Postreplication DNA Repair Research Articles

The yeast Shu complex couples error‐free post‐replication repair to homologous recombination

DNA post-replication repair (PRR) functions to bypass replication-blocking lesions and prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA. While translesion synthesis has been well characterized, little is known about the molecular events involved in error-free bypass, although it has been assumed that homologous recombination (HR) is required for such a mode of lesion bypass. We undertook a genome-wide synthetic genetic array screen for novel genes involved in error-free PRR and observed evidence of genetic interactions between error-free PRR and HR. Furthermore, this screen identified and assigned four genes, CSM2, PSY3, SHU1 and SHU2, whose products form a stable Shu complex, to the error-free PRR pathway. Previous studies have indicated that the Shu complex is required for efficient HR and that inactivation of any of these genes is able to suppress the severe phenotypes of top3 and sgs1. We confirmed and further extended some of the reported observations and demonstrated that error-free PRR mutations are also epistatic to sgs1. Based on the above analyses, we propose a model in which error-free PRR utilizes the Shu complex to recruit HR to facilitate template switching, followed by double-Holliday junction resolution by Sgs1-Top3. This mechanism appears to be conserved throughout eukaryotes.

Utility of DNA postreplication repair protein Rad6B in neoadjuvant chemotherapy response

Neoadjuvant chemotherapy is a standard therapy for patients with locally advanced breast cancer (LABC) and is increasingly used for early stage operable breast cancer. Not all patients benefit from it, and reliable markers for predicting response are needed. The cytotoxic effects of chemotherapy are mediated by induction of DNA damage in tumor cells. There is evidence that resistance to chemotherapy is related to enhanced repair of DNA lesions. The postreplication DNA repair (PRR) or translesion synthesis backup DNA repair pathway is critical for cell viability, conferring tolerance to DNA damaging drugs, and maintenance of genomic integrity. However, despite its importance in conferring tolerance to a variety of DNA damaging drugs including cytotoxic chemotherapy, the involvement of this backup repair pathway in chemotherapy response has not been studied. The Rad6B protein is a fundamental component of PRR. We have shown previously that the ability of breast cells to tolerate chemotherapeutic drugs correlates with Rad6B expression levels and PRR capacity. To determine whether Rad6B expression/distribution can be used singly or in combination with p53, Mdr-1/PgP, PCNA or beta-catenin as predictors of response to neoadjuvant chemotherapy, we analyzed posttreatment samples from 20 patients with LABC in a retrospective study. Only preferential Rad6B nuclear localization was associated with response to neoadjuvant chemotherapy. Nuclear exclusion with cytoplasmic overexpression of Rad6B was observed in some patients who failed to respond, but the association with response is not statistically significant. This is the first study to report that the postreplication DNA repair protein Rad6B could be used as an independent marker for determining response to neoadjuvant chemotherapy. This is an exploratory study and larger studies utilizing interim evaluations of Rad6B expression, its subcellular localization and repair activity are required to confirm its utility as a predictor of chemotherapeutic response.

Distinct Functions of MLH3 at Recombination Hot Spots in the Mouse

The four mammalian MutL homologs (MLH1, MLH3, PMS1, and PMS2) participate in a variety of events, including postreplicative DNA repair, prevention of homeologous recombination, and crossover formation during meiosis. In this latter role, MLH1-MLH3 heterodimers predominate and are essential for prophase I progression. Previous studies demonstrated that mice lacking Mlh1 exhibit a 90% reduction in crossing over at the Psmb9 hot spot while noncrossovers, which do not result in exchange of flanking markers but arise from the same double-strand break event, are unaffected. Using a PCR-based strategy that allows for detailed analysis of crossovers and noncrossovers, we show here that Mlh3(-/-) exhibit a 85-94% reduction in the number of crossovers at the Psmb9 hot spot. Most of the remaining crossovers in Mlh3(-/-) meiocytes represent simple exchanges similar to those seen in wild-type mice, with a small fraction (6%) representing complex events that can extend far from the initiation zone. Interestingly, we detect an increase of noncrossovers in Mlh3(-/-) spermatocytes. These results suggest that MLH3 functions predominantly with MLH1 to promote crossovers, while noncrossover events do not require these activities. Furthermore, these results indicate that approximately 10% of crossovers in the mouse are independent of MLH3, suggesting the existence of alternative crossover pathways in mammals.

Recognition of forked and single‐stranded DNA structures by human RAD18 complexed with RAD6B protein triggers its recruitment to stalled replication forks

Post-replication DNA repair facilitates the resumption of DNA synthesis upon replication fork stalling at DNA damage sites. Despite the importance of RAD18 and polymerase eta (Poleta) for post-replication repair (PRR), the molecular mechanisms by which these factors are recruited to stalled replication forks are not well understood. We present evidence that human RAD18 complexed with RAD6B protein preferentially binds to forked and single-stranded DNA (ssDNA) structures, which are known to be localized at stalled replication forks. The SAP domain of RAD18 (residues 248-282) is crucial for binding of RAD18 complexed with RAD6B to DNA substrates. RAD18 mutated in the SAP domain fails to accumulate at DNA damage sites in vivo and does not guide DNA Poleta to stalled replication forks. The SAP domain is also required for the efficient mono-ubiquitination of PCNA. The SAP domain mutant fails to suppress the ultraviolet (UV)-sensitivity of Rad18-knockout cells. These results suggest that RAD18 complexed with RAD6B is recruited to stalled replication forks via interactions with forked DNA or long ssDNA structures, a process that is required for initiating PRR.

Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination

Human helicase-like transcription factor (HLTF) is frequently inactivated in colorectal and gastric cancers. Here, we show that HLTF is a functional homologue of yeast Rad5 that promotes error-free replication through DNA lesions. HLTF and Rad5 share the same unique structural features, including a RING domain embedded within a SWI/SNF helicase domain and an HIRAN domain. We find that inactivation of HLTF renders human cells sensitive to UV and other DNA-damaging agents and that HLTF complements the UV sensitivity of a rad5Delta yeast strain. Also, similar to Rad5, HLTF physically interacts with the Rad6-Rad18 and Mms2-Ubc13 ubiquitin-conjugating enzyme complexes and promotes the Lys-63-linked polyubiquitination of proliferating cell nuclear antigen at its Lys-164 residue. A requirement of HLTF for error-free postreplication repair of damaged DNA is in keeping with its cancer-suppression role.

Cohesins Functionally Associate with CTCF on Mammalian Chromosome Arms

Cohesins mediate sister chromatid cohesion, which is essential for chromosome segregation and postreplicative DNA repair. In addition, cohesins appear to regulate gene expression and enhancer-promoter interactions. These noncanonical functions remained unexplained because knowledge of cohesin-binding sites and functional interactors in metazoans was lacking. We show that the distribution of cohesins on mammalian chromosome arms is not driven by transcriptional activity, in contrast to S. cerevisiae. Instead, mammalian cohesins occupy a subset of DNase I hypersensitive sites, many of which contain sequence motifs resembling the consensus for CTCF, a DNA-binding protein with enhancer blocking function and boundary-element activity. We find cohesins at most CTCF sites and show that CTCF is required for cohesin localization to these sites. Recruitment by CTCF suggests a rationale for noncanonical cohesin functions and, because CTCF binding is sensitive to DNA methylation, allows cohesin positioning to integrate DNA sequence and epigenetic state.

Damage‐induced reactivation of cohesin in postreplicative DNA repair

Cohesin establishes sister-chromatid cohesion during S phase to ensure proper chromosome segregation in mitosis. It also facilitates postreplicative homologous recombination repair of DNA double-strand breaks by promoting local pairing of damaged and intact sister chromatids. In G2 phase, cohesin that is not bound to chromatin is inactivated, but its reactivation can occur in response to DNA damage. Recent papers by Koshland's and Sjögren's groups describe the critical role of the known cohesin cofactor Eco1 (Ctf7) and ATR checkpoint kinase in damage-induced reactivation of cohesin, revealing an intricate mechanism that regulates sister-chromatid pairing to maintain genome integrity.

Requirement of Nse1, a Subunit of the Smc5-Smc6 Complex, for Rad52-Dependent Postreplication Repair of UV-Damaged DNA in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, postreplication repair (PRR) of UV-damaged DNA occurs by a Rad6-Rad18- and an Mms2-Ubc13-Rad5-dependent pathway or by a Rad52-dependent pathway. The Rad5 DNA helicase activity is specialized for promoting replication fork regression and template switching; previously, we suggested a role for the Rad5-dependent PRR pathway when the lesion is located on the leading strand and a role for the Rad52 pathway when the lesion is located on the lagging strand. In this study, we present evidence for the requirement of Nse1, a subunit of the Smc5-Smc6 complex, in Rad52-dependent PRR, and our genetic analyses suggest a role for the Nse1 and Mms21 E3 ligase activities associated with this complex in this repair mode. We discuss the possible ways by which the Smc5-Smc6 complex, including its associated ubiquitin ligase and SUMO ligase activities, might contribute to the Rad52-dependent nonrecombinational and recombinational modes of PRR.

The Protein Degradation Response of Saccharomyces cerevisiae to Classical DNA-Damaging Agents

Genome wide experiments indicate both proteasome- and vacuole-mediated protein degradation modulate sensitivity to classical DNA-damaging agents. Here, we show that global protein degradation is significantly increased upon methyl methanesulfonate (MMS) exposure. In addition, global protein degradation is similarly increased upon exposure to 4-nitroquinoline-N-oxide (4NQO) and UV and, to a lesser extent, tert-butyl hydroperoxide. The proteasomal inhibitor MG132 decreases both MMS-induced and 4NQO-induced protein degradation, while addition of the vacuolar inhibitor phenylmethanesulfonyl fluoride does not. The addition of both inhibitors grossly inhibits cell growth upon MMS exposure over and above the growth inhibition induced by MMS alone. The MMS-induced protein degradation response remains unchanged in several ubiquitin-proteasome and vacuolar mutants, presumably because these mutants are not totally deficient in either essential pathway. Furthermore, MMS-induced protein degradation is independent of Mec1, Mag1, Rad23, and Rad6, suggesting that the protein degradation response is not transduced through the classical Mec1 DNA damage response pathway or through repair intermediates generated by the base excision, nucleotide excision, or postreplication-DNA repair pathways. These results identify the regulation of protein degradation as an important factor in the recovery of cells from toxicity induced by classical DNA-damaging agents.

The spectrum of spontaneous mutations caused by deficiency in proteasome maturase Ump1 in Saccharomyces cerevisiae

Ump1 is responsible for maturation of the catalytic core of the 26S proteasome. Dysfunction of Ump1 causes an increase in the frequency of spontaneous mutations in Saccharomyces cerevisiae. In this study we analyze the spectrum of mutations occurring spontaneously in yeast deficient in Ump1 by use of the SUP4-o system. Single base substitutions predominate among the mutations analyzed (73 of the 91 alterations examined). Two major classes are GC to TA transversions and GC to AT transitions ( approximately 50 and approximately 30% of base substitutions, respectively). Besides base substitutions, almost all the major types of sequence alterations are represented. The specificity and distribution of mutations occurring in the ump1 strain are unique compared to the spectra previously established for other yeast mutators. However, the profile of mutations arising in this strain is similar to that observed in wild type. The same similarity has previously been reported for yeast deficient in Mms2, a protein involved in Rad6-dependent postreplication DNA repair (PRR). The specificity of the mutator effect caused by ump1 is discussed in light of the proposed role of the proteasome activity in the regulation of the PRR mechanisms.

Werner Helicase-interacting Protein 1 Binds Polyubiquitin via Its Zinc Finger Domain

DNA repair is regulated on many levels by ubiquitination. In order to identify novel connections between DNA repair pathways and ubiquitin signaling, we used mass spectrometry to identify proteins that interact with lysine 6-linked polyubiquitin chains. From this proteomic screen, we identified the DNA repair protein WRNIP1 (Werner helicase-interacting protein 1), along with nucleosome assembly protein 1, as novel ubiquitin-interacting proteins. We found that a small zinc finger domain at the N terminus of WRNIP1 is sufficient and necessary for noncovalent ubiquitin binding. This ubiquitin-binding zinc finger (UBZ) domain binds polyubiquitin but not monoubiquitin and appears to show no specificity for polyubiquitin chain linkage. A homologous zinc finger domain in RAD18 also binds polyubiquitin, suggesting a wider role for the UBZ domain in DNA repair. The WRNIP1 ubiquitin-binding function, along with its previously established ATPase activity, suggests that WRNIP1 plays a role in the metabolism of ubiquitinated proteins. Supporting this model, deletion of MGS1, the yeast homolog of WRNIP1, slows the rate of ubiquitin turnover, rendering yeast resistant to cycloheximide. We also find that WRNIP1 is heavily modified with ubiquitin and SUMO, revealing complex layers in the involvement of ubiquitin pathway proteins in the regulation of DNA repair. The novel ubiquitin-binding ability of WRNIP1 sheds light on the role of UBZ domain-containing proteins in postreplication DNA repair.

A mutation in EXO1 defines separable roles in DNA mismatch repair and post-replication repair

Replication forks stall at DNA lesions or as a result of an unfavorable replicative environment. These fork stalling events have been associated with recombination and gross chromosomal rearrangements. Recombination and fork bypass pathways are the mechanisms accountable for restart of stalled forks. An important lesion bypass mechanism is the highly conserved post-replication repair (PRR) pathway that is composed of error-prone translesion and error-free bypass branches. EXO1 codes for a Rad2p family member nuclease that has been implicated in a multitude of eukaryotic DNA metabolic pathways that include DNA repair, recombination, replication, and telomere integrity. In this report, we show EXO1 functions in the MMS2 error-free branch of the PRR pathway independent of the role of EXO1 in DNA mismatch repair (MMR). Consistent with the idea that EXO1 functions independently in two separate pathways, we defined a domain of Exo1p required for PRR distinct from those required for interaction with MMR proteins. We then generated a point mutant exo1 allele that was defective for the function of Exo1p in MMR due to disrupted interaction with Mlh1p, but still functional for PRR. Lastly, by using a compound exo1 mutant that was defective for interaction with Mlh1p and deficient for nuclease activity, we provide further evidence that Exo1p plays both structural and catalytic roles during MMR.

Evidence for Small Ubiquitin-like Modifier-dependent Nuclear Import of the Thymidylate Biosynthesis Pathway

Perturbations in folate-mediated one-carbon metabolism increase rates of uracil misincorporation into DNA during replication, impair cellular methylation reactions, and increase risk for neural tube defects and cancer. One-carbon metabolism is compromised by folate deficiency and common genetic polymorphisms. In this study, the mechanism for the preferential partitioning of cytoplasmic serine hydroxymethyltransferase (cSHMT)-derived methylenetetrahydrofolate to de novo thymidylate biosynthesis was investigated. The cSHMT enzyme was shown to interact with UBC9 and was a substrate for UBC9-catalyzed small ubiquitin-like modifier (SUMO) modification in vitro. SUMOylated cSHMT was detected in extracts from S phase MCF-7 cells, and cSHMT was shown to localize to the nucleus and nuclear periphery during the S and G(2)/M phases of the cell cycle. A common single nucleotide polymorphism (L474F-cSHMT) impaired the UBC9-cSHMT interaction and inhibited cSHMT SUMOylation in vitro. The three folate-dependent enzymes that constitute the de novo thymidylate biosynthesis pathway, cSHMT, thymidylate synthase, and dihydrofolate reductase, all contain SUMO modification consensus sequences. Compartmentation of the folate-dependent de novo thymidylate biosynthesis pathway in the nucleus accounts for the preferential partitioning of cSHMT-derived folate-activated one-carbon units into thymidylate biosynthesis; the efficiency of nuclear folate metabolism is likely to be modified by the cSHMT L474F polymorphism.

HULC, a Histone H2B Ubiquitinating Complex, Modulates Heterochromatin Independent of Histone Methylation in Fission Yeast

Heterochromatin in fission yeast is targeted dynamically by opposing chromatin-modifying activities capable of alleviating or promoting transcriptional gene silencing. In this study, we report the biochemical and genetic characterization of a ubiquitin-conjugating enzyme Rhp6 (a homolog of budding yeast Rad6), which has been shown to negatively affect stability of heterochromatic structures. We show that Rhp6 is a component of the multisubunit protein complex (termed HULC) that also contains two RING finger proteins Rfp1 and Rfp2, sharing homology with budding yeast Bre1 protein and a unique serine-rich protein Shf1. HULC is required for ubiquitination of histone H2B at lysine 119 (H2B-K119), and it localizes to heterochromatic sequences. Moreover, our analyses suggest that Rhp6-induced changes in heterochromatic silencing are mediated predominantly through H2B ubiquitination (ubH2B), and they correlate with increased RNA polymerase II levels at repeat elements embedded within heterochromatin domains. Interestingly, heterochromatic derepression caused by Rhp6 occurs independently of the involvement of HULC subunits and ubH2B in methylation of histone H3 at lysine 4 (H3K4me). These analyses implicate ubH2B in modulation of heterochromatin, which has important implications for dynamics and many functions associated with heterochromatic structures.

Simple sequence repeats in mycobacterial genomes

Simple sequence repeats (SSRs) or microsatellites are the repetitive nucleotide sequences of motifs of length 1-6 bp. They are scattered throughout the genomes of all the known organisms ranging from viruses to eukaryotes. Microsatellites undergo mutations in the form of insertions and deletions (INDELS) of their repeat units with some bias towards insertions that lead to microsatellite tract expansion. Although prokaryotic genomes derive some plasticity due to microsatellite mutations they have in-built mechanisms to arrest undue expansions of microsatellites and one such mechanism is constituted by post-replicative DNA repair enzymes MutL, MutH and MutS. The mycobacterial genomes lack these enzymes and as a null hypothesis one could expect these genomes to harbour many long tracts. It is therefore interesting to analyse the mycobacterial genomes for distribution and abundance of microsatellites tracts and to look for potentially polymorphic microsatellites. Available mycobacterial genomes, Mycobacterium avium, M. leprae, M. bovis and the two strains of M. tuberculosis (CDC1551 and H37Rv) were analysed for frequencies and abundance of SSRs. Our analysis revealed that the SSRs are distributed throughout the mycobacterial genomes at an average of 220-230 SSR tracts per kb. All the mycobacterial genomes contain few regions that are conspicuously denser or poorer in microsatellites compared to their expected genome averages. The genomes distinctly show scarcity of long microsatellites despite the absence of a post-replicative DNA repair system. Such severe scarcity of long microsatellites could arise as a result of strong selection pressures operating against long and unstable sequences although influence of GC-content and role of point mutations in arresting microsatellite expansions can not be ruled out. Nonetheless, the long tracts occasionally found in coding as well as non-coding regions may account for limited genome plasticity in these genomes.

Essential Role of T-Cell Factor/β-Catenin in Regulation of Rad6B: A Potential Mechanism for Rad6B Overexpression in Breast Cancer Cells

We have previously shown that the postreplication DNA repair gene Rad6B plays a critical role in the maintenance of genomic integrity of human breast cells. Whereas normal breast cells express low levels of Rad6B, increases in Rad6B expression occur in hyperplasia with overexpression in breast carcinomas. Here, we show that the human Rad6B gene is a transcriptional target of T-cell factor (TCF)-4/beta-catenin/p300. Rad6B promoter activity is subject to negative regulation in normal human MCF10A breast cells whereas it is constitutively active in metastatic MDA-MB-231 breast cancer cells. Derepression and activation of Rad6B promoter in MCF10A cells require coexpression of beta-catenin and p300. Using electrophoresis mobility shift assay, Western blot analysis of electrophoresis mobility shift assay, UV cross-linking, and chromatin immunoprecipitation assay, we show that Rad6B transcriptional repression in MCF10A cells is due to paucity of transcriptionally active beta-catenin assembled on the TCF binding sequence in the Rad6B promoter rather than to a deficit/decreased affinity of TCF-4 for the TCF binding element in Rad6B promoter. Three-dimensional epithelial acini generated in vitro from MCF10A cells cotransfected with beta-catenin and p300 showed beta-catenin expression on the membrane, cytoplasm, and/or nuclei with concomitant Rad6 overexpression, whereas control acini showed beta-catenin on the membranes and negligible Rad6 expression. Immunohistochemical analysis of 12 breast carcinomas showed an approximately 80% correlation between Rad6 and beta-catenin expression, and combined nuclear and cytoplasmic staining of beta-catenin and Rad6 was detected in 25% of the breast carcinomas. In vivo implantation of MCF10A-Rad6B cells produced hyperplastic lesions. These data reveal a potentially important role for transcriptionally active beta-catenin in the regulation of Rad6B gene expression, and link aberrant beta-catenin signaling with transcriptional deregulation of Rad6B and breast cancer development.

A novel function of DNA polymerase ζ regulated by PCNA

DNA polymerase zeta (Polzeta) participates in translesion DNA synthesis and is involved in the generation of the majority of mutations induced by DNA damage. The mechanisms that license access of Polzeta to the primer terminus and regulate the extent of its participation in genome replication are poorly understood. The Polzeta-dependent damage-induced mutagenesis requires monoubiquitination of proliferating cell nuclear antigen (PCNA) that is triggered by exposure to mutagens. We show that Polzeta contributes to DNA replication and causes mutagenesis not only in response to DNA damage but also in response to malfunction of normal replicative machinery due to mutations in replication genes. These replication defects lead to ubiquitination of PCNA even in the absence of DNA damage. Unlike damage-induced mutagenesis, the Polzeta-dependent spontaneous mutagenesis in replication mutants is reduced in strains defective in both ubiquitination and sumoylation of Lys164 of PCNA. Additionally, studies of a PCNA mutant defective for functional interactions with Polzeta, but not for monoubiquitination by the Rad6/Rad18 complex demonstrate a role for PCNA in regulating the mutagenic activity of Polzeta separate from its modification at Lys164.

The function of recombinations occurring in the process of DNA replication in Escherichia coli

In a number of works dealing with the relationship between replication and recombination in bacteria, it is assumed that recombinations permit the replication forks to resume moving after having stopped at the damage sites of the template DNA. As an evidence for recombination occurring during DNA replication, the involvement in this process of proteins RuvABC and RecG, providing processing of the Holliday junctions after recombination, is considered. However, it has been shown that these proteins are not essential for resuming DNA synthesis after an exposure of bacteria to UV light. These data cast doubt on the necessity of recombination for reactivation of replication initiated in the oriC region. Studying recombination in tandem duplications in Escherichia coli showed that during replication, unequal crossing over occurs between direct DNA repeats of sister chromosomes. In wild strains, this crossing over results in tandem duplications, thereby enhancing the expression of certain genes. Thus, recombination of two types occurs during DNA replication: unequal crossing over leading to duplications and homologous exchange, responsible for post-replication DNA repair. The unequal exchange constitutes a component of SOS response of the cell to deterioration of the environment.

MLH1-deficient tumor cells are resistant to lipoplatin, but retain sensitivity to lipoxal

Lipoplatin, currently under phase III evaluation, is a novel liposomal cisplatin formulation highly effective against cancers. Lipoplatin has eliminated or reduced the systemic toxicity frequently seen for cisplatin. The objective of the present study was to determine whether the cytotoxic effect of lipoplatin is dependent on the functional integrity of DNA mismatch repair (MMR), a post-replicative DNA repair machinery implicated in cell cycle control and apoptosis. Clonogenic data revealed a significant (P<0.05) 2-fold resistance to lipoplatin of HCT116 human colorectal adenocarcinoma cells lacking MLH1, one of five proteins crucial to MMR function, as compared to MLH1-expressing HCT116 cells. In addition, MLH1-deficient cells were at least 3-fold less susceptible to apoptosis (DNA fragmentation) than MLH1-proficient cells. However, proteolytic processing of caspase-3, caspase-7 and poly(ADP-ribose)polymerase-1 following lipoplatin treatment was comparable in MLH1-deficient cells and -proficient cells. Furthermore, MLH1-deficient cells retained the ability to attenuate cell cycle progression past the G2/M checkpoint following lipoplatin treatment. In conclusion, our results indicate that the lipoplatin-sensitive phenotype of MLH1-proficient cells correlated with increased apoptosis which may occur via caspase-independent pathways. They also suggest that the integrity of MMR function is a relevant determinant accounting for the cytotoxicity of lipoplatin. However, this does not seem to apply to lipoxal, a novel liposomal formulation of oxaliplatin, because MLH1-deficient cells were as sensitive to lipoxal as MLH1-proficient cells.

Mating type regulation of cellular tolerance to DNA damage is specific to the DNA post‐replication repair and mutagenesis pathway

In order to help further define DNA post-replication repair (PRR), a conditional synthetic lethal screen was employed to identify new genes involved in the PRR pathway. A synthetic lethal screen with the mms2 mutation resulted in the recovery of two suppressor mutations responsible for regulating PRR. The recovered suppressors are the mating type genes and SIR3. Indeed, controlled expression of both mating type genes or deletion of SIR3 rescued the conditional synthetic lethal mutant phenotypes. Furthermore, comprehensive analyses suggest that mating type heterozygosity confers tolerance to a broad range of DNA damage, and that this effect is limited to all PRR pathway mutations, but does not apply to base excision repair, nucleotide excision repair or recombination repair mutants. In addition, the tolerance conferred to PRR mutants as a result of mating type heterozygosity is dependent on a functional homologous recombination but not the non-homologous end-joining pathway. Thus, mating type status appears to be responsible for signalling DNA content and possibly cell cycle stage, allowing the cell to select the most efficient means to repair the DNA damage.