Mutations in Recombinational Repair and in Checkpoint Control Genes Suppress the Lethal Combination of Srs2δ with Other DNA

Mutations in Recombinational Repair and in Checkpoint Control Genes Suppress the Lethal Combination of srs2⌬ With Other DNA Repair Genes in Saccharomyces cerevisiae

Hannah L. Klein Department of Biochemistry and Kaplan Cancer Center, New York University School of Medicine, New York, New York 10016 Manuscript received September 5, 2000 Accepted for publication November 8, 2000

ABSTRACT The SRS2 gene of Saccharomyces cerevisiae encodes a DNA helicase that is active in the postreplication repair pathway and homologous recombination. srs2 mutations are lethal in a rad54⌬ background and cause poor growth or lethality in rdh54⌬, rad50⌬, mre11⌬, xrs2⌬, rad27⌬, sgs1⌬, and top3⌬ backgrounds. Some of these genotypes are known to be defective in double-strand break repair. Many of these lethalities or poor growth can be suppressed by mutations in other genes in the DSB repair pathway, namely rad51, rad52, rad55, and rad57, suggesting that inhibition of recombination at a prior step prevents formation of a lethal intermediate. Lethality of the srs2⌬ rad54⌬ and srs2⌬ rdh54⌬ double mutants can also be rescued by mutations in the DNA damage checkpoint functions RAD9, RAD17, RAD24, and MEC3, indicating that the srs2 rad54 and srs2 rdh54 mutant combinations lead to an intermediate that is sensed by these checkpoint functions. When the checkpoints are intact the cells never reverse from the arrest, but loss of the checkpoints releases the arrest. However, cells do not achieve wild-type growth rates, suggesting that unrepaired damage is still present and may lead to chromosome loss.

HE SRS2 gene was first identified through domi- tant diploid strains as compared to mutant haploid Tnant mutations that suppressed trimethoprim sensi- strains (Aboussekhra et al. 1989). This is unusual and tivity of rad6 and rad18 mutants (Lawrence and Chris- has been interpreted as lethal recombination events tensen 1979). It was again recovered as a suppressor of occurring between homologous chromosomes in the damage sensitivity of the rad18 mutant (Aboussekhra et absence of Srs2 function (Aboussekhra et al. 1989). al. 1989) and as a mutant that showed increased sponta- Thus in the mutant the haploid has a hyperrecombina- neous mitotic gene conversion (Rong et al. 1991). These tion phenotype while in the diploid some of the interho- studies placed SRS2 in the RAD6 epistasis group for molog recombination events are lethal, while other re- DNA repair, in an error-free postreplication repair path- combination events such as intragenic recombination way. The sequence of the SRS2 gene revealed high ho- are increased. To make the situation even more compli- mology to the bacterial repair gene uvrD, encoding heli- cated, the suppression of rad6 and rad18 mutants by case II (Aboussekhra et al. 1989). Subsequently, it was srs2 mutation is thought to occur by the channeling of demonstrated that the Srs2 protein has DNA helicase repair substrates into the RAD52 recombination repair activity (Rong and Klein 1993). While the biochemical pathway (Aboussekhra et al. 1989; Rong et al. 1991). activity of SRS2 indicates a function in DNA metabolic Fourth, in meiosis the gene is required for full spore and %50ف activity, the precise role of the gene has remained enig- viability, and in its absence spore viability is matic. map distances for the LEU2-HIS4 interval and the HIS4- First, it is not clear why srs2 suppressor alleles are MAT interval are reduced 2-fold (Palladino 1991). semidominant for the suppressor effect of rad6 or rad18 Further clues as to the functional role of SRS2 have DNA damage sensitivity phenotypes. Second, SRS2 hap- come from identification of double mutant combina- loid mutants have a 5- to 10-fold increased sensitivity to tions with srs2 mutants that are lethal or exhibit ex- UV damage (Aboussekhra et al. 1989; Palladino and tremely poor growth and from suppressors of those srs2 Klein 1992), which is significant but is not of the magni- mutant phenotypes. srs2 rad50 double mutants grow tude of other UV-sensitive mutants. Haploid srs2 mu- very poorly (Rong et al. 1991; Palladino and Klein tants are not sensitive to X-rays while diploid mutants 1992). This most likely reflects an essential role for are sensitive (Aboussekhra et al. 1989). Third, all the RAD50 in recombinational repair involving sister chro- mitotic DNA damage sensitivities are enhanced in mu- matids (Saeki et al. 1980) and suggests that SRS2 functions in G2 repair in haploids. The diploid DNA damage sensitivity and lethal recombination of the srs2 mutant Address for correspondence: Department of Biochemistry, New York University School of Medicine, 550 First Ave., New York, NY 10016. is suppressed by semidominant mutations in RAD51 E-mail: [email protected] (Aboussekhra et al. 1992), which encodes a RecA-like

Genetics 157: 557–565 (February 2001) 558 H. L. Klein protein that functions in recombinational repair (Shi- MATERIALS AND METHODS nohara et al. 1992), again placing SRS2 in a step in Yeast strains: Parental strains used in this study are listed homologous recombination that may occur after the in Table 1. All strains are isogenic and are in the W303 back- RAD51-mediated step, although an alternate interpreta- ground (W303 genotype: leu2-3, 112 his3-11, 15 ade2-1 ura3-1 tion involves negative regulation of homologous recom- trp1-1 can1-1 RAD5). sgs1 disruption strains were a gift from bination pathways by SRS2. srs2 mutants also suppress Rodney Rothstein. The mre11 and exo1 strains were a gift from Lorraine Symington. The rad9, rad17, rad24, and mec3 strains nonnull semidominant alleles of RAD51 (Milne et al. were a gift from Ted Weinert. Strains, if needed, were con- 1995; Chanet et al. 1996). This has been interpreted verted to the RAD5 version of W303 through crosses. The rad54 as a function for the Srs2 helicase in reversing abortive point mutation strains have been described (Petukhova et recombination intermediates. al. 1999). All double and triple mutant strains were derived from crosses using the parental single mutant strains listed in We have previously reported that srs2 mutations are Table 1. All recombination and checkpoint mutants are null lethal in a rad54 null mutant background (Palladino alleles with the exception of the two indicated rad54 point and Klein 1992). This lethality is suppressed by muta- mutants and the one indicated mre11 point mutant. tions in RAD51, RAD52, RAD55, and RAD57 (Schild Media and growth conditions: Standard media were pre- 1995). These genes function in the recombinational pared as described (Sherman et al. 1986). All strains were grown on solid media at 30Њ. repair pathway of yeast and are required for homologous recombination (Saeki et al. 1980). These results implicate SRS2 in recombinational repair, although RESULTS epistasis analysis does not place SRS2 in the RAD52 repair pathway. Nonetheless, there is an interaction be- srs2 and rad54: We have previously reported that the tween SRS2 and RAD52 as null alleles of SRS2 are able srs2 rad54 double null mutant is lethal (Palladino and to suppress nonnull alleles of RAD52 (Kaytor et al. Klein 1992). This lethality is suppressed by loss of the 1995; Milne et al. 1995; Schild 1995). Although the homologous recombination functions of RAD51, RAD52, mechanism of suppression is not understood, it may RAD55,orRAD57 (Schild 1995 and this study). However, reflect a need for the Srs2 protein to stabilize and pro- not all mutations of genes involved in homologous recom- mote recombination between substrates with limited bination or repair of a double-strand break rescue the srs2 rad54 lethality. The RAD1 gene encodes an endonu- homology (Paques and Haber 1997). The rad52 alleles clease that is active in nucleotide excision repair and may attempt recombination between sequences of re- some recombination reactions (Klein 1988; Schiestl duced homology and this reduced fidelity recombina- and Prakash 1988; Thomas and Rothstein 1989; tion requires SRS2. In the absence of functional Srs2p, Siede et al. 1993; Tomkinson et al. 1993; Ivanov and aberrant recombination would be prevented and the Haber 1995; Kirkpatrick and Petes 1997). However, rad52 alleles would be suppressed. a rad1 null mutation was not able to rescue the srs2⌬ Additional evidence for SRS2 functioning in recombi- rad54⌬ strain. nation comes from studies of the interaction between RAD54 encodes a protein of the SNF2/SW12 family SRS2 and SGS1. SGS1 encodes a DNA helicase related and contains DNA helicase consensus motifs (Eisen et to the RecQ family of helicases (Gangloff et al. 1994; al. 1995). The protein has demonstrated in vitro ATPase Watt et al. 1995; Lu et al. 1996). sgs1 mutant strains are activity and promotes Rad51-mediated strand exchange characterized by increased genomic instability (Watt (Petukhova et al. 1998) via a change in DNA double et al. 1996; Yamagata et al. 1998). Although the single helix conformation (Petukhova et al. 1999). Two point null allele mutant strains exhibit normal growth, the mutations in the first helicase consensus domain, double mutant srs2 sgs1 is inviable or grows extremely rad54K341R and rad54K341A, have been shown to abol- poorly (Lee et al. 1999; Gangloff et al. 2000). The ish ATP hydrolysis and Rad51-promoted homologous growth defect of the srs2 sgs1 double mutant can be DNA pairing as well as mitotic recombination (Petu- suppressed by mutations in the RAD51, RAD55,or khova et al. 1999). Both of these mutations in a srs2⌬ RAD57 genes (Gangloff et al. 2000), indicating that background are lethal, showing that the essential func- the growth defect is caused by attempted homologous tion provided by Rad54 protein in a srs2⌬ strain requires recombination that cannot be completed when both the ATPase activity. Srs2p and Sgs1p are missing. Our studies on rad54 rdh54 diploid double mutants To gain additional insight into the biological role of had suggested that the double mutant poor growth was SRS2, we have examined other DNA recombination due to the accumulation of DNA damage through at- functions for synthetic interactions with a srs2 null allele tempted recombination that was sensed by the DNA strain. We have also asked what types of mutants sup- damage checkpoint functions. This finding prompted press srs2 phenotypes. The results link SRS2 to homolo- us to speculate that the srs2 rad54 lethality might be due gous recombination that is associated with DNA replica- to the accumulation of DNA damage from attempted tion and DNA damage that is sensed by DNA damage recombination that was sensed by the DNA damage checkpoints. checkpoint functions. Therefore the role of the check- srs2 Suppressors 559

TABLE 1 Strain list

Strains Genotype HKY590-1D MATa srs2::HIS3 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY590-6D MAT␣ srs2::HIS3 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY723-6C MATa srs2::TRP1 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY604-17C MATa rad50::hisG leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY603-15B MATa xrs2::URA3 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY596-1A MATa rad54::LEU2 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY596-2B MAT␣ rad54::LEU2 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY624 MATa rad54::HIS3 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY837 MATa rad54K314A leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY838 MATa rad54K341R leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY615-1A MAT␣ rad1::LEU2 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY1039-4D MAT␣ rad51::HIS3 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY614-10B MAT␣ rad52::TRP1 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY597-2C MAT␣ rad55::LEU2 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY598-8B MAT␣ rad57::LEU2 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY606-1A MATa top3::LEU2 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY606-4D MAT␣ top3::LEU2 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY619-3C MATa rad27::TRP1 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY845-1A MATa rad9::HIS3 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY843-1B MATa rad17::LEU2 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY844-11A MATa rad24::TRP1 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 HKY846-2A MATa mec3::TRP1 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 1958-10A MAT␣ sgs1::URA3 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 LSY796-3D MATa sgs1::URA3 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 LSY624 MATa exo1::URA3 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 LSY569 MAT␣ mre11::LEU2 leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 LSY726 MAT␣ mre11H125N::URA3::mre11D56N leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 All strains are in the W303 RAD5 background. Strain 1958-10A was received from Rodney Rothstein. Strains LSY796-3D, LSY624, LSY726, and LSY569 were received from Lorraine Symington. point functions in preventing srs2 rad54 cells from prog- related sequences, but the single mutants have unique ressing through the cell cycle was assessed by determin- effects on mitotic recombination and meiotic viability ing the growth phenotype of triple mutants. Triple (Klein 1997; Shinohara et al. 1997). We have pre- mutants were segregated in crosses. An example of such viously reported that, while in contrast to the srs2 rad54 a cross is shown in Figure 1. In Figure 1A growth of haploid mutant lethality the srs2 rdh54 haploid mutant colonies of the genotype srs2 rad54 rad17, srs2 rad54 shows normal growth, lethality is exhibited in the srs2 rad24,orsrs2 rad54 mec3 is compared to a wild-type rdh54 diploid (Klein 1997). Moreover, this lethality is strain. While the triple mutant strains grow slower than suppressed by mutation in any one of the recombination wild type and throw off colonies of varying sizes, these repair genes RAD51, RAD52, RAD55,orRAD57 (Klein genotypes are viable. In contrast, a rad9 mutation is 1997). Similar to the suppression of srs2 rad54 by DNA unable to rescue the srs2 rad54 double mutant (Figure damage checkpoint mutations, the srs2 rdh54 diploid 1B). Figure 1, B and C compares the ability of a rad51 lethality is suppressed by mutations in RAD17, RAD24, mutation vs. a rad17 mutation to suppress the srs2 rad54 or MEC3, but not by mutation of the RAD9 gene (Ta- lethality. It can be seen that rad51 is more effective in ble 2). suppressing the srs2 rad54 lethality, suggesting that the srs2 and rad50/xrs2/mre11: The Rad50, Xrs2, and checkpoint mutants suppress the double mutant by re- Mre11 proteins function in a complex to protect DNA lease from growth arrest, but DNA damage is still pres- ends in end-joining reactions and in meiotic recombina- ent. Further support for this statement comes from plat- tion (for a review see Haber 1998). The RAD50, XRS2, ing efﬁciencies. One hundred unbudded cells of the and MRE11 genes are not required for mitotic homolog srs2 rad54 rad17 and srs2 rad54 rad51 genotypes were recombination, but are required for recombination be- micromanipulated to fresh YPD plates. A total of 50/ tween sister chromatids (Saeki et al. 1980). The srs2 null 100 srs2 rad54 rad17 cells grew to visible colonies whereas allele mutant has a synthetic growth interaction with 93/100 srs2 rad54 rad51 cells grew to visible colonies. null allele mutations in RAD50, XRS2,orMRE11 (see srs2 and rdh54: The RAD54 and RDH54 genes have Figure 2A) and this is suppressed by mutations in the 560 H. L. Klein

2B and Table 3). The plating efficiency of 100 micromanipulated unbudded cells from a spore colony of the srs2 rad50 genotype was 49/100 while the plating efficiency of 100 micromanipulated unbudded cells from a spore colony of the srs2 rad50 rad17 genotype was 34/100. All colonies that grew were extremely small compared to a wild-type strain. MRE11 encodes a protein with single-stranded endo- nuclease activity (Moreau et al. 1999). Mutation of consensus motifs in the phosphodiesterase domains abol- ishes the nuclease activity, but has little effect on mitotic growth, recombination, end-joining or telomere length (Moreau et al. 1999). The effect of mre11-nuclease-deficient alleles on growth of srs2 strains was examined (Figure 2A). The Mre11 nuclease activity is not required for viability of srs2 mutants. Since the double null allele growth defect is rescued by recombination repair mutations, this suggests that the poor growth is due to attempted recombination. The fact that the srs2 mre11- nuclease-deficient strain is viable indicates that recombinational repair is not impaired in this strain. RAD50, XRS2, and MRE11 are also required for telomere length control and in the mutants telomeres are shortened (Kironmai and Muniyappa 1997; Le et al. 1999). Since double mutants of srs2 combined with either rad50, xrs2,ormre11 show poor growth, one possi- bility is that telomere length is greatly altered in the double mutant. This was excluded by first showing that srs2 strains have normal telomere lengths and then ob- serving that srs2 rad50 strains have telomeres of the rad50 characteristic length (H. Klein and R. Boguslav- sky, data not shown). srs2 and exo1: The EXO1 gene encodes an exonuclease that functions in mitotic recombination (Fiorentini et al. 1997). The exo1 mutant when combined with a mutant in nuclease involved in DNA replication is lethal (Tishkoff et al. 1997a). This property is shared with genes involved in recombinational repair (Symington Figure 1.—Suppression of srs2⌬ rad54⌬ lethality through 1998). Since the srs2 mutant also has genetic interac- loss of homologous recombination functions or DNA damage checkpoint functions. Five tetrads from crosses indicated tions with the same set of genes, the phenotype of the above each picture are shown. (A) Suppression by rad17⌬, double mutant srs2 exo1 was determined. The double rad24⌬,ormec3⌬ is shown. Strains of each genotype were mutant srs2 exo1 was fully viable, showing that neither generated by crossing a srs2⌬ rad17⌬/rad24⌬/mec3⌬ strain to gene is required for any essential function when the a rad54⌬ strain. Growth is slightly reduced compared to a other gene is defective. wild-type control strain. (B) The ability of a rad9⌬ mutation to suppress is compared to the ability of a rad17⌬ mutation to srs2 sgs1 and srs2 top3: SGS1 encodes a DNA helicase suppress. srs2⌬ rad54⌬ double mutants are outlined in circles of the same polarity as the Srs2 DNA helicase (Lu et al. while the triple mutants srs2⌬ rad54⌬ rad9⌬/rad17⌬ are out- 1996; Bennett et al. 1998). It has been reported that lined in squares. It can be seen that the srs2⌬ rad54⌬ rad17⌬ the double mutant srs2 sgs1 is lethal or grows extremely strain is viable but grows more slowly than other viable geno- poorly (Lee et al. 1999; Gangloff et al. 2000). This types. (C) The ability of a rad51⌬ mutation to suppress srs2⌬ rad54⌬ is shown. Circles indicate the srs2⌬ rad54⌬ double growth phenotype is due to attempted recombination mutants while the triple mutants srs2⌬ rad54⌬ rad51⌬ are as mutations in the recombination repair genes RAD51, outlined in squares. It can been seen that a rad51⌬ mutation RAD52, RAD55,orRAD57 rescue the growth defect. We is a stronger suppressor than the DNA damage checkpoint have confirmed these observations. sgs1 cells have been mutations. found to be partially defective for an intra-S checkpoint for DNA damage that acts in parallel with the RAD24 recombination repair genes (Table 3). However, the checkpoint function (Frei and Gasser 2000). There- srs2 rad50 poor growth was not suppressed by mutation fore, it was of interest to examine the phenotype of the of the DNA damage checkpoint function RAD17 (Figure srs2 sgs1 rad24 triple mutant. We found that the rad24 srs2 Suppressors 561

TABLE 2 Summary of srs2 interactions with rad54 or rdh54 mutants

Suppressed by Suppressed by rad51, rad52, Suppressed rad17, rad24, Suppressed Strain Phenotype rad55,orrad57 a by rad1 or mec1a by rad9 srs2⌬ rad54⌬ Lethal Yes No Yes No srs2⌬ rad54K341R Lethal ND ND ND ND srs2⌬ rad54K341A Lethal ND ND ND ND srs2⌬ rdh54⌬ Haploid is viable Yes, for diploid No, for diploid Yes, for diploid No, for diploid Diploid is lethal ND, not done. a Suppression was tested for each mutant individually. mutant did not suppress the lethality or poor growth Top3 protein, which has type I topoisomerase activity. of the srs2 sgs1 double mutant (Figure 3). Suppression Moreover, the slow growth phenotype of the top3 mutant was also measured by plating efficiency of micromanipu- is suppressed by a sgs1 mutation (Gangloff et al. 1994). lated unbudded cells from spore colonies onto fresh Therefore we examined the phenotype of the srs2 top3 growth medium. The srs2 strain had a high plating effi- double mutant. The double mutant is lethal (Fig- ciency (47/50) while the sgs1 mutant plating efficiency ure 4) and this lethality is also suppressed by loss of the was moderate (36/50). Although spores of the srs2 sgs1 recombination repair pathway through mutations in RAD- genotype were viable, the growth was slow and the plat- 51, RAD52, RAD55,orRAD57 (Figure 4 and Table 3). ing efficiency was 0/50. The presence of a rad24 muta- srs2 and rad27: RAD27 encodes an exonuclease that tion in this genotype increased the plating efficiency to is involved in the completion of lagging strand synthesis 31/100 (srs2 sgs1 rad24 genotype), but the colonies grew (Harrington and Lieber 1994; Reagan et al. 1995; very slowly and never reached wild-type size. Sommers et al. 1995). rad27 mutants have elevated rates The Sgs1 protein of yeast interacts physically with the of recombination between direct repeats (Sommers et al. 1995; Tishkoff et al. 1997b; Symington 1998) among the multiple mutant phenotypes and rad27 mutants are lethal when combined with mutations in genes involved in double-strand break repair and homologous recombination (Sommers et al. 1995; Vallen and Cross 1995; Tishkoff et al. 1997b; Symington 1998). Therefore the phenotype of the double mutant srs2 rad27 was determined. The double mutant was inviable (Figure 5), un- dergoing 8–10 cell divisions before arresting as enlarged doublet cells or lysing, providing additional support for a role of SRS2 in homologous recombination and double-strand break repair. A rad51 mutation was not able to suppress the double mutant lethality, but this is most likely due to the lethality of the rad51 rad27 genotype (Tishkoff et al. 1997b; Symington 1998). The DNA damage checkpoint gene RAD17 also was not able to rescue the srs2 rad27 double mutant lethality when mutant, but this could be due to lethality of the rad27 rad17 genotype. RAD17 is part of the DNA damage checkpoint that also includes the RAD9 and MEC1 genes. rad27 rad9 and rad27 mec1 double mutants have been reported to Figure 2.—srs2⌬ interaction with mre11 mutants and a be lethal (Vallen and Cross 1995). rad50⌬ mutant. (A) Left, five tetrads from the cross of a srs2⌬ strain to a mre11⌬ null allele strain. The double mutants srs2⌬ ⌬ mre11 are indicated by the circles and are inviable. Right, DISCUSSION five tetrads from the cross of a srs2⌬ strain to a mre11H125N- mre11D56N nonnull allele strain. The double mutants, indi- All of the double mutant interactions with srs2 and cated by circles, are fully viable. (B) A rad17 mutation does suppression by defects in homologous recombination not suppress the poor growth phenotype of a srs2⌬ rad50⌬ double mutant. A cross of srs2⌬ rad17⌬ by rad50⌬ is shown. or the DNA damage checkpoint are summarized in Ta- Circles indicate the srs2⌬ rad50⌬ double mutants while the bles 2 and 3. The findings would indicate that Srs2p triple mutants srs2⌬ rad50⌬ rad17⌬ are outlined in squares. acts in recombination after commitment to repair via 562 H. L. Klein

TABLE 3 Summary of srs2 interactions with repair mutants

Strain Phenotype Mutant suppression of poor growth/lethality srs2⌬ rad50⌬ Very poor growth Suppressed by rad51, not suppressed by rad17 srs2⌬ xrs2⌬ Very poor growth Suppressed by rad51 srs2⌬ mre11⌬ Very poor growth Suppressed by rad51 srs2⌬ mre11H125N-mre11D56N Normal growth srs2⌬ exo1⌬ Normal growth srs2⌬ sgs1⌬ Lethal Suppressed by rad51, not suppressed by rad1 or rad24 srs2⌬ top3⌬ Lethal Suppressed by rad51, rad52, rad55, rad57, not suppressed by rad1 srs2⌬ rad27⌬ Lethal Not suppressed by rad51 or rad17 Not all recombination repair or checkpoint function mutants were tested. The genes tested are indicated. homologous recombination. If homologous recombina- ficity as to the action of Srs2p in repair as diploid srs2 tion is prevented by inhibiting the initial steps of form- mutants show a greatly increased damage sensitivity and ing a Rad51 filament on single-stranded DNA, then srs2 it is the attempted homologous recombination between rad54 or srs2 rad50/xrs2/mre11 lethality/poor growth homolog chromosomes, not sister chromatids, that is can be prevented. This suggests, first, that there exist lethal in the absence of Srs2 helicase (Aboussekhra et other pathways in the cell to repair the spontaneous al. 1989, 1992). damage that occurs in these double mutants and, sec- Studies of srs2 mutants as suppressors of nonnull al- ond, that once a cell is committed to repair damage leles of RAD51 and RAD52 have provided an alternative through homologous recombination, through action of interpretation of the role of Srs2p in homologous re- Rad51p, there is no alternate repair option available. combination (Kaytor et al. 1995; Milne et al. 1995; Experiments on in vivo repair of substrates containing Chanet et al. 1996). These articles have suggested two a double-strand break indicate an important role for regulatory functions for SRS2. First, the Srs2 helicase is the Srs2 helicase in the repair process (Paques and proposed to antagonize the action of Rad51p and Haber 1997). The lethality of haploid strains bearing Rad52p in homologous recombination. Through such srs2 and rad54 mutations shows that Srs2p can have an an activity Srs2p would have an antirecombinase func- important role in repair of spontaneous DNA damage, but in srs2 single mutants there is some substrate speci-

Figure 4.—Suppression of srs2⌬ top3⌬ inviability by a rad51⌬ mutation. Five tetrads from the cross of a srs2⌬ rad51⌬ strain to a top3⌬ strain are shown. The double mutants srs2⌬ Figure 3.—A rad24⌬ mutation does not suppress the poor top3⌬ are indicated by circles and are inviable. The triple growth phenotype of srs2⌬ sgs1⌬ strains. Five tetrads from the mutants srs2⌬ top3⌬ rad51⌬ are indicated by squares and are cross of a srs2⌬ rad24⌬ strain to a sgs1⌬ strain are shown. The viable, although they grow slower than other genotypes. The double mutants srs2⌬ sgs1⌬ are indicated by circles and are unmarked small colonies carry the top3⌬ allele. The reduced inviable or grow very poorly. The triple mutants srs2⌬ sgs1⌬ growth of the top3⌬ strain is not rescued by a rad51⌬ mutation, rad24⌬ are indicated by squares and also are inviable or grow probably accounting for the slower growth of the triple mu- very poorly. tant. srs2 Suppressors 563

Rad54 proteins triggers an irreversible arrest that is sensed by DNA damage checkpoint functions. Loss of the checkpoint functions allows cells to grow, but full viability as measured by plating efficiency and colony size is not restored, suggesting that unrepaired damage remains or that chromosome loss is occurring. This cannot be measured in haploid cells for essential chromosomes. The data thus far show that mutations in RAD17, RAD24, and MEC3 can partially suppress the growth arrest, but loss of RAD9 function cannot. Why there is a differential suppression by this group of DNA damage checkpoint genes is not clear, but for some types of DNA damage RAD9 acts additively to RAD17/ RAD24/MEC3 in sensing DNA damage (Lydall and Weinert 1995; de la Torre-Ruiz et al. 1998). However, in the DNA damage arrest of rad54 rdh54 mutants, no Figure 5.—Lethality of the srs2⌬ rad27⌬ genotype. Five additive effect of a rad9 and a rad24 mutation has been tetrads from the cross of a srs2⌬ strain to a rad27⌬ strain are seen in suppression of poor growth (H. Klein, unpub- shown. The double mutants srs2⌬ rad27⌬ are indicated by circles and are inviable. lished observations). The Sgs1 helicase is a component of the S-phase checkpoint response acting upstream of the Rad53 ki- tion and could also control use of the homologous re- nase (Frei and Gasser 2000). This response has been combination pathway to repair a spontaneous damaged shown to act in parallel to the checkpoint response substrate. Srs2p would determine which lesions could that requires RAD24 (Frei and Gasser 2000). The Srs2 be processed through homologous recombination. helicase has also been shown to be involved in an Therefore Srs2p would inhibit homologous recombina- S-phase checkpoint response that is dependent on tion prior to the commitment to repair by homologous RAD17 and RAD24 (Liberi et al. 2000). The observation recombination. In this scheme one would then suggest that the srs2 sgs1 double mutant poor growth is sup- that, in the absence of Srs2p, substrates are inappropri- pressed by mutations in recombination repair functions, ately targeted for repair through the homologous re- but not by a mutation in the RAD24 checkpoint func- combination pathway. When Rad54p is also absent, the tion, may indicate that the double mutant defect is due processing of substrates through homologous recombi- to attempted recombination, which can trigger a check- nation becomes an irreversible lethal action. Rescue point response. Recombination may be an attempt to through loss of Rad51p, Rad52p, Rad55p, or Rad57p repair DNA damage that has its origin in a replication is thought to happen by inhibiting the initial steps of defect (Frei and Gasser 2000). Mutation of RAD24 in homologous recombination, prior to the Rad54p-medi- the srs2 sgs1 double mutant may result in a defect in two ated step (Petukhova et al. 1998). Rescue through loss parallel checkpoint response pathways, with the result of of DNA damage checkpoints implies that the cells are continued poor growth. Alternatively, loss of the RAD24 arrested due to the accumulation of unrepaired DNA checkpoint function may permit the srs2 sgs1 cells to damage that is sensed by these checkpoint genes. progress through the cell cycle without repairing DNA Other studies have suggested that the Srs2 helicase lesions that have become lethal. Since a rad24 mutation can act at a later step in recombination (Rong et al. can suppress the lethality of the srs2 rad54 mutant, but 1991; Paques and Haber 1997). Whether the helicase not the srs2 sgs1 mutant, this would suggest that the acts in concert with Rad54 protein is not known. How- DNA intermediate, presumably related to attempted re- ever, under either model the basic scenario for suppres- combination, that occurs in each of these cell types is sion remains the same, namely, preventing recombina- different. The difference may be the presence of an tion at an early step rescues lethal mutant combinations. unrepaired double-strand break or a structure that can The second regulatory function for Srs2p has been be shunted into an alternate repair pathway. proposed to be repression of a repair pathway that paral- It has been suggested that the Sgs1 helicase can act lels the RAD52 recombination repair pathway (Kaytor on different substrates, controlled by the action of the et al. 1995). This remains conjectural, although the re- topoisomerases I and III and the Srs2 DNA helicase cent finding of multiple repair pathways and the RAD59 (Duno et al. 2000). In this regard the srs2 sgs1 poor gene, which is related to RAD52 (Rattray and Syming- growth/lethality is proposed to be similar to the srs2 ton 1995; Bai and Symington 1996), provides support top3 lethality as the alternative helicase/topoisomerase for this idea. activities of Sgs1/Top3 and Srs2/Top1 are disrupted in The arrest of haploid cells through loss of Srs2 and these double mutants. What determines the specificity 564 H. L. Klein of the target substrate is not known, but must be linked characterization of the Sgs1 DNA helicase activity of Saccharomyces cerevisiae. J. Biol. Chem. 273: 9644–9650. to recombinational repair. Chanet, R., M. Heude, A. Adjiri, L. Maloisel and F. Fabre, 1996 Srs2 helicase has some interaction with Rad50p/ Semidominant mutations in the yeast Rad51 protein and their Xrs2p/Mre11p although whether this is merely genetic relationships with the Srs2 helicase. Mol. Cell. Biol. 16: 4782– 4789. or also has a physical basis is not known. We have not de la Torre-Ruiz, M. A., C. M. Green and N. F. Lowndes, 1998 detected any interaction with these proteins from two- RAD9 and RAD24 define two additive, interacting branches of hybrid screens (H. Klein, unpublished observations). the DNA damage checkpoint pathway in budding yeast normally required for Rad53 modification and activation. EMBO J. 17: However, the genetic interaction does not involve the 2687–2698. Mre11p nuclease function, suggesting that Srs2p may Duno, M., B. Thomsen, O. Westergaard, L. Kreci and C. Bendixen, be targeted to open up broken ends by the Rad50p/ 2000 Genetic analysis of the Saccharomyces cerevisiae Sgs1 helicase defines an essential function for the Sgs1-Top3 complex in the Xrs2p/Mre11p complex. The failure of a rad17 muta- absence of SRS2 or TOP1. Mol. Gen. Genet. 264: 89–97. tion to suppress the poor growth phenotype of the srs2 Eisen, J. A., K. S. Sweder and P. C. Hanawalt, 1995 Evolution of rad50 mutant can be interpreted in two ways. Loss of the SNF2 family of proteins: subfamilies with distinct sequences and functions. Nucleic Acids Res. 23: 2715–2723. the RAD17 checkpoint function may permit srs2 rad50 Fiorentini, P., K. N. Huang, D. X. Tishkoff, R. D. Kolodner and cells to progress through the cell cycle with unrepaired L. S. Symington, 1997 Exonuclease I of Saccharomyces cerevi- damage, with continued poor growth due to some type siae functions in mitotic recombination in vivo and in vitro. Mol. Cell. Biol. 17: 2764–2773. of genomic instability. Alternatively, the DNA damage Frei, C., and S. M. Gasser, 2000 The yeast Sgs1p helicase acts up- that accumulates in the srs2 rad50 mutant may not be a stream of Rad53p in the DNA replication checkpoint and colocal- target for the DNA damage checkpoints that act through izes with Rad53p in S-phase-specific foci. Genes Dev. 14: 81–96. Gangloff, S., J. P. McDonald, C. Bendixen, L. Arthur and R. RAD17. 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