Budding yeast mms4 is epistatic with rad52 and the function of Mms4 can be replaced by a bacterial Holliday junction resolvase

MMS4 of Saccharomyces cerevisiae was originally identified as the gene responsible for one of the collection of methyl methanesulfonate (MMS)-sensitive mutants, mms4. Recently it was identified as a synthetic lethal gene with an SGS1 mutation. Epistatic analyses revealed that MMS4 is involved in a pathway leading to homologous recombination requiring Rad52 or in the recombination itself, in which SGS1 is also involved. MMS sensitivity of mms4 but not sgs1, was suppressed by introducing a bacterial Holliday junction (HJ) resolvase, RusA. The frequencies of spontaneously occurring unequal sister chromatid recombination (SCR) and loss of marker in the rDNA in haploid mms4 cells and interchromosomal recombination between heteroalleles in diploid mms4 cells were essentially the same as those of wild-type cells. Although UV- and MMS-induced interchromosomal recombination was defective in sgs1 diploid cells, hyper-induction of interchromosomal recombination was observed in diploid mms4 cells, indicating that the function of Mms4 is dispensable for this type of recombination.     

SMC6 is required for MMS-induced interchromosomal and sister chromatid recombinations in Saccharomyces cerevisiae

SMC6 (RHC18) in Saccharomyces cerevisiae, which is a homologue of the Schizosaccharomyces pombe rad18+ gene and essential for cell viability, encodes a structural maintenance of chromosomes (SMC) family protein. In contrast to the rest of the SMC family of proteins, Smc1-Smc4, which are the components of cohesin or condensin, little is known about Smc6. In this study, we generated temperature sensitive (ts) smc6 mutants of budding yeast and characterized their properties. One ts-mutant, smc6-56, ceased growth soon after up-shift to a non-permissive temperature, arrested in the late S and G2/M phase, and gradually lost viability. smc6-56 cells at a permissive temperature showed a higher sensitivity than wild-type cells to various DNA damaging agents including methyl methanesulfonate (MMS). The rad52 smc6-56 double mutant showed a sensitivity to MMS similar to that of the rad52 single mutant, indicating that Smc6 is involved in a pathway that requires Rad52 to function. Moreover, no induction of interchromosomal recombination and sister chromatid recombination was observed in smc6-56 cells, which occurred in wild-type cells upon exposure to MMS.     

Involvement of SGS1 in DNA damage-induced heteroallelic recombination that requires RAD52 in Saccharomyces cerevisiae

The SGS1 gene of Saccharomyces cerevisiae is homologous to the genes that are mutated in Bloom's syndrome and Werner's syndrome in humans. Disruption of SGS1 results in high sensitivity to methyl methanesulfonate (MMS), poor sporulation, and a hyper-recombination phenotype including recombination between heteroalleles. In this study, we found that SGS1 forms part of the RAD52 epistasis group when cells are exposed to MMS. Exposure to DNA-damaging agents causes a striking, Rad52-dependent, increase in heteroallelic recombination in wild-type cells, but not in sgs1 disruptants. However, in the absence of DNA damage, the frequency of heteroallelic recombination in sgs1 disruptants was several-fold higher than in wild-type cells, as described previously. These results imply a function for Sgs1: it acts to suppress spontaneous heteroallelic recombination, and to promote DNA damage-induced heteroallelic recombination.     

Primary mouse fibroblasts deficient for c-Fos, p53 or for both proteins are hypersensitive to UV light and alkylating agent-induced chromosomal breakage and apoptosis

The important regulatory proteins, c-Fos and p53 are induced by exposure of cells to a variety of DNA damaging agents. To investigate their role in cellular defense against genotoxic compounds, we comparatively analysed chromosomal aberrations and apoptosis induced by ultraviolet (UV-C) light and the potent alkylating agent methyl methanesulfonate (MMS) in primary diploid mouse fibroblasts knockout for either c-Fos or p53, or double knockout for both genes. We show that c-Fos and p53 deficient fibroblasts are more sensitive than the corresponding wild-type cells as to the induction of chromosomal aberrations and apoptosis. Double knockout fibroblasts lacking both c-Fos and p53 are viable and were even more sensitive, showing additivity of the chromosomal breakage effects observed in the single knockouts. Regarding the endpoint apoptosis, double knockout fibroblasts displayed a sensitivity similar to c-Fos and p53 deficient cells. The data indicate that (a) both c-Fos and p53 are involved in cellular protection against the clastogenic effect of genotoxic agents, (b) p53 is not required for induction of apoptosis by UV light and MMS, but rather prevents fibroblasts from undergoing apoptotic cell death upon DNA damage, and (c) c-Fos and p53 seem to act independently in determining genotoxic resistance, which is hypothesized to be achieved by impaired DNA repair or differential cell cycle check point control.     

Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair

Ubiquitin-conjugating enzyme variant (UEV) proteins resemble ubiquitin-conjugating enzymes (E2s) but lack the defining E2 active-site residue. The MMS2-encoded UEV protein has been genetically implicated in error-free postreplicative DNA repair in Saccharomyces cerevisiae. We show that Mms2p forms a specific heteromeric complex with the UBC13-encoded E2 and is required for the Ubc13p-dependent assembly of polyubiquitin chains linked through lysine 63. A ubc13 yeast strain is UV sensitive, and single, double, and triple mutants of the UBC13, MMS2, and ubiquitin (ubiK63R) genes display a comparable phenotype. These findings support a model in which an Mms2p/Ubc13p complex assembles novel polyubiquitin chains for signaling in DNA repair, and they suggest that UEV proteins may act to increase diversity and selectivity in ubiquitin conjugation.     

The ability of Sgs1 to interact with DNA topoisomerase III is essential for damage-induced recombination

SGS1 encodes a protein having DNA helicase activity, and a mutant allele of SGS1 was identified as a suppressor of the slow growth phenotype of top3 mutants. In this study, we examined whether Sgs1 prevents formation of DNA double strand breaks (DSBs) or is involved in DSB repair following exposure to methyl methanesulfonate (MMS). An analysis by pulsed-field gel electrophoresis and epistasis analyses indicated that Sgs1 is required for DSB repair that involves Rad52. In addition, analyses on the relationship between Sgs1 and proteins involved in DSB repair suggested that Sgs1 and Mre11 function via independent pathways both of which require Rad52. In sgs1 mutants, interchromosomal heteroallelic recombination and sister chromatid recombination (SCR) were not induced upon exposure to MMS, though both were induced in wild type cells, indicating the involvement of Sgs1 in heteroallelic recombination and SCR. Surprisingly, the ability of Sgs1 to bind to DNA topoisomerase III (Top3) was absolutely required for the induction of heteroallelic recombination and SCR and suppression of MMS sensitivity but its helicase activity was not, suggesting that Top3 plays a more important role in both recombinations than the DNA helicase activity of Sgs1.     

Human MMS21/NSE2 is a SUMO ligase required for DNA repair

DNA repair is required for the genomic stability and well-being of an organism. In yeasts, a multisubunit complex consisting of SMC5, SMC6, MMS21/NSE2, and other non-SMC proteins is required for DNA repair through homologous recombination. The yeast MMS21 protein is a SUMO ligase. Here we show that the human homolog of MMS21 is also a SUMO ligase. hMMS21 stimulates sumoylation of hSMC6 and the DNA repair protein TRAX. Depletion of hMMS21 by RNA interference (RNAi) sensitizes HeLa cells toward DNA damage-induced apoptosis. Ectopic expression of wild-type hMMS21, but not its ligase-inactive mutant, rescues this hypersensitivity of hMMS21-RNAi cells. ATM/ATR are hyperactivated in hMMS21-RNAi cells upon DNA damage. Consistently, hMMS21-RNAi cells show an increased number of phospho-CHK2 foci. Finally, we show that hMMS21-RNAi cells show a decreased capacity to repair DNA lesions as measured by the comet assay. Our findings suggest that the human SMC5/6 complex and the SUMO ligase activity of hMMS21 are required for the prevention of DNA damage-induced apoptosis by facilitating DNA repair in human cells.     

Chl1 and Ctf4 are required for damage-induced recombinations

Deletion mutants of CHL1 or CTF4, which are required for sister chromatid cohesion, showed higher sensitivity to the DNA damaging agents methyl methanesulfonate (MMS), hydroxyurea (HU), phleomycin, and camptothecin, similar to the phenotype of mutants of RAD52, which is essential for recombination repair. The levels of Chl1 and Ctf4 associated with chromatin increased considerably after exposure of the cells to MMS and phleomycin. Although the activation of DNA damage checkpoint did not affected in chl1 and ctf4 mutants, the repair of damaged chromosome was inefficient, suggesting that Chl1 and Ctf4 act in DNA repair. In addition, MMS-induced sister chromatid recombination in haploid cells, and, more importantly, MMS-induced recombination between homologous chromosomes in diploid cells were impaired in these mutants. Our results suggest that Chl1 and Ctf4 are directly involved in homologous recombination repair rather than acting indirectly via the establishment of sister chromatid cohesion.     

Studies on temperature-sensitive mutants of Chinese hamster ovary cells affected in DNA synthesis

DNA synthesis in two mutants of Chinese hamster overy cells, ts 13A and ts 15C, which were temperature sensitive for growth, was found to be shut off rapidly at the nonpermissive temperature. The mutants did not complement each other and the ts lesion was not located on the X chromosome. Both isolates were found to be considerably more sensitive to the alkylating agents, ethylmethanesulfonate (EMS) and methylmethanesulfonate (MMS), as compared to the parental cells, but showed normal sensitivity to UV irradiation. The mutants also showed interesting differences in their response to EMS-induced mutation frequencies at the ouabain-resistant and thioguanine-resistant loci. At high survival (50%) the frequencies of mutations at these genetic loci were markedly low in the ts mutants as compared to the parental cells. In ts+ revertants isolated from the mutants, the ts phenotype and the increased sensitivity to EMS and MMS were affected simultaneously, indicating that both these characteristics resulted from a single genetic lesion.     

Rad52 sumoylation and its involvement in the efficient induction of homologous recombination

The protein Rad52 is a key player in various types of homologous recombination and is essential to maintenance of genomic integrity. Although evidence indicates that Rad52 is modified by SUMO, the physiological relevance of this sumoylation remains unclear. Here, we identify the conditions under which Rad52 sumoylation is induced, and clarify the role of this modification in homologous recombination. Oligomerization of Rad52 was a prerequisite for sumoylation, and the modification occurred in the cell proceeding S phase being exposed to the DNA-damaging agent methyl methanesulfonate (MMS). Following exposure to MMS, sumoylated Rad52 accumulated in rad51 cells, but not in the recombination-related gene mutants, rad54, rad55, rad59, sgs1, or srs2. The accumulation of sumoylated Rad52 was suppressed in rad51 cells expressing Rad51-K191R, an ATPase-defective protein presumed to be recruited to ssDNA. Although the sumoylation defective mutant rad52-3KR (K10R/K11R/K220R) showed no defect in mating-type switching, which did not lead to Rad52 sumoylation in wild-type cells, the mutant did demonstrate a partial defect in MMS-induced interchromosomal homologous recombination.     

Saccharomyces Cerevisiae Rad52 Alleles Temperature-Sensitive for the Repair of DNA Double-Strand Breaks

We have screened for mutations of the Saccharomyces cerevisiae RAD52 gene which confer a temperature-sensitive (ts) phenotype with respect to either the repair of DNA lesions caused by methyl methanesulfonate (MMS) or the recombination of an intrachromosomal recombination reporter. We were readily able to isolate alleles ts for the repair of lesions caused by MMS but were unable to find alleles with a severe ts deficiency in intrachromosomal recombination. We extensively characterized four strains conferring ts growth on MMS agar. These strains also exhibit ts survival when exposed to γ-radiation or when the HO endonuclease is constitutively expressed. Although none of the four alleles confers a severe ts defect in intrachromosomal recombination, two confer significant defects in tests of mitotic, interchromosomal recombination carried out in diploid strains. The mutant diploids sporulate, but the two strains with defects in interchromosomal recombination have reduced spore viability. Meiotic recombination is not depressed in the two diploids with reduced spore viability. Thus, in the two strains with reduced spore viability, defects in mitotic and meiotic recombination do not correlate. Sequence analysis revealed that in three of the four ts alleles the causative mutations are in the first one-third of the open reading frame while the fourth is in the C-terminal third.     

Structural and functional conservation of error-free DNA postreplication repair in Schizosaccharomyces pombe

DNA postreplication repair (PRR) is a cellular process by which cells survive replication-blocking lesions without removing the lesion. In the budding yeast Saccharomyces cerevisiae, MMS2 plays a key role in the error-free PRR pathway: the mms2 null mutant displays an increased spontaneous mutation rate and sensitivity to a variety of DNA damaging agents. In contrast, its human homologs appear to play a different role. In order to address whether the MMS2-mediated PRR pathway is conserved in eukaryotes, we isolated a Schizosaccharomyces pombe cDNA homologous to MMS2, which we named spm2(+). Using spm2(+) as a bait in a yeast two-hybrid screen, we identified a fission yeast cDNA homologous to UBC13 from various species and named it spu13(+). Two-hybrid analysis confirmed physical interaction between Spm2 and Spu13, and between Spm2 and budding yeast Ubc13. Genetic analysis shows that both spm2(+) and spu13(+) are able to functionally complement the corresponding budding yeast mutants. Furthermore, deletion of either spm2(+), spu13(+) or both genes from fission yeast results in an increased sensitivity to DNA damaging agents, suggesting that spm2(+) and spu13(+) indeed function in PRR. The fact that the spm2(-) spu13(-) double mutant showed sensitivity similar to that of the single mutant indicates that these two gene products act at the same step. Hence, our data strongly support the hypothesis that the PRR function mediated by UBC13-MMS2 is conserved throughout eukaryotes.     

The DNA excision repair system of the highly radioresistant bacterium Deinococcus radiodurans is facilitated by the pentose phosphate pathway

Deinococcus radiodurans is highly resistant to radiation and mutagenic chemicals. Mutants defective in the putative glucose-6-phosphate dehydrogenase gene (zwf-) and the aldolase gene (fda-) were generated by homologous recombination. These mutants were used to test the cells' resistance to agents that cause dimer formation and DNA strand breaks. The zwf - mutants were more sensitive to agents that induce DNA excision repair, such as UV irradiation and H2O2, but were as resistant to DNA strand break-causing agents such as methylmethanesulphonic acid (MMS) and mitomycin C (MMC) as the wild-type cells. Analysis of the cytoplasmic fraction of zwf- cells showed that the concentrations of inosine monophosphate (IMP) and uridine monophosphate (UMP) were only 30% of those found in the wild-type cells. The fda- mutants were slightly more resistant to UV light and H2O2. Results suggested that the deinococcal pentose phosphate pathway augmented the DNA excision repair system by providing cells with adequate metabolites for the DNA mismatch repair.     

Mec1-dependent phosphorylation of Mms21 modulates its SUMO ligase activity

The SUMO ligase Mms21, which is a subunit of the Smc5/6 complex, is required for DNA repair. Here we present results showing that Mms21 was phosophorylated during S-phase in a manner dependent on the DNA damage kinase Mec1. Phosphorylation of Mms21 occurred in unchallenged cells, but was more abundant in the presence of DNA damaging agents. Mass spectrometry identified five phosphorylated serines organized in two regions of Mms21, and two C-terminal serines, S260 and S261, formed part of a Mec1/Tel1 consensus motif. Nonphosphorylatable substitutions of the C-terminal serines, inactivation of Mec1 or removal of the Mms21 C-terminus all abolished Mms21 phosphorylation. Additionally, strains carrying Mms21 phosphoablative alleles displayed reduced SUMO ligase activity, sensitivity to MMS and an increased rate of chromosome loss in the presence of MMS. We propose that one function of S260 S261 phosphorylation is to positively regulate the SUMO ligase activity of Mms21 and thereby promote genomic stability.     

Saccharomyces cerevisiae RAD53 (CHK2) but not CHK1 is required for double-strand break-initiated SCE and DNA damage-associated SCE after exposure to X rays and chemical agents

Saccharomyces cerevisiae RAD53 (CHK2) and CHK1 control two parallel branches of the RAD9-mediated pathway for DNA damage-induced G(2) arrest. Previous studies indicate that RAD9 is required for X-ray-associated sister chromatid exchange (SCE), suppresses homology-directed translocations, and is involved in pathways for double-strand break repair (DSB) repair that are different than those controlled by PDS1. We measured DNA damage-associated SCE in strains containing two tandem fragments of his3, his3-Delta5' and his3-Delta3'::HOcs, and rates of spontaneous translocations in diploids containing GAL1::his3-Delta5' and trp1::his3-Delta3'::HOcs. DNA damage-associated SCE was measured after log phase cells were exposed to methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4-NQO), UV, X rays and HO-induced DSBs. We observed that rad53 mutants were defective in MMS-, 4-NQO, X-ray-associated and HO-induced SCE but not in UV-associated SCE. Similar to rad9 pds1 double mutants, rad53 pds1 double mutants exhibited more X-ray sensitivity than the single mutants. rad53 sml1 diploid mutants exhibited a 10-fold higher rate of spontaneous translocations compared to the sml1 diploid mutants. chk1 mutants were not deficient in DNA damage-associated SCE after exposure to DNA damaging agents or after DSBs were generated at trp1::his3-Delta5'his3-Delta3'::HOcs. These data indicate that RAD53, not CHK1, is required for DSB-initiated SCE, and DNA damage-associated SCE after exposure to X-ray-mimetic and UV-mimetic chemicals.     

Cellular and genetic studies in three UV-sensitive Chinese hamster mutants

Three UV sensitive (UV^s) mutants (CHO43RO, CHO423PV, CHO30PV), characterized by different levels of reduction in their ability to perform unscheduled DNA synthesis (UDS), were analysed for spontaneous and UV-induced frequency of chromosomal aberrations and for sensitivity to alkylating agents. The baseline frequency of chromosomal aberrations was in the normal range, whereas after UV irradiation a positive correlation between the degree of UV sensitivity and the rate of chromosomal breakage was observed. Survival experiments after mutagen exposure indicated that the UV^s clones are characterized by different levels of hypersensitivity to bifunctional alkylating agents whereas the sensitivity to monofunctional alkylating agents is in the normal range. Genetic analysis performed by measuring the survival after UV in hybrids produced by fusing UV^s cells with wild-type or UV^s cells belonging to the six Chinese hamster complementation groups, indicated that the three clones carry recessive mutations and belong to c.g. 2. These findings suggest that defects in the same gene may result in different degrees of phenotypic alterations.Abbreviations CG complementation group - EMS ethyl methane sulfonate - MMS methyl methane sulfonate - MMC mitomycin C - UV ultraviolet - UDS unscheduled DNA synthesis     

The SUMO isopeptidase Ulp2p is required to prevent recombination-induced chromosome segregation lethality following DNA replication stress

SUMO conjugation is a key regulator of the cellular response to DNA replication stress, acting in part to control recombination at stalled DNA replication forks. Here we examine recombination-related phenotypes in yeast mutants defective for the SUMO de-conjugating/chain-editing enzyme Ulp2p. We find that spontaneous recombination is elevated in ulp2 strains and that recombination DNA repair is essential for ulp2 survival. In contrast to other SUMO pathway mutants, however, the frequency of spontaneous chromosome rearrangements is markedly reduced in ulp2 strains, and some types of rearrangements arising through recombination can apparently not be tolerated. In investigating the basis for this, we find DNA repair foci do not disassemble in ulp2 cells during recovery from the replication fork-blocking drug methyl methanesulfonate (MMS), corresponding with an accumulation of X-shaped recombination intermediates. ulp2 cells satisfy the DNA damage checkpoint during MMS recovery and commit to chromosome segregation with similar kinetics to wild-type cells. However, sister chromatids fail to disjoin, resulting in abortive chromosome segregation and cell lethality. This chromosome segregation defect can be rescued by overproducing the anti-recombinase Srs2p, indicating that recombination plays an underlying causal role in blocking chromatid separation. Overall, our results are consistent with a role for Ulp2p in preventing the formation of DNA lesions that must be repaired through recombination. At the same time, Ulp2p is also required to either suppress or resolve recombination-induced attachments between sister chromatids. These opposing defects may synergize to greatly increase the toxicity of DNA replication stress.