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

Corresponding author: Hannah L. Klein, New York University School of Medicine, 550 First Ave., New York, NY 10016., hannah.klein{at}med.nyu.edu (E-mail)

Communicating editor: L. S. SYMINGTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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.

THE SRS2 gene was first identified through dominant mutations that suppressed trimethoprim sensitivity of rad6 and rad18 mutants (LAWRENCE and CHRISTENSEN 1979 ). It was again recovered as a suppressor of damage sensitivity of the rad18 mutant (ABOUSSEKHRA et al. 1989 ) and as a mutant that showed increased spontaneous mitotic gene conversion (RONG et al. 1991 ). These studies placed SRS2 in the RAD6 epistasis group for DNA repair, in an error-free postreplication repair pathway. The sequence of the SRS2 gene revealed high homology to the bacterial repair gene uvrD, encoding helicase II (ABOUSSEKHRA et al. 1989 ). Subsequently, it was demonstrated that the Srs2 protein has DNA helicase activity (RONG and KLEIN 1993 ). While the biochemical activity of SRS2 indicates a function in DNA metabolic activity, the precise role of the gene has remained enigmatic.

First, it is not clear why srs2 suppressor alleles are semidominant for the suppressor effect of rad6 or rad18 DNA damage sensitivity phenotypes. Second, SRS2 haploid mutants have a 5- to 10-fold increased sensitivity to UV damage (ABOUSSEKHRA et al. 1989 ; PALLADINO and KLEIN 1992 ), which is significant but is not of the magnitude of other UV-sensitive mutants. Haploid srs2 mutants are not sensitive to X-rays while diploid mutants are sensitive (ABOUSSEKHRA et al. 1989 ). Third, all the mitotic DNA damage sensitivities are enhanced in mutant diploid strains as compared to mutant haploid strains (ABOUSSEKHRA et al. 1989 ). This is unusual and has been interpreted as lethal recombination events occurring between homologous chromosomes in the absence of Srs2 function (ABOUSSEKHRA et al. 1989 ). Thus in the mutant the haploid has a hyperrecombination phenotype while in the diploid some of the interhomolog recombination events are lethal, while other recombination events such as intragenic recombination are increased. To make the situation even more complicated, the suppression of rad6 and rad18 mutants by srs2 mutation is thought to occur by the channeling of repair substrates into the RAD52 recombination repair pathway (ABOUSSEKHRA et al. 1989 ; RONG et al. 1991 ). Fourth, in meiosis the gene is required for full spore viability, and in its absence spore viability is 50% and map distances for the LEU2-HIS4 interval and the HIS4-MAT interval are reduced 2-fold (PALLADINO 1991 ).

Further clues as to the functional role of SRS2 have come from identification of double mutant combinations with srs2 mutants that are lethal or exhibit extremely poor growth and from suppressors of those srs2 mutant phenotypes. srs2 rad50 double mutants grow very poorly (RONG et al. 1991 ; PALLADINO and KLEIN 1992 ). This most likely reflects an essential role for RAD50 in recombinational repair involving sister chromatids (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 is suppressed by semidominant mutations in RAD51 (ABOUSSEKHRA et al. 1992 ), which encodes a RecA-like protein that functions in recombinational repair (SHINOHARA et al. 1992 ), again placing SRS2 in a step in homologous recombination that may occur after the RAD51-mediated step, although an alternate interpretation involves negative regulation of homologous recombination pathways by SRS2. srs2 mutants also suppress nonnull semidominant alleles of RAD51 (MILNE et al. 1995 ; CHANET et al. 1996 ). This has been interpreted as a function for the Srs2 helicase in reversing abortive recombination intermediates.

We have previously reported that srs2 mutations are lethal in a rad54 null mutant background (PALLADINO and KLEIN 1992 ). This lethality is suppressed by mutations in RAD51, RAD52, RAD55, and RAD57 (SCHILD 1995 ). These genes function in the recombinational repair pathway of yeast and are required for homologous recombination (SAEKI et al. 1980 ). These results implicate SRS2 in recombinational repair, although epistasis analysis does not place SRS2 in the RAD52 repair pathway. Nonetheless, there is an interaction between SRS2 and RAD52 as null alleles of SRS2 are able to suppress nonnull alleles of RAD52 (KAYTOR et al. 1995 ; MILNE et al. 1995 ; SCHILD 1995 ). Although the mechanism of suppression is not understood, it may reflect a need for the Srs2 protein to stabilize and promote recombination between substrates with limited homology (PAQUES and HABER 1997 ). The rad52 alleles may attempt recombination between sequences of reduced homology and this reduced fidelity recombination requires SRS2. In the absence of functional Srs2p, aberrant recombination would be prevented and the rad52 alleles would be suppressed.

Additional evidence for SRS2 functioning in recombination comes from studies of the interaction between SRS2 and SGS1. SGS1 encodes a DNA helicase related to the RecQ family of helicases (GANGLOFF et al. 1994 ; WATT et al. 1995 ; LU et al. 1996 ). sgs1 mutant strains are characterized by increased genomic instability (WATT et al. 1996 ; YAMAGATA et al. 1998 ). Although the single null allele mutant strains exhibit normal growth, the double mutant srs2 sgs1 is inviable or grows extremely poorly (LEE et al. 1999 ; GANGLOFF et al. 2000 ). The growth defect of the srs2 sgs1 double mutant can be suppressed by mutations in the RAD51, RAD55, or RAD57 genes (GANGLOFF et al. 2000 ), indicating that the growth defect is caused by attempted homologous recombination that cannot be completed when both Srs2p and Sgs1p are missing.

To gain additional insight into the biological role of SRS2, we have examined other DNA recombination functions for synthetic interactions with a srs2 null allele strain. We have also asked what types of mutants suppress srs2 phenotypes. The results link SRS2 to homologous recombination that is associated with DNA replication and DNA damage that is sensed by DNA damage checkpoints.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains:
Parental strains used in this study are listed in Table 1. All strains are isogenic and are in the W303 background (W303 genotype: leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-1 RAD5). sgs1 disruption strains were a gift from Rodney Rothstein. The mre11 and exo1 strains were a gift from Lorraine Symington. The rad9, rad17, rad24, and mec3 strains were a gift from Ted Weinert. Strains, if needed, were converted to the RAD5 version of W303 through crosses. The rad54 point mutation strains have been described (PETUKHOVA et al. 1999 ). All double and triple mutant strains were derived from crosses using the parental single mutant strains listed in Table 1. All recombination and checkpoint mutants are null alleles with the exception of the two indicated rad54 point mutants and the one indicated mre11 point mutant.

Media and growth conditions:
Standard media were prepared as described (SHERMAN et al. 1986 ). All strains were grown on solid media at 30°.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

srs2 and rad54:
We have previously reported that the srs2 rad54 double null mutant is lethal (PALLADINO and KLEIN 1992 ). This lethality is suppressed by loss of the homologous recombination functions of RAD51, RAD52, RAD55, or RAD57 (SCHILD 1995 and this study). However, not all mutations of genes involved in homologous recombination or repair of a double-strand break rescue the srs2 rad54 lethality. The RAD1 gene encodes an endonuclease that is active in nucleotide excision repair and some recombination reactions (KLEIN 1988 ; SCHIESTL and PRAKASH 1988 ; THOMAS and ROTHSTEIN 1989 ; SIEDE et al. 1993 ; TOMKINSON et al. 1993 ; IVANOV and HABER 1995 ; KIRKPATRICK and PETES 1997 ). However, a rad1 null mutation was not able to rescue the srs2 rad54 strain.

RAD54 encodes a protein of the SNF2/SW12 family and contains DNA helicase consensus motifs (EISEN et al. 1995 ). The protein has demonstrated in vitro ATPase activity and promotes Rad51-mediated strand exchange (PETUKHOVA et al. 1998 ) via a change in DNA double helix conformation (PETUKHOVA et al. 1999 ). Two point mutations in the first helicase consensus domain, rad54K341R and rad54K341A, have been shown to abolish ATP hydrolysis and Rad51-promoted homologous DNA pairing as well as mitotic recombination (PETUKHOVA et al. 1999 ). Both of these mutations in a srs2 background are lethal, showing that the essential function provided by Rad54 protein in a srs2 strain requires the ATPase activity.

Our studies on rad54 rdh54 diploid double mutants had suggested that the double mutant poor growth was due to the accumulation of DNA damage through attempted recombination that was sensed by the DNA damage checkpoint functions. This finding prompted us to speculate that the srs2 rad54 lethality might be due to the accumulation of DNA damage from attempted recombination that was sensed by the DNA damage checkpoint functions. Therefore the role of the checkpoint functions in preventing srs2 rad54 cells from progressing through the cell cycle was assessed by determining the growth phenotype of triple mutants. Triple mutants were segregated in crosses. An example of such a cross is shown in Fig 1. In Fig 1A growth of colonies of the genotype srs2 rad54 rad17, srs2 rad54 rad24, or srs2 rad54 mec3 is compared to a wild-type strain. While the triple mutant strains grow slower than wild type and throw off colonies of varying sizes, these genotypes are viable. In contrast, a rad9 mutation is unable to rescue the srs2 rad54 double mutant (Fig 1B). Fig 1B and Fig C compares the ability of a rad51 mutation vs. a rad17 mutation to suppress the srs2 rad54 lethality. It can be seen that rad51 is more effective in suppressing the srs2 rad54 lethality, suggesting that the checkpoint mutants suppress the double mutant by release from growth arrest, but DNA damage is still present. Further support for this statement comes from plating efficiencies. One hundred unbudded cells of the srs2 rad54 rad17 and srs2 rad54 rad51 genotypes were micromanipulated to fresh YPD plates. A total of 50/100 srs2 rad54 rad17 cells grew to visible colonies whereas 93/100 srs2 rad54 rad51 cells grew to visible colonies.

View larger version (47K): In this window In a new window Download PPT slide   Figure 1. Suppression of srs2 rad54 lethality through loss of homologous recombination functions or DNA damage checkpoint functions. Five tetrads from crosses indicated above each picture are shown. (A) Suppression by rad17, rad24, or mec3 is shown. Strains of each genotype were generated by crossing a srs2 rad17/rad24/mec3 strain to a rad54 strain. Growth is slightly reduced compared to a wild-type control strain. (B) The ability of a rad9 mutation to suppress is compared to the ability of a rad17 mutation to suppress. srs2 rad54 double mutants are outlined in circles while the triple mutants srs2 rad54 rad9/rad17 are outlined in squares. It can be seen that the srs2 rad54 rad17 strain is viable but grows more slowly than other viable genotypes. (C) The ability of a rad51 mutation to suppress srs2 rad54 is shown. Circles indicate the srs2 rad54 double mutants while the triple mutants srs2 rad54 rad51 are outlined in squares. It can been seen that a rad51 mutation is a stronger suppressor than the DNA damage checkpoint mutations.

srs2 and rdh54:
The RAD54 and RDH54 genes have related sequences, but the single mutants have unique effects on mitotic recombination and meiotic viability (KLEIN 1997 ; SHINOHARA et al. 1997 ). We have previously reported that, while in contrast to the srs2 rad54 haploid mutant lethality the srs2 rdh54 haploid mutant shows normal growth, lethality is exhibited in the srs2 rdh54 diploid (KLEIN 1997 ). Moreover, this lethality is suppressed by mutation in any one of the recombination repair genes RAD51, RAD52, RAD55, or RAD57 (KLEIN 1997 ). Similar to the suppression of srs2 rad54 by DNA damage checkpoint mutations, the srs2 rdh54 diploid lethality is suppressed by mutations in RAD17, RAD24, or MEC3, but not by mutation of the RAD9 gene (Table 2).

srs2 and rad50/xrs2/mre11:
The Rad50, Xrs2, and Mre11 proteins function in a complex to protect DNA ends in end-joining reactions and in meiotic recombination (for a review see HABER 1998 ). The RAD50, XRS2, and MRE11 genes are not required for mitotic homolog recombination, but are required for recombination between sister chromatids (SAEKI et al. 1980 ). The srs2 null allele mutant has a synthetic growth interaction with null allele mutations in RAD50, XRS2, or MRE11 (see Fig 2A) and this is suppressed by mutations in the recombination repair genes (Table 3). However, the srs2 rad50 poor growth was not suppressed by mutation of the DNA damage checkpoint function RAD17 (Fig 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.

View larger version (58K): In this window In a new window Download PPT slide   Figure 2. srs2 interaction with mre11 mutants and a 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, five tetrads from the cross of a srs2 strain to a mre11H125N-mre11D56N nonnull allele strain. The double mutants, indicated by circles, are fully viable. (B) A rad17 mutation does not suppress the poor growth phenotype of a srs2 rad50 double mutant. A cross of srs2 rad17 by rad50 is shown. Circles indicate the srs2 rad50 double mutants while the triple mutants srs2 rad50 rad17 are outlined in squares.

MRE11 encodes a protein with single-stranded endonuclease activity (MOREAU et al. 1999 ). Mutation of consensus motifs in the phosphodiesterase domains abolishes 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 (Fig 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, or mre11 show poor growth, one possibility 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 observing that srs2 rad50 strains have telomeres of the rad50 characteristic length (H. KLEIN and R. BOGUSLAVSKY, 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 1998 ). Since the srs2 mutant also has genetic interactions with the same set of genes, the phenotype of the double mutant srs2 exo1 was determined. The double mutant srs2 exo1 was fully viable, showing that neither gene is required for any essential function when the other gene is defective.

srs2 sgs1 and srs2 top3:
SGS1 encodes a DNA helicase of the same polarity as the Srs2 DNA helicase (LU et al. 1996 ; BENNETT et al. 1998 ). It has been reported that the double mutant srs2 sgs1 is lethal or grows extremely poorly (LEE et al. 1999 ; GANGLOFF et al. 2000 ). This growth phenotype is due to attempted recombination as mutations in the recombination repair genes RAD51, RAD52, RAD55, or RAD57 rescue the growth defect. We have confirmed these observations. sgs1 cells have been found to be partially defective for an intra-S checkpoint for DNA damage that acts in parallel with the RAD24 checkpoint function (FREI and GASSER 2000 ). Therefore, it was of interest to examine the phenotype of the srs2 sgs1 rad24 triple mutant. We found that the rad24 mutant did not suppress the lethality or poor growth of the srs2 sgs1 double mutant (Fig 3). Suppression was also measured by plating efficiency of micromanipulated unbudded cells from spore colonies onto fresh growth medium. The srs2 strain had a high plating efficiency (47/50) while the sgs1 mutant plating efficiency was moderate (36/50). Although spores of the srs2 sgs1 genotype were viable, the growth was slow and the plating efficiency was 0/50. The presence of a rad24 mutation in this genotype increased the plating efficiency to 31/100 (srs2 sgs1 rad24 genotype), but the colonies grew very slowly and never reached wild-type size.

View larger version (125K): In this window In a new window Download PPT slide   Figure 3. A rad24 mutation does not suppress the poor growth phenotype of srs2 sgs1 strains. Five tetrads from the cross of a srs2 rad24 strain to a sgs1 strain are shown. The double mutants srs2 sgs1 are indicated by circles and are inviable or grow very poorly. The triple mutants srs2 sgs1 rad24 are indicated by squares and also are inviable or grow very poorly.

The Sgs1 protein of yeast interacts physically with the Top3 protein, which has type I topoisomerase activity. Moreover, the slow growth phenotype of the top3 mutant is suppressed by a sgs1 mutation (GANGLOFF et al. 1994 ). Therefore we examined the phenotype of the srs2 top3 double mutant. The double mutant is lethal (Fig 4) and this lethality is also suppressed by loss of the recombination repair pathway through mutations in RAD-51, RAD52, RAD55, or RAD57 (Fig 4 and Table 3).

View larger version (118K): In this window In a new window Download PPT slide   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 top3 are indicated by circles and are inviable. The triple mutants srs2 top3 rad51 are indicated by squares and are viable, although they grow slower than other genotypes. The unmarked small colonies carry the top3 allele. The reduced growth of the top3 strain is not rescued by a rad51 mutation, probably accounting for the slower growth of the triple mutant.

srs2 and rad27:
RAD27 encodes an exonuclease that is involved in the completion of lagging strand synthesis (HARRINGTON and LIEBER 1994 ; REAGAN et al. 1995 ; SOMMERS et al. 1995 ). rad27 mutants have elevated rates 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 (Fig 5), undergoing 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 be lethal (VALLEN and CROSS 1995 ).

View larger version (127K): In this window In a new window Download PPT slide   Figure 5. Lethality of the srs2 rad27 genotype. Five tetrads from the cross of a srs2 strain to a rad27 strain are shown. The double mutants srs2 rad27 are indicated by circles and are inviable.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

All of the double mutant interactions with srs2 and suppression by defects in homologous recombination or the DNA damage checkpoint are summarized in Table 2 and Table 3. The findings would indicate that Srs2p acts in recombination after commitment to repair via homologous recombination. If homologous recombination is prevented by inhibiting the initial steps of forming a Rad51 filament on single-stranded DNA, then srs2 rad54 or srs2 rad50/xrs2/mre11 lethality/poor growth can be prevented. This suggests, first, that there exist other pathways in the cell to repair the spontaneous damage that occurs in these double mutants and, second, that once a cell is committed to repair damage through homologous recombination, through action of Rad51p, there is no alternate repair option available. Experiments on in vivo repair of substrates containing a double-strand break indicate an important role for the Srs2 helicase in the repair process (PAQUES and HABER 1997 ). The lethality of haploid strains bearing srs2 and rad54 mutations shows that Srs2p can have an important role in repair of spontaneous DNA damage, but in srs2 single mutants there is some substrate specificity as to the action of Srs2p in repair as diploid srs2 mutants show a greatly increased damage sensitivity and it is the attempted homologous recombination between homolog chromosomes, not sister chromatids, that is lethal in the absence of Srs2 helicase (ABOUSSEKHRA et al. 1989 , ABOUSSEKHRA et al. 1992 ).

Studies of srs2 mutants as suppressors of nonnull alleles of RAD51 and RAD52 have provided an alternative interpretation of the role of Srs2p in homologous recombination (KAYTOR et al. 1995 ; MILNE et al. 1995 ; CHANET et al. 1996 ). These articles have suggested two regulatory functions for SRS2. First, the Srs2 helicase is proposed to antagonize the action of Rad51p and Rad52p in homologous recombination. Through such an activity Srs2p would have an antirecombinase function and could also control use of the homologous recombination pathway to repair a spontaneous damaged substrate. Srs2p would determine which lesions could be processed through homologous recombination. Therefore Srs2p would inhibit homologous recombination prior to the commitment to repair by homologous recombination. In this scheme one would then suggest that, in the absence of Srs2p, substrates are inappropriately targeted for repair through the homologous recombination pathway. When Rad54p is also absent, the processing of substrates through homologous recombination becomes an irreversible lethal action. Rescue through loss of Rad51p, Rad52p, Rad55p, or Rad57p is thought to happen by inhibiting the initial steps of homologous recombination, prior to the Rad54p-mediated step (PETUKHOVA et al. 1998 ). Rescue through loss of DNA damage checkpoints implies that the cells are arrested due to the accumulation of unrepaired DNA damage that is sensed by these checkpoint genes.

Other studies have suggested that the Srs2 helicase can act at a later step in recombination (RONG et al. 1991 ; PAQUES and HABER 1997 ). Whether the helicase acts in concert with Rad54 protein is not known. However, under either model the basic scenario for suppression remains the same, namely, preventing recombination at an early step rescues lethal mutant combinations.

The second regulatory function for Srs2p has been proposed to be repression of a repair pathway that parallels the RAD52 recombination repair pathway (KAYTOR et al. 1995 ). This remains conjectural, although the recent finding of multiple repair pathways and the RAD59 gene, which is related to RAD52 (RATTRAY and SYMINGTON 1995 ; BAI and SYMINGTON 1996 ), provides support for this idea.

The arrest of haploid cells through loss of Srs2 and 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 additive effect of a rad9 and a rad24 mutation has been seen in suppression of poor growth (H. KLEIN, unpublished observations).

The Sgs1 helicase is a component of the S-phase checkpoint response acting upstream of the Rad53 kinase (FREI and GASSER 2000 ). This response has been shown to act in parallel to the checkpoint response that requires RAD24 (FREI and GASSER 2000 ). The Srs2 helicase has also been shown to be involved in an S-phase checkpoint response that is dependent on RAD17 and RAD24 (LIBERI et al. 2000 ). The observation that the srs2 sgs1 double mutant poor growth is suppressed by mutations in recombination repair functions, but not by a mutation in the RAD24 checkpoint function, may indicate that the double mutant defect is due to attempted recombination, which can trigger a checkpoint response. Recombination may be an attempt to repair DNA damage that has its origin in a replication defect (FREI and GASSER 2000 ). Mutation of RAD24 in the srs2 sgs1 double mutant may result in a defect in two parallel checkpoint response pathways, with the result of continued poor growth. Alternatively, loss of the RAD24 checkpoint function may permit the srs2 sgs1 cells to progress through the cell cycle without repairing DNA lesions that have become lethal. Since a rad24 mutation can suppress the lethality of the srs2 rad54 mutant, but not the srs2 sgs1 mutant, this would suggest that the DNA intermediate, presumably related to attempted recombination, that occurs in each of these cell types is different. The difference may be the presence of an unrepaired double-strand break or a structure that can be shunted into an alternate repair pathway.

It has been suggested that the Sgs1 helicase can act on different substrates, controlled by the action of the topoisomerases I and III and the Srs2 DNA helicase (DUNO et al. 2000 ). In this regard the srs2 sgs1 poor growth/lethality is proposed to be similar to the srs2 top3 lethality as the alternative helicase/topoisomerase activities of Sgs1/Top3 and Srs2/Top1 are disrupted in these double mutants. What determines the specificity of the target substrate is not known, but must be linked to recombinational repair.

Srs2 helicase has some interaction with Rad50p/Xrs2p/Mre11p although whether this is merely genetic or also has a physical basis is not known. We have not detected any interaction with these proteins from two-hybrid screens (H. KLEIN, unpublished observations). However, the genetic interaction does not involve the Mre11p nuclease function, suggesting that Srs2p may be targeted to open up broken ends by the Rad50p/Xrs2p/Mre11p complex. The failure of a rad17 mutation to suppress the poor growth phenotype of the srs2 rad50 mutant can be interpreted in two ways. Loss of the RAD17 checkpoint function may permit srs2 rad50 cells to progress through the cell cycle with unrepaired damage, with continued poor growth due to some type of genomic instability. Alternatively, the DNA damage that accumulates in the srs2 rad50 mutant may not be a target for the DNA damage checkpoints that act through RAD17.

We have no information as to when the spontaneous damage is occurring in the srs2 rad54 strain and in the other double mutant combinations that exhibit poor or no growth. SRS2 is induced in expression at the beginning of S phase and is induced in G2 by DNA damage (HEUDE et al. 1995 ), suggesting that these phases of the cell cycle may be more prone to DNA damage resulting from loss of Srs2 protein. In the case of the srs2 rad27 lethality, it is likely that the damage occurs during S phase as RAD27 is required for DNA replication (HARRINGTON and LIEBER 1994 ; REAGAN et al. 1995 ; SOMMERS et al. 1995 ). If the damage that is targeted for repair by Srs2 helicase, either as a catalytic function in repair or through regulation of which substrate is repaired by the DSB repair pathway, occurs during S phase, this would reinforce the close link between DNA replication and recombination and also bring into play a link between DNA replication and nonrecombination (non-DSB repair) repair modes.

	ACKNOWLEDGMENTS

I thank Rodney Rothstein, Lorraine Symington, and Ted Weinert for strains; and Revekka Boguslavsky for technical assistance. This work was supported by U.S. Public Health Service grant GM53738.

Manuscript received September 5, 2000; Accepted for publication November 8, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ABOUSSEKHRA, A., R. CHANET, Z. ZGAGA, C. CASSIER-CHAUVAT, and M. HEUDE et al., 1989  RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene. Nucleic Acids Res. 17:7211-7219[Abstract/Free Full Text].

ABOUSSEKHRA, A., R. CHANET, A. ADJIRI, and F. FABRE, 1992  Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins. Mol. Cell. Biol. 12:3224-3234[Abstract/Free Full Text].

BAI, Y. and L. S. SYMINGTON, 1996  A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae.. Genes Dev. 10:2025-2037[Abstract/Free Full Text].

BENNETT, R. J., J. A. SHARP, and J. C. WANG, 1998  Purification and characterization of the Sgs1 DNA helicase activity of Saccharomyces cerevisiae.. J. Biol. Chem. 273:9644-9650[Abstract/Free Full Text].

CHANET, R., M. HEUDE, A. ADJIRI, L. MALOISEL, and F. FABRE, 1996  Semidominant mutations in the yeast Rad51 protein and their relationships with the Srs2 helicase. Mol. Cell. Biol. 16:4782-4789[Abstract].

DE LA TORRE-RUIZ, M. A., C. M. GREEN, and N. F. LOWNDES, 1998  RAD9 and RAD24 define two additive, interacting branches of the DNA damage checkpoint pathway in budding yeast normally required for Rad53 modification and activation. EMBO J. 17:2687-2698[Medline].

DUNO, M., B. THOMSEN, O. WESTERGAARD, L. KRECI, and C. BENDIXEN, 2000  Genetic analysis of the Saccharomyces cerevisiae Sgs1 helicase defines an essential function for the Sgs1-Top3 complex in the absence of SRS2 or TOP1.. Mol. Gen. Genet. 264:89-97[Medline].

EISEN, J. A., K. S. SWEDER, and P. C. HANAWALT, 1995  Evolution of the SNF2 family of proteins: subfamilies with distinct sequences and functions. Nucleic Acids Res. 23:2715-2723[Abstract/Free Full Text].

FIORENTINI, P., K. N. HUANG, D. X. TISHKOFF, R. D. KOLODNER, and L. S. SYMINGTON, 1997  Exonuclease I of Saccharomyces cerevisiae functions in mitotic recombination in vivo and in vitro. Mol. Cell. Biol. 17:2764-2773[Abstract].

FREI, C. and S. M. GASSER, 2000  The yeast Sgs1p helicase acts upstream of Rad53p in the DNA replication checkpoint and colocalizes with Rad53p in S-phase-specific foci. Genes Dev. 14:81-96[Abstract/Free Full Text].

GANGLOFF, S., J. P. MCDONALD, C. BENDIXEN, L. ARTHUR, and R. ROTHSTEIN, 1994  The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase. Mol. Cell. Biol. 14:8391-8398[Abstract/Free Full Text].

GANGLOFF, S., C. SOUSTELLE, and F. FABRE, 2000  Homologous recombination is responsible for cell death in the absence of the Sgs1 and Srs2 helicases. Nat. Genet. 25:192-194[Medline].

HABER, J. E., 1998  The many interfaces of Mre11. Cell 95:583-586[Medline].

HARRINGTON, J. J. and M. R. LIEBER, 1994  Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair. Genes Dev. 8:1344-1355[Abstract/Free Full Text].

HEUDE, M., R. CHANET, and F. FABRE, 1995  Regulation of the Saccharomyces cerevisiae Srs2 helicase during the mitotic cell cycle, meiosis and after irradiation. Mol. Gen. Genet. 248:59-68[Medline].

IVANOV, E. L. and J. E. HABER, 1995  RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae.. Mol. Cell. Biol. 15:2245-2251[Abstract].

KAYTOR, M. D., M. NGUYEN, and D. M. LIVINGSTON, 1995  The complexity of the interaction between RAD52 and SRS2.. Genetics 140:1441-1442[Medline].

KIRKPATRICK, D. T. and T. D. PETES, 1997  Repair of DNA loops involves DNA-mismatch and nucleotide-excision repair proteins. Nature 387:929-931[Medline].

KIRONMAI, K. M. and K. MUNIYAPPA, 1997  Alteration of telomeric sequences and senescence caused by mutations in RAD50 of Saccharomyces cerevisiae.. Genes Cells 2:443-455[Abstract].

KLEIN, H. L., 1988  Different types of recombination events are controlled by the RAD1 and RAD52 genes of Saccharomyces cerevisiae.. Genetics 120:367-377[Abstract/Free Full Text].

KLEIN, H. L., 1997  RDH54, a RAD54 homologue in Saccharomyces cerevisiae, is required for mitotic diploid-specific recombination and repair and for meiosis. Genetics 147:1533-1543[Abstract].

LAWRENCE, C. W. and R. B. CHRISTENSEN, 1979  Metabolic suppressors of trimethoprim and ultraviolet light sensitivities of Saccharomyces cerevisiae rad6 mutants. J. Bacteriol. 139:866-887[Abstract/Free Full Text].

LE, S., J. K. MOORE, J. E. HABER, and C. W. GREIDER, 1999  RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase. Genetics 152:143-152[Abstract/Free Full Text].

LEE, S. K., R. E. JOHNSON, S. L. YU, L. PRAKASH, and S. PRAKASH, 1999  Requirement of yeast SGS1 and SRS2 genes for replication and transcription. Science 286:2339-2342[Abstract/Free Full Text].

LIBERI, G., I. CHIOLO, A. PELLICIOLI, M. LOPES, and P. PLEVANI et al., 2000  Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1-dependent pathway and Cdk1 activity. EMBO J. 19:5027-5038[Medline].

LU, J., J. R. MULLEN, S. J. BRILL, S. KLEFF, and A. M. ROMEO et al., 1996  Human homologues of yeast helicase. Nature 383:678-679[Medline].

LYDALL, D. and T. WEINERT, 1995  Yeast checkpoint genes in DNA damage processing: implications for repair and arrest. Science 270:1488-1491[Abstract/Free Full Text].

MILNE, G. T., T. HO, and D. T. WEAVER, 1995  Modulation of Saccharomyces cerevisiae DNA double-strand break repair by SRS2 and RAD51.. Genetics 139:1189-1199[Abstract].

MOREAU, S., J. R. FERGUSON, and L. S. SYMINGTON, 1999  The nuclease activity of Mre11 is required for meiosis but not for mating type switching, end joining, or telomere maintenance. Mol. Cell. Biol. 19:556-566[Abstract/Free Full Text].

PALLADINO, F., 1991 Genetic characterization of the HPR5 helicase gene of Saccharomyces cerevisiae. Ph.D. Thesis, New York University, New York.

PALLADINO, F. and H. L. KLEIN, 1992  Analysis of mitotic and meiotic defects in Saccharomyces cerevisiae SRS2 DNA helicase mutants. Genetics 132:23-37[Abstract].

PAQUES, F. and J. E. HABER, 1997  Two pathways for removal of nonhomologous DNA ends during double-strand break repair in Saccharomyces cerevisiae.. Mol. Cell. Biol. 17:6765-6771[Abstract].

PETUKHOVA, G., S. STRATTON, and P. SUNG, 1998  Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins. Nature 393:91-94[Medline].

PETUKHOVA, G., S. VAN KOMEN, S. VERGANO, H. KLEIN, and P. SUNG, 1999  Yeast Rad54 promotes Rad51-dependent homologous DNA pairing via ATP hydrolysis-driven change in DNA double helix conformation. J. Biol. Chem. 274:29453-29462[Abstract/Free Full Text].

RATTRAY, A. J. and L. S. SYMINGTON, 1995  Multiple pathways for homologous recombination in Saccharomyces cerevisiae.. Genetics 139:45-56[Abstract].

REAGAN, M. S., C. PITTENGER, W. SIEDE, and E. C. FRIEDBERG, 1995  Characterization of a mutant strain of Saccharomyces cerevisiae with a deletion of the RAD27 gene, a structural homolog of the RAD2 nucleotide excision repair gene. J. Bacteriol. 177:364-371[Abstract/Free Full Text].

RONG, L. and H. L. KLEIN, 1993  Purification and characterization of the SRS2 DNA helicase of the yeast Saccharomyces cerevisiae.. J. Biol. Chem. 268:1252-1259[Abstract/Free Full Text].

RONG, L., F. PALLADINO, A. AGUILERA, and H. L. KLEIN, 1991  The hyper-gene conversion hpr5-1 mutation of Saccharomyces cerevisiae is an allele of the SRS2/RADH gene. Genetics 127:75-85[Abstract].

SAEKI, T., I. MACHIDA, and S. NAKAI, 1980  Genetic control of diploid recovery after gamma-irradiation in the yeast Saccharomyces cerevisiae.. Mutat. Res. 73:251-265[Medline].

SCHIESTL, R. H. and S. PRAKASH, 1988  RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination. Mol. Cell. Biol. 8:3619-3626[Abstract/Free Full Text].

SCHILD, D., 1995  Suppression of a new allele of the yeast RAD52 gene by overexpression of RAD51, mutations in srs2 and ccr4, or mating-type heterozygosity. Genetics 140:115-127[Abstract].

SHERMAN, F., G. R. FINK and J. B. HICKS, 1986 Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SHINOHARA, A., H. OGAWA, and T. OGAWA, 1992  Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell 69:457-470[Medline].

SHINOHARA, M., E. SHITA-YAMAGUCHI, J. M. BUERSTEDDE, H. SHINAGAWA, and H. OGAWA et al., 1997  Characterization of the roles of the Saccharomyces cerevisiae RAD54 gene and a homologue of RAD54, RDH54/TID1, in mitosis and meiosis. Genetics 147:1545-1556[Abstract].

SIEDE, W., A. S. FRIEDBERG, and E. C. FRIEDBERG, 1993  Evidence that the Rad1 and Rad10 proteins of Saccharomyces cerevisiae participate as a complex in nucleotide excision repair of UV radiation damage. J. Bacteriol. 175:6345-6347[Abstract/Free Full Text].

SOMMERS, C. H., E. J. MILLER, B. DUJON, S. PRAKASH, and L. PRAKASH, 1995  Conditional lethality of null mutations in RTH1 that encodes the yeast counterpart of a mammalian 5'- to 3'-exonuclease required for lagging strand DNA synthesis in reconstituted systems. J. Biol. Chem. 270:4193-4196[Abstract/Free Full Text].

SYMINGTON, L. S., 1998  Homologous recombination is required for the viability of rad27 mutants. Nucleic Acids Res. 26:5589-5595[Abstract/Free Full Text].

THOMAS, B. J. and R. ROTHSTEIN, 1989  The genetic control of direct-repeat recombination in Saccharomyces: the effect of rad52 and rad1 on mitotic recombination at GAL10, a transcriptionally regulated gene. Genetics 123:725-738[Abstract/Free Full Text].

TISHKOFF, D. X., A. L. BOERGER, P. BERTRAND, N. FILOSI, and G. M. GAIDA et al., 1997a  Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2.. Proc. Natl. Acad. Sci. USA 94:7487-7492[Abstract/Free Full Text].

TISHKOFF, D. X., N. FILOSI, G. M. GAIDA, and R. D. KOLODNER, 1997b  A novel mutation avoidance mechanism dependent on S. cerevisiae RAD27 is distinct from DNA mismatch repair. Cell 88:253-263[Medline].

TOMKINSON, A. E., A. J. BARDWELL, L. BARDWELL, N. J. TAPPE, and E. C. FRIEDBERG, 1993  Yeast DNA repair and recombination proteins Rad1 and Rad10 constitute a single-stranded-DNA endonuclease. Nature 362:860-862[Medline].

VALLEN, E. A. and F. R. CROSS, 1995  Mutations in RAD27 define a potential link between G1 cyclins and DNA replication. Mol. Cell. Biol. 15:4291-4302[Abstract].

WATT, P. M., E. J. LOUIS, R. H. BORTS, and I. D. HICKSON, 1995  Sgs1: a eukaryotic homolog of E. coli RecQ that interacts with topoisomerase II in vivo and is required for faithful chromosome segregation. Cell 81:253-260[Medline].

WATT, P. M., I. D. HICKSON, R. H. BORTS, and E. J. LOUIS, 1996  SGS1, a homologue of the Bloom's and Werner's syndrome genes, is required for maintenance of genome stability in Saccharomyces cerevisiae.. Genetics 144:935-945[Abstract].

YAMAGATA, K., J. KATO, A. SHIMAMOTO, M. GOTO, and Y. FURUICHI et al., 1998  Bloom's and Werner's syndrome genes suppress hyperrecombination in yeast sgs1 mutant: implication for genomic instability in human diseases. Proc. Natl. Acad. Sci. USA 95:8733-8738[Abstract/Free Full Text].

This article has been cited by other articles:

				I. Chiolo, M. Saponaro, A. Baryshnikova, J.-H. Kim, Y.-S. Seo, and G. Liberi The Human F-Box DNA Helicase FBH1 Faces Saccharomyces cerevisiae Srs2 and Postreplication Repair Pathway Roles Mol. Cell. Biol., November 1, 2007; 27(21): 7439 - 7450. [Abstract] [Full Text] [PDF]

				J. C. Hope, L. D. Cruzata, A. Duvshani, J. Mitsumoto, M. Maftahi, and G. A. Freyer Mus81-Eme1-Dependent and -Independent Crossovers Form in Mitotic Cells during Double-Strand Break Repair in Schizosaccharomyces pombe Mol. Cell. Biol., May 15, 2007; 27(10): 3828 - 3838. [Abstract] [Full Text] [PDF]

				K. H. Schmidt and R. D. Kolodner Suppression of spontaneous genome rearrangements in yeast DNA helicase mutants PNAS, November 28, 2006; 103(48): 18196 - 18201. [Abstract] [Full Text] [PDF]

				Y.-C. Lo, K. S. Paffett, O. Amit, J. A. Clikeman, R. Sterk, M. A. Brenneman, and J. A. Nickoloff Sgs1 regulates gene conversion tract lengths and crossovers independently of its helicase activity. Mol. Cell. Biol., June 1, 2006; 26(11): 4086 - 4094. [Abstract] [Full Text] [PDF]

				S. Raynard, W. Bussen, and P. Sung A Double Holliday Junction Dissolvasome Comprising BLM, Topoisomerase III{alpha}, and BLAP75 J. Biol. Chem., May 19, 2006; 281(20): 13861 - 13864. [Abstract] [Full Text] [PDF]

				I. Chiolo, W. Carotenuto, G. Maffioletti, J. H. J. Petrini, M. Foiani, and G. Liberi Srs2 and Sgs1 DNA Helicases Associate with Mre11 in Different Subcomplexes following Checkpoint Activation and CDK1-Mediated Srs2 Phosphorylation Mol. Cell. Biol., July 1, 2005; 25(13): 5738 - 5751. [Abstract] [Full Text] [PDF]

				K. Suzuki, A. Kato, Y. Sakuraba, and H. Inoue Srs2 and RecQ homologs cooperate in mei-3-mediated homologous recombination repair of Neurospora crassa Nucleic Acids Res., March 30, 2005; 33(6): 1848 - 1858. [Abstract] [Full Text] [PDF]

G. Liberi, G. Maffioletti, C. Lucca, I. Chiolo, A. Baryshnikova, C. Cotta-Ramusino, M. Lopes, A. Pellicioli, J. E. Haber, and M. Foiani Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase Genes & Dev., February 1, 2005; 19(3): 339 - 350. [Abstract] [Full Text]