cells as well as of cells treated with 100 mm HU for 100 min. Between 150 and 300 cells wereexamined for every strain for either condition (HU or no HU) during this experiment.

to 45% of all S/G2/M top3� shu1� cells (Figure 2B). binational repair centers, or foci (Lisby et al. 2004).Replication forks that have arrested due to the actionThis effect is not caused by a change in cell cycle distri-

bution of the strain, as mutation of SHU1 does not of HU are maintained and stabilized by processes thatare checkpoint dependent. When cellular checkpointrescue the abnormal cell cycle distribution of the top3�

mutant (see inset in Figure 2B). We conclude that the mechanisms are perturbed, such as by mutation of theMEC1 gene, HU causes replication fork collapse (LopesSHU1 gene contributes to genomic instability and in-

creased formation of Rad52-YFP foci in the absence of et al. 2001; Cha and Kleckner 2002). In mec1 cells,HU treatment results in a great increase in Rad52 foci,topoisomerase III.

In addition to cell cycle stage and mutation of DNA probably reflecting the formation of recombinationalrepair complexes at collapsed replication forks (Lisbymetabolism genes, Rad52 focus formation is affected by

genotoxic agents, such as HU, which inhibit replication et al. 2004). Mutation of SGS1 causes HU sensitivity, pre-sumably because of the dual role of Sgs1 in the intra-Sfork progression by depleting dNTP pools. There is a

great decrease in Rad52-YFP foci in wild-type cells checkpoint activation and in replication fork restart(Frei and Gasser 2000; Cobb et al. 2003). As describedtreated with HU, suggesting that active progression of

replication forks is necessary for the formation of recom- above, deletion of any of the SHU genes suppresses


sgs1� HU sensitivity (Figure 1C). Thus, we examinedif deletion of SGS1 and/or SHU1 perturb the normaldynamics of Rad52-YFP foci after exposure to HU. Theresults of these experiments are shown in Figure 2C.We find that deletion of SHU1 does not significantlyaffect Rad52-YFP focus formation in either untreatedor HU-treated cells, consistent with the observation thatthe shu mutants are not HU sensitive. Mutation of SGS1causes an increased incidence of spontaneous Rad52-YFP foci, particularly in the S/G2/M cells (M. Wagner,G. Price and R. Rothstein, unpublished results). Dele-tion of SHU1 in the sgs1� strain slightly suppresses in-creased spontaneous Rad52-YFP foci (Figure 2C). In thewild-type strain, treatment with 100 mm HU reducesthe focus-containing proportion of S/G2/M cells to 1%(Lisby et al. 2004; Figure 2C). This effect of HU onRad52-YFP focus formation is perturbed by deletion ofSGS1, with �22% of S/G2/M sgs1� cells containingRad52-YFP foci after HU treatment. These are mostlylarge-budded cells and cells that appear arrested withthe nucleus positioned between the mother and thebud (Figure 2C; data not shown). Deletion of SHU1 inthe sgs1� strain reduces the proportion of S/G2/M cellsthat contain foci after HU treatment from 22% to 8%,reflecting the suppression of sgs1� HU sensitivity byshu1�.

Genetic analysis of Shu function: We have shown thatthe Shu proteins function in the same pathway of MMS-induced DNA damage resistance (Figure 1D). Next, weasked how they interact genetically with other genesinvolved in MMS resistance. Because of the genetic in-teraction between the shu mutants and mutation of SGS1on HU, we tested whether they have a similar relation-ship upon exposure to MMS. Thus, we analyzed theresponse of sgs1� shu1� and sgs1� csm2� double mu-tants to increasing concentrations of MMS comparedto that of the single mutants (Figure 3A). Interestingly,we observed an additive effect of the mutations on thereduction in cell survival, indicating that, for repair ofMMS-induced DNA damage, Sgs1 and the Shu proteinsfunction in separate pathways. Figure 3.—The SHU genes are in the RAD52 epistasis group

for MMS resistance. All mutations shown are gene deletions.Several lines of evidence suggest that the Shu pro-When comparing the data shown in A to those in B, noteteins’ function may be linked to HR. First, deletion ofthe difference in MMS concentrations. (A) Survival curvesPSY3 was isolated in a screen for MMS-sensitive mutants showing that SGS1 and the SHU genes are in different epistasis

and analyzed for genetic interactions with other mutants groups for MMS resistance. Deletion of SGS1 and either SHU1known to be involved in MMS resistance (Hanway et or CSM2 results in additional MMS sensitivity compared to

any of the single mutants. (B) Survival curves showing thatal. 2002). The only mutation found to be epistatic torad52� shu1� and rad52� csm2� double mutants are no morepsy3� was rad52�. Second, similarly to mutations in HRMMS sensitive than the rad52� single mutant. At the highestgenes, shu mutants suppress top3 slow growth. Finally, MMS concentration used in these experiments (0.015%), dele-

recovery from HU-induced replication arrest in sgs1� tion of SHU1 or CSM2 results in a two- to threefold decreasemutants is thought to be a recombination-linked pro- in survival compared to the wild-type strain.cess. We have observed that this process is affected byshu mutations both on the level of cell survival and onthe level of Rad52-YFP focus formation (Figures 1C and ous concentrations of MMS (Figure 3B). Indeed, we find

that rad52� shu1� and rad52� csm2� double mutants are2C). Thus, we examined the genetic interactions amongshu1�, csm2�, and rad52� mutations by measuring the no more sensitive to MMS than rad52� single mutants,

i.e., that rad52� is epistatic to mutation of SHU1 or CSM2appropriate single- and double-mutant survival in vari-


was affected by deletion of either SHU1 or CSM2 (datanot shown). Increased rDNA recombination has beenlinked to a shortened life span in yeast cells, e.g., in thesgs1� strain (Sinclair et al. 1997). Consistent with wild-type rDNA recombination rates, the shu mutants exhibita normal life span (A. Falcon and J. Aris, unpublishedresults). We also measured recombination at the locuscontaining SUP4-o, the gene encoding a tyrosine tRNAochre suppressor. This locus contains five -sequences(derived from long terminal repeats of the yeast Tytransposon) that are positioned as direct and inverted

Figure 4.—Shu1-YFP-YFP exhibits predominantly nuclear repeats (for details on the locus see Rothstein et al.localization. Differential interference contrast (DIC), YFP, and1987; Shor et al. 2002). Recombination between theseCFP images of a representative field of cells expressing Shu1--repeats by gene conversion (GC) between misalignedYFP-YFP and Nup49-CFP proteins are shown. Nup49 is a nu-

clear envelope protein. The Shu1-YFP-YFP fluorescent signal is sister chromatids or by single-strand annealing (SSA)strongest within the Nup49-CFP-defined nuclear boundaries. can result in deletion of the region containing SUP4-o.

Insertion of the URA3 gene next to SUP4-o can facilitatedetection and quantification of these deletion events

for MMS sensitivity. This observation provides further as they result in the simultaneous loss of both genessupport for the notion that the Shu proteins’ function (Rothstein et al. 1987; Shor et al. 2002). Both top3�is linked to HR. and sgs1� mutants exhibit hyperrecombination at

The Shu proteins are predominantly nuclear: Many SUP4-o, with the degree of the defect greater in theproteins involved in recombinational DNA repair, when top3� strain (Shor et al. 2002). We find that deletiontagged with fluorescent markers, show diffuse nuclear of SHU1 partially suppresses sgs1� and top3� SUP4-olocalization but can relocalize to discrete foci either

hyperrecombination, consistent with the observationspontaneously or upon DNA damage (Lisby et al. 2001,

that shu mutants also suppress other defects of top3� and2003; Shor et al. 2002). To determine whether thissgs1� strains (Figure 5A). Interestingly, in an otherwisepattern holds true for the Shu proteins, we C-terminallywild-type background, deletion of SHU1 or CSM2 resultstagged each of them with YFP at the native chromosomalin a three- to sevenfold increase in the rate of SUP4-olocus using a cloning-free allele replacement methodrecombination (Figure 5A). Overall, we conclude thatand analyzed their localization using a fluorescent mi-the Shu proteins affect certain kinds of recombinationcroscope (Reid et al. 2002). Shu2-YFP and Csm2-YFPevents, such as those leading to deletion formation atdid not yield a signal detectable above the background.the SUP4-o locus, both in an otherwise wild-type back-However, Shu1-YFP and Psy3-YFP did produce a detect-ground and in the absence of Sgs1 or Top3.able, albeit very faint, fluorescent signal that colocalized

In addition to measuring the rate of recombinationwith 4,6-diamidino-2-phenylindole dye, indicating nu-at the SUP4-o locus, it is possible to analyze the structureclear localization (data not shown). To enhance theof the locus in the recombinants by PCR and restrictionsignal of the Shu1-YFP fusion, we introduced anotherenzyme digestion. In this manner, seven SUP4-o dele-copy of YFP at the Shu1-YFP C terminus. The Shu1-YFP-tion classes can be distinguished (Rothstein et al. 1987;YFP construct fully complements MMS sensitivity of theShor et al. 2002). On the basis of the sequence andshu1� strain (data not shown). To precisely delineaterelative positioning of the -sequences in the recombi-the position of nuclei in Shu1-YFP-YFP-containing cells,nants, it was concluded that five of these classes arisewe transformed them with a plasmid containing Nup49-via GC and two via direct repeat recombination by SSACFP, a marker for the nuclear envelope. As shown in(Rothstein et al. 1987). In the wild-type strain, GC isFigure 4, the Shu1-YFP-YFP signal is indeed predomi-the predominant mechanism of deletion formation. HRnantly nuclear. However, we did not observe any Shu1-mutants rad51�, rad52�, rad54�, rad55�, and rad57� areYFP-YFP foci, either spontaneously or in response toseverely impaired in performing GC, so virtually allMMS treatment.SUP4-o deletions generated in these strains belong toMutation of SHU1 or CSM2 affects recombination atthe SSA classes (Shor et al. 2002). On the other hand,the SUP4-o locus: The epistatic relationship between rad-the relative proportions of GC and SSA classes are not52 and the shu mutants suggests a possible function ofsignificantly affected by deletion of either SGS1 or TOP3the Shu proteins in recombination. Thus, we measured(Shor et al. 2002). We analyzed the SUP4-o locus struc-recombination rates in the shu1� and csm2� mutantsture in a number of independently generated recombi-at several loci. Both spontaneous and MMS-inducednants in the shu1� and csm2� strains. We find that themarker loss at the rDNA locus and recombination be-spectrum of SUP4-o deletion classes is not significantlytween leu2 heteroalleles positioned on homologous

chromosomes in the diploid were measured and neither affected by mutation of SHU1 or CSM2 (Figure 5B).


Figure 6.—Deletion of SHU1 or CSM2 results in a mutatorphenotype. The forward mutation rates of the CAN1 gene areshown. The strains shown are meiotic segregants of diploidsESD41, ESD44, and ESD49. (A) Spontaneous CAN1 mutationrates. rad52 is epistatic to shu1 and csm2 : CAN1 mutation ratein the rad52� shu1� and rad52� csm2� double mutants issimilar to that in the single mutants. This graph also showsthat the mutator phenotype of shu1� and csm2� mutants isdependent on the REV3 gene. (B) MMS-induced CAN1 muta-tion rates. Treatment with 0.01% MMS stimulates the CAN1

Figure 5.—Effect of deletion of SHU1 or CSM2 on recombi- mutation rate �35-fold in the wild-type and the mutant strains.nation at the SUP4-o locus in an otherwise wild-type back-ground, as well as in sgs1� and top3� mutants. Diploids ESD45,ESD47, and ESD48 produced the haploid strains shown. (A)

tivity, suggesting that the Shu proteins’ function is con-shu1� and csm2� mutants show a three- to sevenfold increasein the SUP4-o recombination rate. Deletion of SHU1 partially nected to HR. To address whether the mutator pheno-suppresses the hyperrecombination phenotype of sgs1� and type of the shu mutants is due to a HR defect, wetop3� mutants. (B) shu1� and csm2� mutants do not affect measured the CAN1 forward mutation rate in rad52�,SUP4 deletion class distribution. n, number of independently

shu1�, csm2�, rad52� shu1�, and rad52� csm2� mutants.generated SUP4 recombinants tested for each strain.The mutation rates that we obtained for the single mu-tants are similar in magnitude to the ones published byother groups (Swanson et al. 1999; Huang et al. 2003;Genetic characterization of the shu mutator pheno-Figure 6A). Interestingly, the mutation rates of thetype: A study of new genes involved in protecting therad52� shu1� and rad52� csm2� double mutants aregenome from mutagenesis demonstrated that shu1�,similar to those of the single mutants, indicating anshu2�, psy3�, and csm2� strains exhibit increased spon-epistatic relationship between rad52 and the shu muta-taneous forward mutation rates at the CAN1 locustions (Figure 6A). Thus, we conclude that the mutator(Huang et al. 2003). The so-called “mutator” phenotypephenotype of the shu� strains is due to a defect in ais characteristic of mutations that impair any one ofRAD52-dependent process, such as HR.several pathways involved in error-free repair of sponta-

In HR mutants, the majority of mutations introducedneous DNA damage, such as base excision repair (BER),into the CAN1 gene are produced by translesion synthe-the error-free branch of postreplicative repair (PRR),sis (TLS), an error-prone DNA repair process carriedor HR (Swanson et al. 1999; Broomfield and Xiaoout by the low-fidelity polymerase � (Pol �) (Morey et2002). Elimination of the HR pathway by deletion ofal. 2003). Pol �, a complex of Rev3p and Rev7p, canRAD52 increases the CAN1 forward mutation rate ap-insert nucleotides across damaged bases or fill in gapsproximately sevenfold, which is similar to the rate ob-left by the replicative polymerase unable to replicateserved in the shu� strains (Swanson et al. 1999; Huangacross a DNA lesion (Murakumo 2002). To test whetheret al. 2003; Figure 6A). We have shown that mutation

of RAD52 is epistatic to shu1� and csm2� for MMS sensi- TLS is responsible for the increased mutation rate ob-


Figure 7.—Deletion of SHU1 alters Rad52-YFP focus dynamics after DNA damage by MMS. (A) Deletion of SHU1 results inan increase in Rad52-YFP foci upon MMS treatment. Representative samples of wild-type and shu1� cells treated with 0.05%MMS are shown. (B) The graph shows the response of the wild-type strain and the shu1� mutant to increasing concentrationsof MMS. The percentages of S/G2/M (i.e., all budded) cells containing Rad52-YFP foci are plotted. Fluorescent images werecaptured after 100 min of growth in the presence of MMS. At least 150 cells were analyzed for each strain under each condition.(C) Time-lapse analyses of Rad52-YFP foci in cells exposed to 0.01% MMS for 100 min. Duration of individual foci is plotted onthe x-axis vs. the number of foci that lasted for that amount of time. While images of cells were captured every 5 min, the dataare plotted in 10-min intervals to reduce complexity. Thus, in the graph, the bars over the 10-min mark correspond to thenumber of foci that lasted �10 min, the bars over the 20-min mark correspond to the number of foci that lasted between 10and 20 min, etc. The median duration of Rad52-YFP foci is increased from 15 min in the wild-type strain to 25 min in the shu1�mutant. The inset shows the data from untreated cells, where the median duration of Rad52-YFP foci is unchanged by shu1�mutation.

served in the shu mutants, we asked whether the mutator ship indicates that mutation of the SHU genes affectsa RAD52-dependent process, presumably HR. As men-phenotype of the shu1� strain is REV3 dependent. We

combined rev3� with shu1� and measured the CAN1 tioned above, Rad52-YFP focus dynamics are altered bymutations that affect HR and genome stability, such asmutation rate in the double mutant. Indeed, we found

that deletion of REV3 virtually abrogates the mutator top3� and sgs1�, and by exposure to DNA-damagingagents. We find that mutation of SHU1 does not signifi-phenotype of the shu1� mutant, indicating that most

of the mutations introduced into the CAN1 gene in the cantly affect Rad52-YFP focus formation in untreatedcells (Figure 2B). However, since shu mutants are MMSshu1� strain are produced by TLS (Figure 6A).

Certain mutants of the PRR pathway exhibit a sponta- sensitive, we considered the possibility that their effecton Rad52-YFP focus dynamics could become evidentneous mutator phenotype but are defective for damage-

induced mutagenesis (Cassier-Chauvat and Fabre upon MMS treatment. Thus, we treated wild-type andshu1� cells expressing Rad52-YFP with increasing con-1991; Giot et al. 1997). Thus, we measured the rate of

MMS-induced CAN1 forward mutation in the shu1� and centrations of MMS and determined the percentageof cells containing Rad52-YFP foci. In agreement withcsm2� mutants, as well as in the wild-type strain (Figure

6B). We found that treatment with 0.01% MMS in- published data, we find that MMS treatment causes anincrease in the number of Rad52-YFP foci in wild-typecreases the rate of mutagenesis �35-fold in all three

strains tested, indicating that mutation of SHU1 or CSM2 cells (Lisby et al. 2003; Figure 7B). Interestingly, muta-tion of SHU1 exacerbates this effect: MMS-treated shu1�does not impair the cellular ability to induce mutagene-

sis in response to DNA damage by MMS. cells form more Rad52-YFP foci than do wild-type cells,especially at higher concentrations of the drug (FigureRad52 foci dynamics after DNA damage are affected

by mutation of SHU1: Genetic analyses of MMS sensitiv- 7, A and B).How can this observation be explained in light of theity and the mutator phenotype of the shu mutants place

them in the rad52 epistasis group. This genetic relation- epistatic relationship between shu and rad52 mutants?


Mutation of the SHU genes presumably impairs some phenotype (Figure 1); (2) all four genes are in the sameepistasis group (Figure 1); and (3) the proteins interactaspect of RAD52-dependent recombinational repair. Im-

paired function of the recombinational repair factories, among themselves in the two-hybrid assay (Ito et al.2001; Table 2). We also show that rad52 is epistatic toor foci, can result in a delay in focus disassembly. Thus,

an increase in the observed number of Rad52-YFP foci the shu mutants for both MMS sensitivity and mutatorphenotype, indicating that mutation of the Shu proteinscould conceivably be due to the fact that individual foci

last longer in MMS-treated shu1� cells than in wild-type impairs a RAD52-dependent process, presumably HR(Figure 3). Finally, we find that the dynamics of Rad52-cells. To test the idea of longer-lasting foci directly,

we performed time-lapse experiments measuring the YFP foci are affected by mutation of SHU1, particularlyafter MMS treatment (Figure 7). We observe an in-duration of Rad52-YFP foci in untreated and MMS-

treated wild-type and shu1� cells. MMS was added to creased percentage of cells with Rad52-YFP foci afterMMS treatment of the shu1� strain, and time-lapse ex-exponentially growing cells to a concentration of 0.01%.

The cells were immediately mounted onto slides and periments suggest that this phenotype is due to theincreased duration of individual foci. Taken together,placed under the fluorescent microscope, with images

taken every 5 min for 5 hr. The acquired time-lapse these data strongly suggest that the Shu proteins’ func-tion is important for error-free repair of DNA lesionsimages were analyzed, noting the time of appearance

and disappearance of a particular Rad52-YFP focus. via HR.The MMS sensitivity and mutator phenotype of theOnly cells that were budding and/or growing were taken

into account (for every condition used, at least 75% of shu mutants suggest an important role for the Shu pro-teins in protecting the genome from spontaneous andcells present at the beginning of the time lapse satisfied

this requirement). The results of these experiments are induced DNA damage and in promoting error-free DNArepair. MMS-induced DNA lesions can be repaired bygraphed in Figure 7C. In agreement with previously

published data, we find that the median duration of error-free mechanisms, such as PRR or HR (which maybe interlinked) or by error-prone TLS involving poly-Rad52-YFP foci in untreated wild-type cells is 10 min

(Lisby et al. 2003). Deletion of SHU1 does not affect merase � (Barbour and Xiao 2003). Since several re-pair systems can work on the same lesion, mutationthis Rad52-YFP property. Treatment with MMS increases

the median duration of the foci in wild-type cells to 15 of one system increases the load on the others. Thisprinciple is illustrated by the observations that mutationmin. Interestingly, we find that the foci do persist longer

in MMS-treated shu1� cells (the median is 25 min) than of BER or TLS leads to an increase in recombination,while mutation of BER or HR causes a mutator pheno-in wild-type cells. This observation supports the hypothe-

sis that an increase in the overall number of Rad52- type (Swanson et al. 1999). Consistent with this pattern,mutation of the SHU genes causes a mutator phenotypeYFP foci in MMS-treated shu1� cells is due to a longer

persistence of the foci, reflecting the importance of that is dependent on Pol �, showing that when the Shupathway is abolished, the load on TLS is increased. Im-Shu1 in the timely completion of recombinational re-

pair. portantly, we find that mutation of RAD52 is epistaticto mutation of the SHU genes for both MMS sensitivityand the mutator phenotype. This epistatic relationship

DISCUSSIONsuggests that the Shu proteins are involved in error-freerepair via HR.We have isolated four genes, SHU1, SHU2, PSY3, and

CSM2, as mutational suppressors of top3� slow growth. In a previous article, we showed that HR is “toxic” inthe top3� mutants (Shor et al. 2002). We now showMutation of any of these genes also partially rescues

other defects associated with loss of TOP3 or SGS1, such that toxicity of HR genes in the top3� mutant can bealleviated by mutation of the SHU genes. Previously, weas sensitivity to genotoxic agents, increased formation of

Rad52-YFP foci, and hyperrecombination at the SUP4-o hypothesized that, in the absence of Top3, Sgs1 createsa recombinogenic intermediate that is acted upon bylocus (Figures 1, 2, and 5). Mutation of these genes in

an otherwise wild-type background causes sensitivity to HR proteins, causing hyperrecombination, genome in-stability, and slow growth. This model is complicated byMMS and an increased rate of forward mutation in the

CAN1 gene (Hanway et al. 2002; Huang et al. 2003; the notion that HR is also thought to act upstream ofSgs1 (Gangloff et al. 2000; Wu et al. 2001). Thus, takingFigures 1 and 6). CSM2 was originally identified as a

mutation affecting meiotic chromosome segregation into account the genetic data showing that SHU muta-tions impair some aspect of HR, several nonmutually(Rabitsch et al. 2001) and PSY3 as a gene important for

platinum resistance (Wu et al. 2004). These observations exclusive reasons for suppression of top3� by the shumutants exist. For example, in the wild-type strain, theindicate that the four proteins function in DNA metabo-

lism and are presumably involved in error-free repair Shu proteins may be involved in generating the sub-strate for Sgs1. Thus, in the shu mutants, the require-of spontaneous and induced DNA lesions. Moreover,

several lines of evidence suggest that the four proteins ment for a functional Sgs1-Top3 pathway may be at leastpartially bypassed. Alternatively, the Shu proteins mayfunction together in vivo : (1) the mutants have a similar


be involved in the processing of the Sgs1-generated in- Finally, it is conceivable that the Shu-YFPs do form fociupon DNA damage, but these foci are short lived (i.e.,termediate in the top3� mutant with detrimental conse-

quences. In the shu mutants, this intermediate may be under 5 min) and disappear by the time the cells arevisualized under the microscope.channeled into other pathways for resolution, account-

ing for the suppression of top3� defects. Proteins that have established roles in HR have beenassigned these roles both by analyses of their biochemi-The observation that mutation of the SHU genes res-

cues sgs1� HU sensitivity indicates that Shu protein cal activities on DNA substrates in vitro and by measuringrecombination rates at various chromosomal loci in thefunction is detrimental in the absence of Sgs1 when

replication forks pause en masse. The reasons for sgs1� mutants. Mutation of RAD52 affects several kinds of HR,such as heteroallelic recombination between homolo-HU sensitivity are probably manifold since several roles

have been proposed for Sgs1 during DNA replication. gous chromosomes in the diploid, GC, SSA, and break-induced replication (BIR; reviewed in Symington 2002).Sgs1 contributes to both the intra-S checkpoint and the

stabilization of DNA polymerases at stalled forks (Frei At the SUP4-o locus, recombination is virtually elimi-nated in the rad52� mutant, and the few recombinantsand Gasser 2000; Cobb et al. 2003). Also, Sgs1 is thought

to function in maturation and/or resolution of recombi- that arise belong to the SSA classes (Rothstein et al.1987; Shor et al. 2002). On the other hand, mutationnation intermediates at replication forks, preventing

excessive recombination that can be initiated at stalled of RAD51 causes a drastic reduction in GC, but SSA andBIR can still take place. Interestingly, the overall rate offorks and cause genome rearrangement (Kaliraman et

al. 2001; Fabre et al. 2002; Fricke and Brill 2003). We SUP4 recombination increases severalfold in the rad51�mutant, but all the recombinants belong to the SSAimagine that the reasons for the shu suppression of sgs1

HU sensitivity largely mirror the ones proposed above classes (Shor et al. 2002). This observation is explainedby the idea that, in the wild-type strain, SUP4-o recombi-with respect to suppression of top3 slow growth. In sgs1

shu mutants, recombination intermediates that are nor- nation occurs exclusively via GC using the sister chroma-tid as substrate, virtually always resulting in accuratemally resolved by Sgs1 may not form as readily or may

be channeled into another, less detrimental, pathway repair and generating very few deletions. Eliminationof GC by rad51� channels all DNA lesions into thefor resolution. This hypothesis is supported by the par-

tial rescue of increased Rad52-YFP foci in HU-treated SSA pathway, accounting for the observed increase indeletion formation (McDonald and Rothstein 1994).sgs1� cells by mutation of SHU1 (Figure 2C).

Although the epistatic relationship between rad52 Similarly, deletion formation at the SUP4-o locus is in-creased three- to sevenfold in the shu mutants comparedand the shu mutations indicates that the Shu proteins

are important for some aspect of HR, they clearly do to the wild-type strain. However, unlike the situation inthe rad51� mutant, distribution of recombinant classesnot play a direct or essential role in this process. This

conclusion is illustrated by several significant differ- is not significantly altered in the shu mutants, indicatingthat both GC and SSA can still occur. We conclude that,ences between the shu phenotype and the phenotype of

bona fide recombination mutants, like rad51� or rad52�. in the absence of Shu proteins, HR substrates and/orintermediates are processed in a way that is less accurateOne important difference concerns the mutants’ sensi-

tivity to DNA-damaging agents: unlike HR mutants, the and more rearrangement prone compared to the wild-type strain. Moreover, time-lapse analyses of Rad52-YFPshu mutants are not sensitive to HU and exhibit only

mild gamma-ray sensitivity to high doses of radiation foci in the shu1� mutant suggest that shu mutationsimpair not only the accuracy, but also the efficiency of( 1000 Gy; data not shown). These observations indi-

cate that the Shu proteins are not essential for repair of recombinational repair.In conclusion, we have identified mutations in SHU1,DSBs or of stalled replication forks. Also, unlike proteins

directly involved in HR, the Shu proteins (or, at least, SHU2, PSY3, and CSM2 as suppressors of several defectsassociated with mutation of Top3 or Sgs1, such as hyper-Shu1-YFP and Psy3-YFP, the two Shu-YFPs that produced

a fluorescent signal in our hands) do not form foci recombination at the SUP4-o locus, increased incidenceof Rad52-YFP foci, top3� slow growth and HU and MMSeither spontaneously or in response to gamma-ray or

MMS treatment. There are several possible explanations sensitivity, and sgs1� HU sensitivity. Our results suggestthat the Shu proteins function together in vivo in pro-for this observation. Judging by the low intensity of

fluorescence produced by the Shu-YFP constructs, the tecting the genome from spontaneous and inducedDNA damage. Genetic analyses place the SHU genes inShu proteins are not abundant. Even if there is some

aggregation of these proteins at sites of damage, it may the RAD52 epistasis group, implicating them in HR.However, they do not play a direct or essential role innot produce enough localized fluorescence to qualify

as a “focus.” Alternatively, the Shu proteins may not HR, as evidenced by several notable differences betweenthe shu phenotype and that of bona fide HR mutants.need to aggregate to function, unlike HR protein Rad51,

which forms a nucleo-protein filament on DNA, and The Shu proteins, on the other hand, do play an impor-tant role in error-free DNA repair, since in the absenceRad52, which forms 7- to 11-mer ring structures (Rad-

ding 1993; Stasiak et al. 2000; Kagawa et al. 2002). of Shu proteins DNA lesions are channeled into TLS


de La Torre Ruiz et al., 2001 Topoisomerase III acts upstreamand rearrangement-prone recombinational repair routes.of Rad53p in the S-phase DNA damage checkpoint. Mol. Cell.

We have not been able to identify Shu1, Shu2, Psy3, Biol. 21: 7150–7162.Champoux, J. J., 2001 DNA topoisomerases: structure, function, andand Csm2 homologs in organisms other than closely

mechanism. Annu. Rev. Biochem. 70: 369–413.related yeast species. This lack of homologs could beCobb, J. A., L. Bjergbaek, K. Shimada, C. Frei and S. M. Gasser,

due either to other proteins taking on Shu functions 2003 DNA polymerase stabilization at stalled replication forksrequires Mec1 and the RecQ helicase Sgs1. EMBO J. 22: 4325–in higher eukaryotes or to a high degree of divergence4336.between S. cerevisiae Shu proteins and their counterparts

Constantinou, A., M. Tarsounas, J. K. Karow, R. M. Brosh, V. A.in other organisms. Since the Shu proteins do not con- Bohr et al., 2000 Werner’s syndrome protein (WRN) migrates

Holliday junctions and co-localizes with RPA upon replicationtain easily identifiable motifs that yield informationarrest. EMBO Rep. 1: 80–84.about their molecular roles, biochemical analyses are

Cox, M. M., 2001 Historical overview: searching for replication helpnecessary to understand how the Shu proteins function in all of the rec places. Proc. Natl. Acad. Sci. USA 98: 8173–8180.

Doe, C. L., J. Dixon, F. Osman and M. C. Whitby, 2000 Partialat the molecular level. Also, crystallization and determi-suppression of the fission yeast rqh1(-) phenotype by expressionnation of 3-D protein structure can help find structural,of a bacterial Holliday junction resolvase. EMBO J. 19: 2751–2762.

if not sequence, homologs of the Shu proteins. Erdeniz, N., U. H. Mortensen and R. Rothstein, 1997 Cloning-free PCR-based allele replacement methods. Genome Res. 7:We gratefully acknowledge the gifts of two-hybrid plasmids from1174–1183.the Ito laboratory, of a genomic DNA library from the Yamashita

Fabre, F., A. Chan, W. D. Heyer and S. Gangloff, 2002 Alternatelaboratory, and of plasmid pWJ1323 from Michael Lisby. We thank pathways involving Sgs1/Top3, Mus81/Mms4, and Srs2 preventMarian Carlson, Michael Cox, Catherine Fox, James Keck, Michael formation of toxic recombination intermediates from single-Lisby, Robert Reid, Lorraine Symington, and Marisa Wagner for useful stranded gaps created by DNA replication. Proc. Natl. Acad. Sci.discussions. We also thank John Aris and Alaric Falcon for communica- USA 99: 16887–16892.

Frei, C., and S. M. Gasser, 2000 The yeast Sgs1p helicase acts up-tion of unpublished results. E.S. is indebted to the Craig, Fox, Kimble,stream of Rad53p in the DNA replication checkpoint and colocal-and, especially, Cox laboratories at the University of Wisconsin forizes with Rad53p in S-phase-specific foci. Genes Dev. 14: 81–96.providing laboratory materials and support during 2002 and 2003.

Fricke, W. M., and S. J. Brill, 2003 Slx1-Slx4 is a second structure-This work was supported by a National Science Foundation Graduatespecific endonuclease functionally redundant with Sgs1-Top3.Fellowship to E.S. and by National Institutes of Health grant GM50237Genes Dev. 17: 1768–1778.

to R.R. Fricke, W. M., V. Kaliraman and S. J. Brill, 2001 Mapping theDNA topoisomerase III binding domain of the Sgs1 DNA helicase.J. Biol. Chem. 276: 8848–8855.

Gangloff, S., J. P. McDonald, C. Bendixen, L. Arthur and R.LITERATURE CITEDRothstein, 1994 The yeast type I topoisomerase Top3 interactswith Sgs1, a DNA helicase homolog: a potential eukaryotic reverseAkada, R., J. Yamamoto and I. Yamashita, 1997 Screening and

identification of yeast sequences that cause growth inhibition gyrase. Mol. Cell. Biol. 14: 8391–8398.Gangloff, S., B. de Massy, L. Arthur, R. Rothstein and F. Fabre,when overexpressed. Mol. Gen. Genet. 254: 267–274.

Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman 1999 The essential role of yeast topoisomerase III in meiosisdepends on recombination. EMBO J. 18: 1701–1711.et al., 1998 Current Protocols in Molecular Biology. John Wiley &

Sons, New York. Gangloff, S., C. Soustelle and F. Fabre, 2000 Homologous recom-bination is responsible for cell death in the absence of the Sgs1Barbour, L., and W. Xiao, 2003 Regulation of alternative replica-

tion bypass pathways at stalled replication forks and its effects and Srs2 helicases. Nat. Genet. 25: 192–194.Giot, L., R. Chanet, M. Simon, C. Facca and G. Faye, 1997 Involve-on genome stability: a yeast model. Mutat. Res. 532: 137–155.

Bendixen, C., S. Gangloff and R. Rothstein, 1994 A yeast mating- ment of the yeast DNA polymerase delta in DNA repair in vivo.Genetics 146: 1239–1251.selection scheme for detection of protein-protein interactions.

Nucleic Acids Res. 22: 1778–1779. Haaf, T., E. I. Golub, G. Reddy, C. M. Radding and D. C. Ward,1995 Nuclear foci of mammalian Rad51 recombination proteinBennett, R. J., J. A. Sharp and J. C. Wang, 1998 Purification and

characterization of the Sgs1 DNA helicase activity of Saccharomyces in somatic cells after DNA damage and its localization in synapto-nemal complexes. Proc. Natl. Acad. Sci. USA 92: 2298–2302.cerevisiae. J. Biol. Chem. 273: 9644–9650.

Bennett, R. J., J. L. Keck and J. C. Wang, 1999 Binding specificity Hanway, D., J. K. Chin, G. Xia, G. Oshiro, E. A. Winzeler et al.,2002 Previously uncharacterized genes in the UV- and MMS-determines polarity of DNA unwinding by the Sgs1 protein of S.

cerevisiae. J. Mol. Biol. 289: 235–248. induced DNA damage response in yeast. Proc. Natl. Acad. Sci.USA 99: 10605–10610.Bennett, R. J., M. F. Noirot-Gros and J. C. Wang, 2000 Interaction

between yeast Sgs1 helicase and DNA topoisomerase III. J. Biol. Harmon, F. G., R. J. DiGate and S. C. Kowalczykowski, 1999 RecQhelicase and topoisomerase III comprise a novel DNA strandChem. 275: 26898–26905.

Bischof, O., S. H. Kim, J. Irving, S. Beresten, N. A. Ellis et al., passage function: a conserved mechanism for control of DNArecombination. Mol. Cell 3: 611–620.2001 Regulation and localization of the Bloom syndrome pro-

tein in response to DNA damage. J. Cell Biol. 153: 367–380. Hickson, I. D., 2003 RecQ helicases: caretakers of the genome. Nat.Rev. Cancer 3: 169–178.Broomfield, S., and W. Xiao, 2002 Suppression of genetic defects

within the RAD6 pathway by srs2 is specific for error-free post- Huang, M. E., A. G. Rio, A. Nicolas and R. D. Kolodner, 2003A genomewide screen in Saccharomyces cerevisiae for genes thatreplication repair but not for damage-induced mutagenesis. Nu-

cleic Acids Res. 30: 732–739. suppress the accumulation of mutations. Proc. Natl. Acad. Sci.USA 100: 11529–11534.Brosh, R. M., Jr., J. L. Li, M. K. Kenny, J. K. Karow, M. P. Cooper

et al., 2000 Replication protein A physically interacts with the Ito, T., T. Chiba, R. Ozawa, M. Yoshida, M. Hattori et al., 2001A comprehensive two-hybrid analysis to explore the yeast proteinBloom’s syndrome protein and stimulates its helicase activity. J.

Biol. Chem. 275: 23500–23508. interactome. Proc. Natl. Acad. Sci. USA 98: 4569–4574.James, P., J. Halladay and E. A. Craig, 1996 Genomic librariesCassier-Chauvat, C., and F. Fabre, 1991 A similar defect in UV-

induced mutagenesis conferred by the rad6 and rad18 mutations and a host strain designed for highly efficient two-hybrid selectionin yeast. Genetics 144: 1425–1436.of Saccharomyces cerevisiae. Mutat. Res. 254: 247–253.

Cha, R. S., and N. Kleckner, 2002 ATR homolog Mec1 promotes Kagawa, W., H. Kurumizaka, R. Ishitani, S. Fukai, O. Nureki etal., 2002 Crystal structure of the homologous-pairing domainfork progression, thus averting breaks in replication slow zones.

Science 297: 602–606. from the human Rad52 recombinase in the undecameric form.Mol. Cell 10: 359–371.Chakraverty, R. K., J. M. Kearsey, T. J. Oakley, M. Grenon, M. A.


Kaliraman, V., J. R. Mullen, W. M. Fricke, S. A. Bastin-Shanower alterations in Saccharomyces cerevisiae using adaptamer-mediatedPCR. Methods Enzymol. 350: 258–277.and S. J. Brill, 2001 Functional overlap between Sgs1-Top3

and the Mms4-Mus81 endonuclease. Genes Dev. 15: 2730–2740. Rose, M. D., F. Winston and P. Heiter, 1990 Methods in Yeast Genet-ics: A Laboratory Course Manual. Cold Spring Harbor LaboratoryKarow, J. K., A. Constantinou, J. L. Li, S. C. West and I. D. Hickson,

2000 The Bloom’s syndrome gene product promotes branch Press, Cold Spring Harbor, NY.Rothstein, R., C. Helms and N. Rosenberg, 1987 Concerted dele-migration of Holliday junctions. Proc. Natl. Acad. Sci. USA 97:

6504–6508. tions and inversions are caused by mitotic recombination betweendelta sequences in Saccharomyces cerevisiae. Mol. Cell. Biol. 7: 1198–Khakhar, R. R., J. A. Cobb, L. Bjergbaek, I. D. Hickson and S. M.

Gasser, 2003 RecQ helicases: multiple roles in genome mainte- 1207.Sherman, F., G. R. Fink and J. B. Hicks, 1986 Methods in Yeastnance. Trends Cell Biol. 13: 493–501.

Krejci, L., S. Van Komen, Y. Li, J. Villemain, M. S. Reddy et al., Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Har-bor, NY.2003 DNA helicase Srs2 disrupts the Rad51 presynaptic fila-

ment. Nature 423: 305–309. Shor, E., S. Gangloff, M. Wagner, J. Weinstein, G. Price et al.,2002 Mutations in homologous recombination genes rescueKwan, K. Y., and J. C. Wang, 2001 Mice lacking DNA topoisomerase

IIIbeta develop to maturity but show a reduced mean lifespan. top3 slow growth in Saccharomyces cerevisiae. Genetics 162: 647–662.Sinclair, D. A., K. Mills and L. Guarente, 1997 Accelerated agingProc. Natl. Acad. Sci. USA 98: 5717–5721.

Lea, D. E., and C. A. Coulson, 1949 The distribution in the numbers and nucleolar fragmentation in yeast sgs1 mutants. Science 277:1313–1316.of mutants in bacterial populations. J. Genet. 49: 264–285.

Li, W., and J. C. Wang, 1998 Mammalian DNA topoisomerase IIIal- Sogo, J. M., M. Lopes and M. Foiani, 2002 Fork reversal and ssDNAaccumulation at stalled replication forks owing to checkpointpha is essential in early embryogenesis. Proc. Natl. Acad. Sci.

USA 95: 1010–1013. defects. Science 297: 599–602.Stasiak, A. Z., E. Larquet, A. Stasiak, S. Muller, A. Engel et al.,Lisby, M., R. Rothstein and U. H. Mortensen, 2001 Rad52 forms

DNA repair and recombination centers during S phase. Proc. 2000 The human Rad52 protein exists as a heptameric ring.Curr. Biol. 10: 337–340.Natl. Acad. Sci. USA 98: 8276–8282.

Lisby, M., U. H. Mortensen and R. Rothstein, 2003 Colocalization Stewart, E., C. R. Chapman, F. Al-Khodairy, A. M. Carr and T.Enoch, 1997 rqh1�, a fission yeast gene related to the Bloom’sof multiple DNA double-strand breaks at a single Rad52 repair

centre. Nat. Cell Biol. 5: 572–577. and Werner’s syndrome genes, is required for reversible S phasearrest. EMBO J. 16: 2682–2692.Lisby, M., J. H. Barlow, R. C. Burgess and R. Rothstein, 2004

Choreography of the DNA damage response: spatiotemporal rela- Swanson, R. L., N. J. Morey, P. W. Doetsch and S. Jinks-Robertson,1999 Overlapping specificities of base excision repair, nucleo-tionships among checkpoint and repair proteins. Cell 118: 699–

713. tide excision repair, recombination, and translesion synthesispathways for DNA base damage in Saccharomyces cerevisiae. Mol.Lopes, M., C. Cotta-Ramusino, A. Pellicioli, G. Liberi, P. Plevani

et al., 2001 The DNA replication checkpoint response stabilizes Cell. Biol. 19: 2929–2935.Symington, L. S., 2002 Role of RAD52 epistasis group genes installed replication forks. Nature 412: 557–561.

homologous recombination and double-strand break repair. Mi-Lucca, C., F. Vanoli, C. Cotta-Ramusino, A. Pellicioli, G. Libericrobiol. Mol. Biol. Rev. 66: 630–670.et al., 2004 Checkpoint-mediated control of replisome-fork asso-

Thomas, B. J., and R. Rothstein, 1989 Elevated recombinationciation and signalling in response to replication pausing. Onco-rates in transcriptionally active DNA. Cell 56: 619–630.gene 23: 1206–1213.

van Brabant, A. J., T. Ye, M. Sanz, I. J. German, N. A. Ellis et al.,McDonald, J. P., and R. Rothstein, 1994 Unrepaired hetero-2000 Binding and melting of D-loops by the Bloom syndromeduplex DNA in Saccharomyces cerevisiae is decreased in RAD1helicase. Biochemistry 39: 14617–14625.RAD52-independent recombination. Genetics 137: 393–405.

Veaute, X., J. Jeusset, C. Soustelle, S. C. Kowalczykowski, E. LeMcVey, M., M. Kaeberlein, H. A. Tissenbaum and L. Guarente,Cam et al., 2003 The Srs2 helicase prevents recombination by2001 The short life span of Saccharomyces cerevisiae sgs1 and srs2disrupting Rad51 nucleoprotein filaments. Nature 423: 309–312.mutants is a composite of normal aging processes and mitotic

Wallis, J. W., G. Chrebet, G. Brodsky, M. Rolfe and R. Rothstein,arrest due to defective recombination. Genetics 157: 1531–1542.1989 A hyper-recombination mutation in S. cerevisiae identifiesMichel, B., M. J. Flores, E. Viguera, G. Grompone, M. Seigneura novel eukaryotic topoisomerase. Cell 58: 409–419.et al., 2001 Rescue of arrested replication forks by homologous

Watt, P. M., E. J. Louis, R. H. Borts and I. D. Hickson, 1995 Sgs1:recombination. Proc. Natl. Acad. Sci. USA 98: 8181–8188.a eukaryotic homolog of E. coli RecQ that interacts with topoisom-Morey, N. J., P. W. Doetsch and S. Jinks-Robertson, 2003 Deline-erase II in vivo and is required for faithful chromosome segrega-ating the requirements for spontaneous DNA damage resistancetion. Cell 81: 253–260.pathways in genome maintenance and viability in Saccharomyces

Watt, P. M., I. D. Hickson, R. H. Borts and E. J. Louis, 1996 SGS1,cerevisiae. Genetics 164: 443–455.a homologue of the Bloom’s and Werner’s syndrome genes, isMullen, J. R., V. Kaliraman, S. S. Ibrahim and S. J. Brill, 2001required for maintenance of genome stability in SaccharomycesRequirement for three novel protein complexes in the absencecerevisiae. Genetics 144: 935–945.of the Sgs1 DNA helicase in Saccharomyces cerevisiae. Genetics 157:

Wu, H. I., J. A. Brown, M. J. Dorie, L. Lazzeroni and J. M. Brown,103–118. 2004 Genome-wide identification of genes conferring resis-Murakumo, Y., 2002 The property of DNA polymerase zeta: REV7 tance to the anticancer agents cisplatin, oxaliplatin, and mitomy-is a putative protein involved in translesion DNA synthesis and cin C. Cancer Res. 64: 3940–3948.cell cycle control. Mutat. Res. 510: 37–44. Wu, L., and I. D. Hickson, 2003 The Bloom’s syndrome helicase

Oakley, T. J., and I. D. Hickson, 2002 Defending genome integrity suppresses crossing over during homologous recombination. Na-during S-phase: putative roles for RecQ helicases and topoisomer- ture 426: 870–874.ase III. DNA Repair 1: 175–207. Wu, L., S. L. Davies, P. S. North, H. Goulaouic, J. F. Riou et

Rabitsch, K. P., A. Toth, M. Galova, A. Schleiffer, G. Schaffner al., 2000 The Bloom’s syndrome gene product interacts withet al., 2001 A screen for genes required for meiosis and spore topoisomerase III. J. Biol. Chem. 275: 9636–9644.formation based on whole-genome expression. Curr. Biol. 11: Wu, L., S. L. Davies, N. C. Levitt and I. D. Hickson, 2001 Potential1001–1009. role for the BLM helicase in recombinational repair via a con-

Radding, C. M., 1993 A universal recombination filament. Curr. served interaction with RAD51. J. Biol. Chem. 276: 19375–19381.Biol. 3: 358–360.

Reid, R. J., M. Lisby and R. Rothstein, 2002 Cloning-free genome Communicating editor: M. Lichten

Documents

A Genetic Screen for top3 Suppressors in Saccharomyces ... · The sole RecQ helicase in budding yeast, Sgs1, interacts with Top3 both physically and genetically, and the two proteins