Saccharomyces cerevisiae cells expressing both a- and -mating-type (MAT) genes (termed mating-type heterozygosity) exhibit higher rates of spontaneous recombination and greater radiation resistance than cells expressing only MATa or MAT. MAT heterozygosity suppresses recombination defects of four mutations involved in homologous recombination: complete deletions of RAD55 or RAD57, an ATPase-defective Rad51 mutation (rad51-K191R), and a C-terminal truncation of Rad52, rad52-327. We investigated the genetic basis of MAT-dependent suppression of these mutants by deleting genes whose expression is controlled by the Mata1-Mat2 repressor and scoring resistance to both campothecin (CPT) and phleomycin. Haploid rad55 strains became more damage resistant after deleting genes required for nonhomologous end-joining (NHEJ), a process that is repressed in MATa/MAT cells. Surprisingly, NHEJ mutations do not suppress CPT sensitivity of rad51-K191R or rad52-327. However, rad51-K191R is uniquely suppressed by deleting the RME1 gene encoding a repressor of meiosis or its coregulator SIN4; this effect is independent of the meiosis-specific homolog, Dmc1. Sensitivity of rad52-327 to CPT was unexpectedly increased by the MATa/MAT-repressed gene YGL193C, emphasizing the complex ways in which MAT regulates homologous recombination. The rad52-327 mutation is suppressed by deleting the prolyl isomerase Fpr3, which is not MAT regulated. rad55 is also suppressed by deletion of PST2 and/or YBR052C (RFS1, rad55 suppressor), two members of a three-gene family of flavodoxin-fold proteins that associate in a nonrandom fashion with chromatin. All three recombination-defective mutations are made more sensitive by deletions of Rad6 and of the histone deacetylases Rpd3 and Ume6, although these mutations are not themselves CPT or phleomycin sensitive.

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DNA repair in budding yeast is strongly influenced by the cell's mating status. Saccharomyces cells can be of three mating types: those able to mate expressing only MATa or only MAT and nonmating cells expressing both MATa and MAT. MATa/MAT diploid cells are more radiation resistant and recombination proficient than diploids expressing only MATa or MAT (FRIIS and ROMAN 1968; HEUDE and FABRE 1993; FASULLO and DAVE 1994; LOWELL et al. 2003). A similar increase in radioresistance and resistance to radiomimetic drugs is seen in haploid cells that express both mating-type alleles. Coexpression of MATa and MAT, either in diploids or in haploids, leads to the formation of the Mata1-Mat2 corepressor that turns off the expression of haploid-specific genes and induces expression of diploid-specific genes. The most striking effect of a1–2 repression is a severe disabling of nonhomologous end-joining (NHEJ) by the repression of NEJ1 (ÅSTRÖM et al. 1999; LEE et al. 1999; FRANK-VAILLANT and MARCAND 2001; KEGEL et al. 2001; OOI and BOEKE 2001; VALENCIA et al. 2001). Whether any of the other targets of a1–2 repression affect homologous recombination (HR) is not known.

A double-strand break (DSB) can be repaired by two largely independent processes: HR and NHEJ. NHEJ requires genes encoding the yeast Ku proteins, YKU70 and YKU80, as well as DNA ligase 4 (DNL4) and its associated factors, LIF1 and NEJ1 (BOULTON and JACKSON 1996a,b; MILNE et al. 1996; SCHÄR et al. 1997; TEO and JACKSON 1997; WILSON et al. 1997; HERRMANN et al. 1998; KEGEL et al. 2001; OOI and BOEKE 2001; VALENCIA et al. 2001). NHEJ in budding yeast also depends on the genes encoding the MRX (MRE11, RAD50, and XRS2) complex (MOORE and HABER 1996; TSUKAMOTO et al. 1996). In competition experiments, most DSBs appear to be repaired by HR; for example, 10% of HO endonuclease-induced DSBs were repaired by NHEJ and the remaining 90% by HR (gene conversion) (VALENCIA et al. 2001).

HR depends on genes of the RAD52 epistasis group (PÂQUES and HABER 1999), including the RAD51, RAD52, RAD55, and RAD57 genes discussed here. Here, we investigated cell-type effects on homologous recombination by studying the basis for the DNA-damage suppression of three HR mutants, the null mutant, rad55, and partially defective rad51-K191R and rad52327 alleles. Previous studies have shown that DNA-damage sensitivity of all three mutations can be suppressed by MAT heterozygosity either in diploids or in haploids in which the opposite MAT allele is introduced on a plasmid (LOVETT and MORTIMER 1987; SCHILD 1995; MORGAN et al. 2002).

The RAD55 and RAD57 genes encode for proteins that share some homology with the bacterial RecA protein and with Rad51 (LOVETT 1994; JOHNSON and SYMINGTON 1995); these genes are considered to be paralogs of RAD51 in yeast (SYMINGTON 2002). Rad55 and Rad57 form a heterodimer that interacts with Rad51, and together with Rad52, help in the loading of Rad51 to single-stranded DNA (SUNG 1997a,b; SUGAWARA et al. 2003; WOLNER et al. 2003). Unlike rad51 strains, rad55 and rad57 mutants are sensitive to -irradiation at 23° or below, but are near-wild type at 30° or 37°. At lower temperatures they also show a defect in mitotic recombination, similar to rad51 (JOHNSON and SYMINGTON 1995; SIGNON et al. 2001). The defect in both mitotic recombination and repair is suppressed by MAT heterozygosity (LOVETT and MORTIMER 1987; LOVETT 1994; HAYS et al. 1995; JOHNSON and SYMINGTON 1995).

The Rad51-K191R mutant protein can bind but not hydrolyze ATP. Expression of Rad51-K191R from a low-copy plasmid shows partial complementation of the DNA repair and recombination defects of the null rad51 strain, and its overexpression shows full complementation of rad51 (SHINOHARA et al. 1992; SUNG and STRATTON 1996). MORGAN et al. (2002) found that a haploid rad51-K191R strain is as sensitive to low doses of ionizing radiation (IR) as a rad51 strain. It is similarly defective in spontaneous and DNA-damage-induced mitotic recombination, but unlike a rad51 strain, rad51-K191R shows no defect in meiotic recombination. Moreover, a haploid rad51-K191R strain expressing both MATa and MAT information is more resistant to -irradiation than a haploid strain expressing only one MAT allele. The sensitivity of rad51-K191R to IR can also be suppressed by overexpression of the mutant itself or of RAD54. This effect was specific to the rad51-K191R allele since neither a rad51-K191A nor a rad51 strain was affected by mating-type heterozygosity (MORGAN et al. 2002).

The rad52-327 allele removes the last 327 amino acids of Rad52, including a domain that interacts with Rad51. This allele is less sensitive to MMS-induced DNA damage than a rad52 strain. rad52-327 can be partially complemented for MMS sensitivity by expression of RAD51 from a high-copy plasmid (ASLESON et al. 1999). Also, unlike rad52, rad52-327 does not decrease spontaneous mitotic recombination but rather increases it and is less defective in meiotic recombination (RESNICK et al. 1986; BOUNDY-MILLS and LIVINGSTON 1993), which suggests that this allele still retains some Rad52 functions. A recent article reporting a similar Rad52 C-terminal deletion, rad52-329, suggests that the mutant protein can carry out Rad51-independent modes of recombination (TSUKAMOTO et al. 2003). Two other C-terminal truncations, rad52-2 and rad52-20 alleles (SCHILD 1995), behave similarly in most recombination and DNA repair tests (BOUNDY-MILLS and LIVINGSTON 1993). The sensitivity of rad52-20 to X-rays can be suppressed by deletion of SIR2, SIR3, or SIR4 genes, which are required to prevent expression of the a1–2 repressor from the genetically silenced HML and HMRa mating-type genes (SCHILD 1995). Similarly, a diploid rad52-20/rad52-20 homozygous at MAT (i.e., MATa/MATa) is more sensitive to DNA damage than a MATa/MAT mutant strain (SCHILD 1995). Phenotypically, rad52-20 and rad52327 mutant alleles are similar (BOUNDY-MILLS and LIVINGSTON 1993; SCHILD 1995). Therefore, we investigated whether rad52-327 can also be suppressed by MAT heterozygosity and whether we could identify a Mata1-Mat2-regulated gene whose repression is responsible for this suppression.

To test how mating-type genes regulate HR, we took advantage of several microarray data sets that have identified haploid- and diploid-specific genes in yeast (GALITSKI et al. 1999; VALENCIA et al. 2001), as well as other unpublished data [Saccharomyces Genome Database (SGD) database at http://db.yeastgenome.org/cgi-bin/SGD]. We took the approach of deleting some of the haploid-specific genes identified thus far in combination with the RAD mutations described above. We assayed the sensitivity of the double-mutant strain to DNA damage caused by the chemotherapeutic agent campothecin (CPT). Camptothecin is an antitumor agent known to create DSBs in cells by catalyzing covalent association between topoisomerase I (Top1) and the phosphate group of the DNA backbone (HSIANG et al. 1989). Here, DNA damage in the form of DSBs is presumed to occur by stalled replication forks and by collisions between transcription complexes and the Top1–DNA complex (HSIANG et al. 1989; WU and LIU 1997).

This mutational analysis revealed that the cell-type effect is different for each RAD mutation studied. We identified known and unknown genes that may affect DNA-damage sensitivity in a cell. When Rad55 functions are absent, defects in the NHEJ pathway of DSBR improved resistance of the mutant cell to CPT-induced DNA damage. However, the absence of NHEJ genes did not affect the DNA-damage sensitivity of a rad52-327 or a rad51-K191R mutant cell to CPT, but other gene deletions were identified that suppress these mutations. Thus, their cell- type effect on DNA-damage sensitivity and repair is not dependent on the same mating-type-regulated genes. This implies that there are several independent targets by which MAT heterozygosity alters DNA repair in yeast.

Yeast strains and plasmids:

Saccharomyces cerevisiae strains used in these experiments were predominantly yeast deletion strains from the Saccharomyces genome deletion project (Research Genetics, Huntsville, AL).

They are isogenic derivatives of BY4741 background (Table 1). Deletion of RAD55 was done using sequences from plasmid pSTL11 that replaces the wild-type copy of the gene with a rad55::LEU2 allele in a BY4741 background. Double- and triple-mutant strains were created by obtaining meiotic segregants from crosses of appropriate haploids, screening the haploid progeny by phenotype and by PCR amplification to confirm gene deletions (Table 1). Some double-mutant strains were made by gene replacement with a PCR-amplified gene deletion marked by the KAN-MX cassette obtained from Research Genetics strains. This gene-disrupting fragment was transformed into single-mutant strains, and G418 resistant colonies were screened for the appropriate disruption by PCR amplification. Strains LSY0977 (MATa rad51-K191R::URA3::rad51-K191R) (MORGAN et al. 2002) and LSY0941 (MATa rad52-327) in the W303 background were kindly provided by Lorraine Symington. When double mutants were made, either by transformation or by crosses, at least two derivatives of the relevant genotypes were tested.

View this table: In this window In a new window   TABLE 1 Yeast strains used in this study

 

Growth conditions and drug sensitivity tests:

Yeast strains were grown in YPD overnight at 30°. Strains were diluted to a concentration of 1 x 10⁷ cells/ml, and five additional 10-fold serial dilutions were made in a sterile 96-well microtiter dish. The dilutions were spotted onto the indicated media and incubated at 30° for 3 days or at 23° for 5 days. Strains were spotted onto YPD plates containing 0.5, 0.6, 0.75, and 1 µg/ml CPT and buffered with 25 mM HEPES, pH 7.2. A stock solution of camptothecin was made by dissolving the chemical in dimethyl sulfoxide at a stock concentration of 4 mg/ml. Control plates contained 0.025% dimethyl sulfoxide and 25 mM HEPES. Strains were also tested on YPD plates containing 10 mM hydroxyurea and 0.3–0.5 µg/ml phleomycin. The sensitivity of yeast cells to camptothecin was also measured by treating exponentially growing cells with increasing amounts of the genotoxic agent. The cultures were grown with shaking at 25° and 30° for 2 hr. The survivors were determined by serially diluting the cultures and plating the appropriate dilutions on YPD plates. Colonies were counted after 3 days of growth at 30°. Sensitivity assays were performed a minimum of three times for each mutant strain.

Chromatin immunoprecipitation:

The association of Pst2-Myc with yeast chromatin was assayed by chromatin immunoprecipitation for selected sites along chromosome III. Quantitative chromatin immunoprecipitation analysis was performed using the ABI PRISM 7000 sequence detection system. The detection dye used was SYBR green (Platinum SYBR green qPCR SuperMix UDG, Invitrogen, San Diego). Real-time PCR was carried out as follows: 95° for 3 min (1 cycle), 95° for 1 min, 52° for 1 min, and 72° for 2 min (45 cycles). The fold enrichment was calculated as described previously (LITT et al. 2001).

Mating-type suppression of the rad55 cold sensitivity to DNA damage is dependent on the repression of NHEJ:

A rad55 mutation renders a cell cold sensitive for DNA damage induced by X-rays and -irradiation as well as for repair of an HO-induced DSB (LOVETT and MORTIMER 1987; JOHNSON and SYMINGTON 1995; SIGNON et al. 2001). This sensitivity can be suppressed by both higher temperatures and MAT heterozygosity, as well as by overexpressing Rad51 (JOHNSON and SYMINGTON 1995). The chemotherapeutic agent CPT can also cause DNA damage in the form of either single- or double-strand breaks (HSIANG et al. 1989). We established that rad55 is sensitive to CPT primarily at lower temperatures, i.e., 23° (Figure 1A and data not shown) and all experiments discussed here were carried out at this temperature. The sensitivity of a rad55 strain to CPT is partially suppressed by MAT heterozygosity, as shown by deleting SIR3 in a haploid rad55 strain, so that cells express HML as well as MATa and HMRa (Figure 1A). We note that previous studies have shown an equivalent suppression of rad55 and the other mutants discussed here either by introducing the opposite MAT allele on a plasmid or by deleting Sir2, Sir3, or Sir4. The suppressive effect of temperature and MAT heterozygosity on the survival of rad55 cells to CPT was confirmed by exposing exponentially growing cells to higher concentrations of this genotoxic agent for several hours and then plating them on YEPD plates (data not shown).

View larger version (89K): In this window In a new window Download PPT slide   FIGURE 1.— Suppression of rad55, rad51-K191R, and rad52-327 by mating-type heterozygosity. Strains carrying the sir3 mutation express MAT2 from HML and MATa1 from HMRa, as well as the genes at MAT, thus creating a phenotypic mating-type heterozygosity. (A) Ten-fold serial dilutions (from left to right) of various mutant strains were examined on YEPD medium containing 0.75 µg/ml CPT as well as on YEPD medium without the drug at 23°. (B) Suppression of rad51-K191R phleomycin sensitivity by deletion of SIR3.

 
Since the expression of both a and information in the cell causes the repression of haploid-specific genes, we investigated the role of 10 such genes in the suppression of DNA damage by MAT heterozygosity (GALITSKI et al. 1999; WYRICK et al. 1999; VALENCIA et al. 2001; SGD database at http://www.yeastgenome.org/). We made double mutants and tested their sensitivity to CPT at 23° by spotting serial dilutions of cultures onto YPD plates containing 0.5–0.75 µg/ml CPT. Although there was no effect from deleting YLR041W, YLR040C, YIL117C, YGL193C, TID1 (also known as RDH54), and RME1, deletion of the haploid-specific gene NEJ1 suppressed the rad55 phenotype at a level comparable to the suppression caused by sir3 (Figure 2A, Table 2).

View larger version (71K): In this window In a new window Download PPT slide   FIGURE 2.— Suppression of rad55 but not rad51-K191R or rad52-327 by NHEJ mutants under different DNA-damaging conditions. Serial dilutions were plated onto YEPD plates containing CPT (A), phleomycin (B), or HU (C). (D) In contrast, the end-joining-deficient nej1 mutation does not suppress the sensitivity of either rad51-K191R or rad52-327. None of the NHEJ mutations by themselves affected CPT sensitivity.

 

View this table: In this window In a new window   TABLE 2 Sensitivities of double-mutant strains to CPT

 
We next asked if deleting other NHEJ proteins also suppressed the sensitivity of rad55 to DNA damage at low temperatures. Indeed, deletion of DNL4 (Figure 2B) as well as LIF1 and YKU70 (not shown) also suppressed this phenotype. We did not test the NHEJ-defective mre11, rad50, or xrs2 deletions, as these deletions, which are also important for HR, were highly sensitive to CPT (data not shown). We conclude that the absence of the NHEJ repair pathway increases the survival of a rad55 strain when exposed to CPT at 23°. These results could explain the effect of mating-type heterozygosity since two of the NHEJ components, NEJ1 and to a lesser extent LIF1, are under the control of the cell mating type (VALENCIA et al. 2001). In fact, a defect in NHEJ is epistatic with MAT heterozygosity for rad55 suppression, as deletion of SIR3 (which allows expression of a- and -genes from unsilenced HML and HMRa) did not further increase the resistance of the rad55 dnl4 strain to CPT (Figure 2B; data not shown for CPT).

The suppression of rad55 by NHEJ mutants is not a general feature of recombination-defective mutations. Deletion of NHEJ functions did not suppress the DNA damage sensitivity of rad51 or rad54 mutations (data not shown). This is consistent with the observation that the effect of MAT heterozygosity on DNA-damage sensitivity is dependent on the function of RAD51, RAD52, and RAD54 genes (SAEKI et al. 1980).

Mating-type effects have been reported on the sensitivity to agents that induce DNA DSBs (HEUDE and FABRE 1993; FASULLO and DAVE 1994; LOVETT 1994; JOHNSON and SYMINGTON 1995; SCHILD 1995). We wanted to know whether MAT heterozygosity could suppress the sensitivity of rad55 to other genotoxic agents such as UV, the radiomimetic drug phleomycin, and hydroxyurea (HU). Previous studies have shown that MAT status had little or no effect on UV sensitivity in Rad⁺ cells (FRIIS and ROMAN 1968). We observed that this was also the case in rad55 cells; deletion of SIR3 or of NHEJ genes, which suppressed CPT sensitivity, failed to alter the UV sensitivity of rad55 cells (data not shown). In contrast to UV, cells that express both MAT alleles were between 10 and 100 times more resistant than their homozygous counterpart to damage induced by phleomycin, which primarily causes double-strand breaks (Figure 2B). Sensitivity to phleomycin damage in a rad55 strain was also suppressed by defects in NHEJ, while NHEJ mutations themselves were not phleomycin sensitive.

Treatment of rad55 and double-mutant strains with HU resulted in a phenotype similar to that seen for CPT-induced DNA damage (Figure 2C). We observed suppression of the HU sensitivity of rad55 by MAT heterozygosity (i.e., by sir3) and by defects in NHEJ, increasing the resistance by >10-fold. Thus suppression of rad55 by NHEJ mutants and MAT heterozygosity extends to other agents that require the RAD52 pathway for the repair of presumed DSBs.

Suppression of rad55 by deleting members of a novel flavodoxin-fold family of chromatin-associated proteins:

From the list of haploid-specific genes described above, we found another gene whose deletion suppresses the rad55 sensitivity to CPT, the gene YDR032C (also known as Pst2) (Figure 3, Table 2). Subsequent Northern blot analysis showed that the level of Pst2 was reduced less than twofold in MATa/MAT cells (data not shown). Nevertheless, pst2 is a suppressor of rad55, increasing resistance >10-fold. The effect of pst2 on CPT sensitivity of rad55 was similar to that seen in deletions of genes involved in NHEJ such as dnl4. A pst2 dnl4 double mutant did not make rad55 more resistant than either single mutant; however, deletion of this gene does not impair NHEJ in yeast (see below).

View larger version (89K): In this window In a new window Download PPT slide   FIGURE 3.— Suppression of rad55's CPT sensitivity by pst2 and ybr052 (rfs1) mutations compared to the NHEJ-defective lif1.

 
Pst2 shows homology with two other yeast proteins, Ycr004c (Ycp4) and Ybr052c. Deletion of YBR052C, whose protein product is 47% identical to Pst2, had a very similar phenotype to pst2; moreover, a pst2 ybr052 double mutant showed a greater degree of suppression of rad55 than either single mutant. Deletion of YCP4, despite encoding a protein 67% identical with Pst2 and 46% identical with Ybr052c, had no effect on rad55 sensitivity; possibly this protein already harbors a point mutation and is nonfunctional. Deletions of PST2 and YBR052C also confer resistance of rad55 to phleomycin (data not shown). We suggest that YBR052C be named RFS1 (Rad fifty-five suppressor).

Published affinity purifications (HO et al. 2002) have suggested that Pst2 interacts with both Yku80 and Xrs2 proteins; given that both Yku70-Yku80 and Mre11-Rad50-Xrs2 are involved in NHEJ, we asked if a triple mutant, pst2 ycp4 rfs1, was defective for NHEJ. To measure NHEJ, we used strain JKM179 in which continuous expression of HO endonuclease creates a DSB at the MAT locus that can be repaired only by imprecise end-joining, because both HML and HMR are deleted (MOORE and HABER 1996). Whereas nej1 reduces NHEJ by almost 200-fold (VALENCIA et al. 2001; Figure 4), the triple mutant pst2 ycp4 rfs1 had no effect (Figure 4). There was also no effect of any single mutant.

View larger version (12K): In this window In a new window Download PPT slide   FIGURE 4.— The absence of the Pst2 gene family (Pst2, Ycp4, and Ybr052) does not affect NHEJ. Survival of cells suffering an HO-endonuclease-induced DSB that can be repaired only by imperfect end-joining is unaffected in a pst2 ycp4 ybr052 mutant, whereas there is an inhibition of NHEJ and low survival in a nej1 strain.

 
Pst2 is 54% identical to the Schizosaccharomyces pombe protein Uhp1 that has been implicated in S. pombe mating-type gene silencing (which is not imposed by the same silencing system as in S. cerevisiae) (NARESH et al. 2003). S. pombe Uhp1 has been reported to be ubiquitylated by a Rad6-dependent process and to associate with histone H2B in vitro (NARESH et al. 2003). Uhp1 was initially reported to have a histone-like protein fold, but later analysis suggests that Uhp1 and the budding yeast Pst2 family share motifs consistent with a flavodoxin fold (http://db.yeastgenome.org/cgi-bin/protein/getDomain?sgdid=S000002439). The role of flavodoxin fold proteins in DNA repair or other DNA-related functions has not been previously demonstrated. We have found no evidence that the S. cerevisiae genes are involved in gene silencing, as a pst2 ycp4 rfs1 strain does not unsilence HML or HMRa.

To investigate the chromosomal properties of the budding yeast proteins, we created a Pst2-Myc derivative to analyze the protein biochemically and by chromatin immunoprecipitation. Expression of Pst2-Myc complemented pst2 in a pst2 rad55 strain (data not shown). Overexpression of PST2-Myc did not further sensitize either wild-type or rad55 strains to CPT (data not shown). On Western blots the 22-kDa protein migrates as a larger protein of 40 kDa (data not shown). In view of the finding that the S. pombe homolog was monoubiquitylated by Rad6, we asked if the unusual mobility of Pst2-Myc was due to post-translational modification. However, after immunoprecipitation and Western blot analysis using anti-ubiquitin and anti-SUMO antibodies, we found no evidence of either ubiquitylation or sumoylation (data not shown). Moreover, the mobility of Pst2-Myc did not change in the absence of the ubiquitin ligase E2-conjugating enzyme Rad6 (data not shown).

Chromatin immunoprecipitation was used to determine if Pst2-Myc was chromatin associated. We analyzed the relative enrichment of DNA fragments across chromosome III. As shown in Figure 5A, there is a nonrandom distribution of Pst2-Myc at a number of chromosomal sites. The most abundant locations include subtelomeric regions on both chromosome III and chromosome VI (data not shown). We examined in more detail the region including and distal to the HMR silent locus. Again, Pst2-Myc abundance was nonuniform. The location of the most abundant sites does not correlate with the abundance of the minor histone variant H2A.Z (Figure 5B). We conclude that Pst2 is likely to be chromatin associated, but its role remains to be elucidated. Although Pst2 has been localized to the cytoplasm by analyzing a GFP-tagged protein (http://yeastgfp.ucsf.edu/getOrf.php?orf=YDR032C), its two-hybrid interaction with the nuclear proteins Ku80 and Xsr2 (HO et al. 2002) and the DNA repair phenotype that we report here suggest that this protein functions in the nucleus in DNA repair.

View larger version (28K): In this window In a new window Download PPT slide   FIGURE 5.— Pst2-Myc associates nonrandomly with chromatin. (A) Chromatin immunoprecipitation of Pst2-Myc at 21 locations along the first 250 kb of chromosome III. (B) Distribution of Pst2-Myc compared with histone variant H2A.Z (Htz1).

 

RAD6 prevents hypersensitivity of rad55 to CPT:

Although Pst2 does not seem to be ubiquitylated, it is possible that Rad6 may affect DNA-damage repair by the monoubiquitylating histone H2B or by other targets. Therefore we tested the effect of deleting RAD6 on sensitivity to CPT. The rad6 deletion itself is not CPT sensitive, but rad6 had a significant sensitizing effect on rad55 (Figure 6). The increased sensitivity of rad6 rad55 was not suppressed by pst2 or rfs1 alone; however, the multiple mutant rad6 rad55 pst2 rfs1 suppressed this hypersensitivity back to the level seen in rad55 pst2 rfs1, a 1000-fold increase in resistance (Figure 6A). These data indicate that that the pst2 and rfs1 mutations affect overlapping, partially redundant functions and that they overcome the hypersensitive CPT phenotype seen in rad55 rad6.

View larger version (87K): In this window In a new window Download PPT slide   FIGURE 6.— (A) CPT sensitivity of rad55 is exacerbated by rad6. This enhanced sensitivity is counteracted by deleting pst2 and ybr052. (B) rad6 also exacerbates the sensitivity of rad51-K191R and rad52-327.

 
We also found that rad6 makes rad52-327 and especially rad51-K191R more sensitive to CPT. It will be interesting to determine the target of Rad6, whose lack of modification does not affect CPT sensitivity by itself but exacerbates all three rad mutants tested here.

Suppression of the DNA-damage sensitivity of a rad51-K191R is linked to the function of a different set of haploid-specific genes:

The rad51-K191R allele of RAD51 encodes for a mutant Rad51 protein that is able to bind ATP but cannot hydrolyze it. This allele retains partial Rad51 activity, and its overexpression suppresses the mitotic recombination and repair defects of a rad51 strain (SUNG and STRATTON 1996). The rad51-K191R strain is as sensitive to DNA damage induced by low doses of IR as a rad51 strain, but the sensitivity of the K191R allele can be suppressed by MAT heterozygosity (MORGAN et al. 2002). rad51-K191R cells were also sensitive to the DNA-damaging agent CPT, and this phenotype was similar to that of rad51 cells (Figure 3, top). MAT heterozygosity created by a sir3 mutation also suppresses the CPT sensitivity of a rad51-K191R mutant strain (Figure 1A). The phleomycin sensitivity of this mutant is also suppressed by MAT heterozygosity (Figure 1B). Surprisingly, rad51-K191R is not suppressed by deletion of NHEJ genes, including nej1 (Figure 2D) and lif1 (data not shown).

We investigated the role of the other haploid-specific genes discussed above in suppressing CPT sensitivity of rad51-K191R. Surprisingly, we found that only the absence of RME1 can rescue the sensitivity of the strain to CPT (Figure 7A), giving a 10-fold increase in resistance. Deletion of RME1 does not affect the other two CPT-sensitive mutations studied here (Table 2); data for rad55 are shown in Figure 7C.

View larger version (80K): In this window In a new window Download PPT slide   FIGURE 7.— Suppression of rad51-K191R. (A) Phleomycin sensitivity of rad51-K191R is suppressed by rme1 (two independent derivatives are shown). The effect of rme1 is independent of the possible induction of the alternative Rad51 homolog, Dmc1, as seen in a rme1 dmc1 strain. (B) CPT sensitivity of rad51-K191R is suppressed by deleting either Rme1 or its corepressor Sin4. (C) Deleting Rme1 does not affect CPT sensitivity of rad55.

 
RME1 is a gene expressed only in haploid cells. It encodes a transcriptional repressor of meiotic genes, such as IME1, a transcriptional activator of meiotic gene expression (COVITZ and MITCHELL 1993). In yeast, there is a meiotic-specific gene, DMC1, that is 45% identical to RAD51 (BISHOP et al. 1992), and both Rad51 and Dmc1 are required for efficient meiotic recombination (BISHOP et al. 1992; SHINOHARA et al. 1997). We asked whether the suppression of the DNA-damage sensitivity of rad51-K191R by rme1 is dependent on the indirect effect of derepressing DMC1 in haploid cells. A rad51-K191R rme1 dmc1 triple mutation was still able to suppress the DNA-damage sensitivity of rad51-K191R (Figure 7A). Thus the suppression of the rad51-K191R phenotype by rme1 is not dependent on possible derepression of DMC1. We note also that Western blot analysis did not show any difference in the abundance of Rad51-K191R in the absence of RME1 (data not shown). This was important, as overexpressing Rad51-K191R will suppress its phenotype (MORGAN et al. 2002).

RME1 both positively and negatively regulates the transcription of other genes (MIZUNO and HARASHIMA 2000; FRENZ et al. 2001; BLUMENTAL-PERRY et al. 2002); its repression of genes also depends on the Sin4 transcription factor. To establish whether RME1's action was likely to repress some target gene, we asked if a sin4 mutation could also suppress rad51-K191R. Indeed it did, especially at 23° (Figure 7B). No microarray survey of RME1-regulated genes is available, but a catalog of SIN4-regulated genes (D. MITRA and D. STILLMAN, unpublished results) was examined. No obvious candidate genes involved in recombination are evident.

Sensitivity to DNA damage of a C-terminal truncation of RAD52 is not suppressed by defects in NHEJ:

A C-terminal truncation of RAD52 (rad52-327) creates sensitivity to DNA damage induced by MMS (ASLESON et al. 1999) and by the DNA-damaging agent CPT (Figure 1A). This allele is not nearly as sensitive to CPT as a rad52 mutant strain (data not shown). Deletion of SIR3 suppressed the CPT sensitivity of a rad52-327strain at 30° (Figure 1A). Surprisingly, deletion of genes involved in the NHEJ pathway (e.g., NEJ1 and LIF1) did not suppress the CPT sensitivity of the rad52-327 allele (Figure 2C and Table 2).

The small ORF YGL193C of unknown function has been shown to be repressed by a1–2 in every microarray screen of mating-type-regulated genes (GALITSKI et al. 1999; WYRICK et al. 1999; VALENCIA et al. 2001). Paradoxically, although YGL193C is a haploid-specific gene, turned off in sir3 strains, its absence enhances rather than suppresses the sensitivity of rad52-327 cells to CPT almost 100-fold (Figure 8B), which is the opposite effect of a sir3 mutation (Figure 1A). This result underscores the complex nature of mating-type regulation of DNA repair.

View larger version (69K): In this window In a new window Download PPT slide   FIGURE 8.— Suppression or sensitization of rad52-327. (A) Suppression of rad52-327 but not of rad51-K191R or rad55 by deleting FPR3. (B) Enhancement of rad52-327 sensitivity by deleting the Mata1-Mat2 repressed gene YGL193.

 

Rad52-327 is also suppressed by deleting the Frp3 proline isomerase:

Recently, Frp3 was identified as an enhancer of the DNA damage checkpoint caused in meiotic cells lacking the recombination protein Dmc1 (HOCHWAGEN et al. 2005); hence frp3 dmc1 mutants are able to complete the meiosis and spore formation even though spores have failed to repair DSBs. On the basis of the early results communicated by A. Hochwagen and A. Amon we had speculated that fpr3 might improve the activity of Rad51 in meiosis, as overexpression of Rad51 will suppress dmc1; however, later work showed that the effect of fpr3 was on the meiotic DNA-damage checkpoint and not on the efficiency of meiotic recombination in dmc1 cells (HOCHWAGEN et al. 2005). We asked if fpr3 would also affect the sensitivity of the three CPT-sensitive mutations analyzed here. Although fpr3 had no effect on either rad55 or rad51-K191R, it did suppress (10-fold) the CPT sensitivity of rad52-327 (Figure 8A). Although FPR3 is apparently not MAT regulated, sir3 fpr3 rad52-327 cells were no more resistant than either single-mutant suppressor (data not shown).

Deletion of histone deacetylases:

The gene expression study of GALITSKI et al. (1999) suggested that the histone deacetylase Rpd3 was repressed in MATa/MAT diploids compared to diploids expressing one mating type. Consequently, we deleted RPD3 and also UME6, which acts with a second histone deacetylase, SIN3. Deletion of either RPD3 or UME6 results in both increases and decreases in expression of a large number of genes, 117 of which are commonly affected (http://www.yeastgenome.org), including induction of a number of genes that are also repressed by RME1. Neither rpd3 nor ume6 by itself affects CPT sensitivity, but their deletions enhance significantly the CPT sensitivity of rad55, rad51-K191R, and rad52-327 cells between 10 and 1000-fold (Figure 9). In general, the effect of ume6 was less extreme than that of rpd3, a finding consistent with the fact that Rpd3 is recruited by Ume6, but Rpd3 affects the expression of many other genes (KURDISTANI et al. 2002).

View larger version (62K): In this window In a new window Download PPT slide   FIGURE 9.— Increased sensitivity of rad55, rad51-K191R, and rad52-327 in the absence of RPD3 (A) or UME6 (B).

 

We began our study with the conviction that MATa/MAT heterozygosity would control one or a few genes whose activity would be responsible for the mating-type-dependent suppression of rad55, rad51-K191R, and rad52-327, all of which are also suppressed by overexpressing Rad51 (or Rad51-K191R). Much to our surprise, we found that each of these three mutations is suppressed by deletions of different genes. rad55 is suppressed by deletion of the MATa/MAT-repressed NEJ1 gene as well as by deletion of other genes involved in NHEJ and also by pst2 and/or rfs1. However, rad51-K191R is suppressed by deleting a different MAT-regulated gene, RME1. The CPT sensitivity of a third mutant, rad52-327, is in fact enhanced by deleting yet another MATa/MAT-repressed gene, YGL193C. This last gene is listed in the Saccharomyces Genome Database as a dubious ORF, but here we show that it has a distinct phenotype. The rad52-327 mutant is also uniquely suppressed by deletion of FPR3, recently implicated in regulating protein phosphatase 1 in meiotic cells (HOCHWAGEN et al. 2005). The suppression by fpr3 reported here is its first reported mitotic phenotype.

When rad55 cells are exposed to CPT or phleomycin, their sensitivity can be partially suppressed by mating-type heterozygosity. We have shown that much of this suppression is explained by the fact that cells expressing both MATa and MAT inactivate NHEJ by repressing NEJ1. This result is supported by a recent finding that vertebrate DT40 cells that are defective in NHEJ are also more resistant to CPT than their wild-type counterparts (ADACHI et al. 2004). In budding yeast, we find no effect of knocking out NHEJ in otherwise wild-type cells for either CPT or phleomycin sensitivity, but there is a clear suppressing effect of knocking out DNL4, LIF1, NEJ1, or YKU70 in rad55 strains. We suggest that CPT leaves many single-strand lesions that are converted during DNA replication into DSBs. Some pairs of DSBs may be ligated together by NHEJ, creating lethal deletions or chromosome rearrangements. CPT promotes gross chromosomal rearrangements in budding yeast (MYUNG and KOLODNER 2003). It is possible that DNA damage induced by CPT in a rad55 mutant strain leads to gene rearrangements such as nonreciprocal translocations that require NHEJ functions. When NHEJ is prevented, the weakened HR machinery, lacking Rad55, is then able to (perhaps slowly) repair the lesions by sister-chromatid recombination, thus improving cell survival.

However, knocking out NHEJ does not explain why MAT heterozygosity improves the CPT sensitivity of rad51-K191R or rad52-327. We suggest that the initial steps of strand invasion may be efficient in these two mutations, so that the DSB ends are committed to HR, although perhaps inefficient in completing the process. Thus, the absence of NHEJ does not affect the outcome. We have previously shown that Rad51-K191R protein is normal in filament formation, as judged by the kinetics of chromatin immunoprecipitation; a similar analysis has not yet been done for Rad52-327. In contrast, rad55, by impairing Rad51 filament formation, may leave DSB ends available for NHEJ to take place.

RME1 is best known for its repression of genes required to launch meiosis, but the result presented here confirms suggestions that it might regulate genes under mating-type control in mitotic cells as well. We find that both rme1 and sin4 partially suppress CPT sensitivity of rad51-K191R but not of rad55 or rad52-327. The simplest explanation for this result would have been that a rme1 mutation allows the expression of the Rad51 homolog, Dmc1, and thus provides an alternative, more functional recombinase protein. However, this is not the case; deleting DMC1 did not eliminate the suppression of rad51-K191R by rme1. Further insight into how deleting RME1 suppresses both CPT and phelomycin sensitivity of rad51-K191R would be aided by a microarray study of RME1-regulated genes, expressed in mitotic cells.

Although it does not seem to be MAT regulated, the prolyl isomerase gene FPR3 was found to be involved in rad52-327 CPT sensitivity. This protein belongs to a very large family of related prolyl isomerases in budding yeast, and their absence has very minor effects on cell viability (BENTON et al. 1994; DAVEY et al. 2000; http://db.yeastgenome.org). It is possible that Fpr3 might act to improve the function of Rad51 protein in the absence of its interaction with the C terminus of Rad52. This suggestion is prompted by the findings that overexpression of Rad51 suppresses rad52-327. However, overexpressing Rad51 also suppresses rad55, whereas fpr3 appears to affect only rad52-327. Moreover, a fpr3 mutation did not improve the ability of dmc1 strains to carry out recombination during meiosis, even though overexpressing Rad51 suppresses dmc1 (HOCHWAGEN et al. 2005). We do not believe that fpr3 acts in mitotic cells to suppress the mitotic DNA-damage checkpoint, because a checkpoint-defective mutation such as rad9 (which is by itself CPT sensitive) does not suppress the CPT sensitivity of rad52-327. Another possibility is that a prolyl isomerase such as Fpr3 acts in the degradation of mutant proteins (DAVEY et al. 2000). Perhaps Rad52-327, a truncated protein—or another protein that depends on Rad52 interaction for its stability—is more stable in the absence of Fpr3. Western blot analysis of Rad52-327 with an N-terminal Rad52-specific antibody (provided by A. Shinohara) did not reveal a difference in abundance of the truncated protein in a fpr3 mutant (data not shown).

The suppressive effect of MAT heterozygosity appears to be dependent on the type of DNA damage. While the CPT sensitivity of a rad55 strain can be suppressed by coexpression of MATa and MAT, the sensitivity of this strain to UV is not affected by MAT heterozygosity. This difference may be explained by the finding that Rad52- and Rad51-dependent homologous recombination does not play an important role in resistance to UV (FRIIS and ROMAN 1968). DNA damage induced by phleomycin and HU in rad55 can also be suppressed by MAT heterozygosity. All of these treatments may lead eventually to the formation of DSBs, but it is possible that single-stranded DNA gaps are also important substrates for the Rad52 pathway.

Finally, we found that deletion of genes encoding the histone deacetylase Rpd3 and Ume6 increase CPT sensitivity of rad55, rad52-327, and rad51-K191R strains, but does not affect otherwise wild-type cells. The basis of this synergy is not understood. Since both these genes control the expression of >100 other genes in yeast, we cannot easily determine whether the effect observed was indirect, by changing the expression of an unknown DNA repair gene, or by direct action at the site of the damage. The role of chromatin changes at the site of a DSB is currently a subject of great interest, with various reports suggesting that damage-induced phosphorylation of histone H2A (DOWNS et al. 2000; REDON et al. 2003), modifications of histone H4, and recruitment of the NuA4 (BIRD et al. 2002), Ino80 (MORRISON et al. 2004), Swi2/Snf5 (CHAI et al. 2005), and RSC (SHIM et al. 2005) chromatin-remodeling complexes all may affect DSB repair, either NHEJ or HR. Where the histone deacetylases fit into this picture will need further investigation.

We are grateful to Lorraine Symington for providing the rad52-327 and rad51-K191R mutant strains as well as for her suggestions about this research. Andreas Hochwagen and Angelica Amon stimulated our interest in FPR3 and kindly provided us with their results prior to publication. Doyel Mitra and David Stillman provided unpublished microarray data for sin4. Susan Lovett and members of the Haber Lab provided valuable commentary. This research was supported by National Institutes of Health grant GM20056 and by Department of Energy grant ER01ER63229.

¹ Present address: Center for Advanced Biotechnology, Boston University, Boston, MA 02215.

² Present address: Department of Biochemistry, Nagasaki University School of Medicine, Sakamoto 1-12-4, Nagasaki 852-8523, Japan.

³ Present address: Sinsheimer Labs, UCSC, Santa Cruz, CA 95064.

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