<p>Both prokaryotic and eukaryotic cells are equipped with DNA repair mechanisms to protect the integrity of their genome in case of DNA damage. In the eukaryotic organism <i>Saccharomyces cerevisiae</i>, MMS2 encodes a ubiquitin-conjugating enzyme variant protein belonging to the RAD6 repair pathway; MMS2 functions in error-free postreplication repair (PRR), a subpathway parallel to REV3 mutagenesis. A mutation in MMS2 does not result in extreme sensitivity to DNA damaging agents; however, deletion of both subpathways of PRR results in a synergistic phenotype. By taking advantage of the synergism between error-free PRR and mutagenesis pathway mutations, a conditional synthetic lethal screen was used to identify novel genes genetically involved in PRR. A synthetic lethal screen was modified to use extremely low doses of MMS that would not affect the growth of single mutants, but would effectively kill the double mutants. Fifteen potential mutants were characterized, of which twelve were identified as known error-prone PRR genes. Characterization of mutations in strains SLM-9 and SLM-11, that are conditionally synthetically lethal with mms2Ä, revealed functions for both checkpoints and mating-type heterozygosity in regulating PRR. Cell cycle checkpoints monitor the integrity of the genome and ensure that cell cycle progression is deferred until chromosome damage is repaired. The checkpoint genes genetically interact with both the error-free and error-prone branches of PRR, potentially for delaying cell cycle progression to allow time for DNA repair, and for signaling the stage of the cell cycle and thus DNA content. Other potential monitors for DNA content are the a1 and á2 proteins encoded by the mating type genes MATa and MATá, respectively. Diploid cells heterozygous for mating type (a/á) show an increased resistance to UV damage and are more recombination-proficient than haploid cells. Haploid PRR mutants expressing both mating type genes show an increased resistance to DNA-damaging agents. This phenomenon is specific to PRR: it was not seen in excision repair-deficient and recombination-deficient mutants tested. The rescuing effect seen in PRR mutants heterozygous for mating type is likely the result of channeling lesions into a recombination repair pathway and away from the non-operational PRR pathway. Both checkpoint and mating type genes play a role in regulating PRR. Almost certainly these genes are required to monitor the cell cycle stage and DNA content to determine the best mechanism to repair the damaged DNA thus preventing genomic instability.</p>

<p>Both prokaryotic and eukaryotic cells are equipped with DNA repair mechanisms to protect the integrity of their genome in case of DNA damage. In the eukaryotic organism <i>Saccharomyces cerevisiae</i>, MMS2 encodes a ubiquitin-conjugating enzyme variant protein belonging to the RAD6 repair pathway; MMS2 functions in error-free postreplication repair (PRR), a subpathway parallel to REV3 mutagenesis. A mutation in MMS2 does not result in extreme sensitivity to DNA damaging agents; however, deletion of both subpathways of PRR results in a synergistic phenotype. By taking advantage of the synergism between error-free PRR and mutagenesis pathway mutations, a conditional synthetic lethal screen was used to identify novel genes genetically involved in PRR. A synthetic lethal screen was modified to use extremely low doses of MMS that would not affect the growth of single mutants, but would effectively kill the double mutants. Fifteen potential mutants were characterized, of which twelve were identified as known error-prone PRR genes. Characterization of mutations in strains SLM-9 and SLM-11, that are conditionally synthetically lethal with mms2Ä, revealed functions for both checkpoints and mating-type heterozygosity in regulating PRR. Cell cycle checkpoints monitor the integrity of the genome and ensure that cell cycle progression is deferred until chromosome damage is repaired. The checkpoint genes genetically interact with both the error-free and error-prone branches of PRR, potentially for delaying cell cycle progression to allow time for DNA repair, and for signaling the stage of the cell cycle and thus DNA content. Other potential monitors for DNA content are the a1 and á2 proteins encoded by the mating type genes MATa and MATá, respectively. Diploid cells heterozygous for mating type (a/á) show an increased resistance to UV damage and are more recombination-proficient than haploid cells. Haploid PRR mutants expressing both mating type genes show an increased resistance to DNA-damaging agents. This phenomenon is specific to PRR: it was not seen in excision repair-deficient and recombination-deficient mutants tested. The rescuing effect seen in PRR mutants heterozygous for mating type is likely the result of channeling lesions into a recombination repair pathway and away from the non-operational PRR pathway. Both checkpoint and mating type genes play a role in regulating PRR. Almost certainly these genes are required to monitor the cell cycle stage and DNA content to determine the best mechanism to repair the damaged DNA thus preventing genomic instability.</p>

In Saccharomyces cerevisiae DNA postreplication repair (PRR) functions to bypass replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA. While translesion synthesis (TLS) 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 (SGA) screen for novel genes involved in PRR and observed evidence of genetic interactions between error-free PRR and HR. We were screening for synthetic lethality which occurs when the combination of two mutations leads to an inviable organism, however, either single mutation allows for cell viability. In addition, we screened for conditionally synthetic lethal interaction which occurs when the combination of two mutations is inviable only in the presence of a DNA-damaging agent. 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 one 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. Null mutations of HR genes including rad51, 52, 54, 55 and 57 are known to confer characteristic synergistic interactions with TLS mutations. To our surprise, null mutations of genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which is also required for HR, are epistatic to TLS mutations. The MRX complex confers an endo/exonuclease activity required for the detection and processing of DNA double-strand breaks (DSBs). Our results suggest that the MRX complex functions in both TLS and error-free PRR and that this function requires the nuclease activity of Mre11. This is in sharp contrast to other known HR genes that only function downstream of error-free PRR. Furthermore, we found that inactivation of SGS1 significantly inhibits proliferating cell nuclear antigen (PCNA) monoubiquitination and is epistatic to mutations in TLS, suggesting that Sgs1 also functions at earlier steps in DNA lesion bypass. We also examined the roles of Sae2 and Exo1, two accessory nucleases involved in DSB resection, in PRR. We found that while Sae2 is primarily required for TLS, Exo1 is exclusively involved in error-free PRR. In light of the distinct and overlapping activities of the above nucleases in the resection of DSBs, we propose that the distinct single-strand nuclease activities of MRX, Sae2 and Exo1 dictate the preference between TLS and error-free PRR for lesion bypass. While both PRR pathways are dependent on the ubiquitination of PCNA, error-free PRR utilizes non-canonical Lys63-linked polyubiquitinated PCNA to signal lesion bypass. This mechanism is dependent on the Mms2-Ubc13 complex being in close proximity to PCNA, a process thought to be dependent on Rad5. Rad5 is a member of the SWI/SNF family of ATPases that contains a RING finger motif characteristic of an E3 Ub ligase. Previous in vitro experiments demonstrated the ability of Rad5 to promote replication fork regression, a function dependent on its helicase/ATPase activity. We therefore created site-specific mutants defective in either Rad5 RING finger or helicase/ATPase activity, or both, in order to examine their genetic interactions with known TLS and error-free PRR genes. Our results indicate that both the Rad5 RING finger motif and the helicase/ATPase activity are exclusively involved in error-free PRR. To our surprise, like the Rad5 RING finger, lack of the helicase/ATPase activity also abolishes the Lys63-linked PCNA polyubiquitin chain formation, suggesting that either the Rad5 helicase/ATPase-promoted replication fork regression signals PCNA polyubiquitination or this domain has a yet unidentified activity. In summary, results obtained from this thesis dissertation have revealed novel mechanisms of yeast PRR in S. cerevisiae, a mechanism that appears to be evolutionarily conserved throughout eukaryotes, from yeast to humans.

In Saccharomyces cerevisiae DNA postreplication repair (PRR) functions to bypass replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA. While translesion synthesis (TLS) 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 (SGA) screen for novel genes involved in PRR and observed evidence of genetic interactions between error-free PRR and HR. We were screening for synthetic lethality which occurs when the combination of two mutations leads to an inviable organism, however, either single mutation allows for cell viability. In addition, we screened for conditionally synthetic lethal interaction which occurs when the combination of two mutations is inviable only in the presence of a DNA-damaging agent. 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 one 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. Null mutations of HR genes including rad51, 52, 54, 55 and 57 are known to confer characteristic synergistic interactions with TLS mutations. To our surprise, null mutations of genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which is also required for HR, are epistatic to TLS mutations. The MRX complex confers an endo/exonuclease activity required for the detection and processing of DNA double-strand breaks (DSBs). Our results suggest that the MRX complex functions in both TLS and error-free PRR and that this function requires the nuclease activity of Mre11. This is in sharp contrast to other known HR genes that only function downstream of error-free PRR. Furthermore, we found that inactivation of SGS1 significantly inhibits proliferating cell nuclear antigen (PCNA) monoubiquitination and is epistatic to mutations in TLS, suggesting that Sgs1 also functions at earlier steps in DNA lesion bypass. We also examined the roles of Sae2 and Exo1, two accessory nucleases involved in DSB resection, in PRR. We found that while Sae2 is primarily required for TLS, Exo1 is exclusively involved in error-free PRR. In light of the distinct and overlapping activities of the above nucleases in the resection of DSBs, we propose that the distinct single-strand nuclease activities of MRX, Sae2 and Exo1 dictate the preference between TLS and error-free PRR for lesion bypass. While both PRR pathways are dependent on the ubiquitination of PCNA, error-free PRR utilizes non-canonical Lys63-linked polyubiquitinated PCNA to signal lesion bypass. This mechanism is dependent on the Mms2-Ubc13 complex being in close proximity to PCNA, a process thought to be dependent on Rad5. Rad5 is a member of the SWI/SNF family of ATPases that contains a RING finger motif characteristic of an E3 Ub ligase. Previous in vitro experiments demonstrated the ability of Rad5 to promote replication fork regression, a function dependent on its helicase/ATPase activity. We therefore created site-specific mutants defective in either Rad5 RING finger or helicase/ATPase activity, or both, in order to examine their genetic interactions with known TLS and error-free PRR genes. Our results indicate that both the Rad5 RING finger motif and the helicase/ATPase activity are exclusively involved in error-free PRR. To our surprise, like the Rad5 RING finger, lack of the helicase/ATPase activity also abolishes the Lys63-linked PCNA polyubiquitin chain formation, suggesting that either the Rad5 helicase/ATPase-promoted replication fork regression signals PCNA polyubiquitination or this domain has a yet unidentified activity. In summary, results obtained from this thesis dissertation have revealed novel mechanisms of yeast PRR in S. cerevisiae, a mechanism that appears to be evolutionarily conserved throughout eukaryotes, from yeast to humans.