<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>

The Chfr checkpoint is a point at which a cell checks whether it is safe to enter mitosis. Chfr is a protein that functions at this particular checkpoint to ensure safe entry into mitosis, but the molecular mechanism by which this protein functions is not entirely clear. The hypothesis in this thesis is that Ubc13, Chfr, and Uev1/Mms2 function together in mitosis. The results were observed using immunocytochemistry, the mitotic shake off procedure, Western blot analysis, and coimmunoprecipitation. High Ubc13, Mms2, and Chfr-Ub levels at the interphase-early prophase transition, indicate that these proteins function together at the Chfr checkpoint. Localization of Chfr to decondensed chromatin in interphase cells and to decondensing chromatin in telophase cells indicates a decondensing function for Chfr. Interaction between Chfr and Ubc13, Chfr and phosphorylated histone H3, as well as Ubc13 and phosphorylated histone H3, further indicates that these proteins may function together at the Chfr checkpoint, because phosphorylated histone H3 is a mitotic protein at that particular point in mitosis. Localization of Chfr, Ubc13, and Mms2 to the centrosomes, indicates that they function together at these sites to target substrates important in centrosome maturation, separation, and spindle formation. Furthermore, there are two molecular states of Chfr: Chfr and Chfr-Ub. Chfr is predominant at late prophase, whereas, Chfr-Ub is predominant at interphase-early prophase. Chfr increases in level upon nocodazole exposure at late prophase to counteract the mitotic stress; and it also looses its ubiquitin signal upon passage into mitosis. High Ubc13 and Mms2 levels coincide with high Chfr-Ub levels at the interphase-early prophase transition, indicating that they function together at the Chfr checkpoint. The ubiquitin signal could be either K-48-linked or K-63-linked in nature. The Chfr, Ubc13, and Mms2 protein complex could function through a self ubiquitination-decondensation-Chfr destruction-recondensation mechanism. Chfr could bind to pH3 and its auto-ubiquitin signal to serve as a bulky modification that hinders chromosome condensation.

Ubiquitylation is an indispensable post-translational modification system in eukaryotic cells that leads to the covalent attachment of a small ubiquitin (Ub) protein onto a target. The traditional and best-characterized role for ubiquitylation is a fundamental regulatory mechanism whereby target proteins are tagged with a characteristic Lys48-linked Ub chain that signals for their elimination through proteasomal degradation. Challenging this conventional wisdom is the finding that some ubiquitylated proteins are modified by Ub chains linked through Lys63, providing a molecular signal that is thought to be structurally and functionally distinct from Lys48-linked Ub chains. Of further interest and significance is that the Lys63-linked Ub chains are apparently synthesized through a novel biochemical mechanism employing a unique complex formed between a true Ub conjugating enzyme (E2), Ubc13, and an E2-variant (Uev), Mms2 (or Uev1A). The goal of this thesis was to employ structural and functional approaches in order to better characterize the Ubc13-Mms2 Lys63-linked Ub conjugation complex. <p>Error-free DNA damage tolerance (DDT) in the budding yeast is dependent on Lys63-linked Ub chains synthesized by Ubc13-Mms2 and thus provided the opportunity to experimentally test the function of the human UBC13 and MMS2 genes in a simple model organism. Human UBC13 and MMS2 were each shown to function in place of their yeast counterparts and in accordance, human Ubc13 was shown to physically interact with yeast Mms2, and vice versa. Two human MMS2 homologs were also tested and it was determined that UEV1A but not UEV1B can function in place of mms2 in yeast DDT. Physical interactions were observed between Ubc13 and Uev1A, but not between Ubc13 and Uev1B, suggesting that Ubc13-Uev complex formation is required for function. <p>In collaboration with a research group at the University of Alberta, crystal structure and NMR data were used to develop a mechanistic model for the conjugation of Lys63-linked Ub chains by the Ubc13-Mms2 heterodimer, whereby the special orientation of two Ub molecules facilitates a specific Ub-Ub linkage via Lys63. In order to help support the in vitro model and to determine how the Ubc13-Mms2 structure relates to biological function, I used a structure-based approach to direct the creation of point mutations within four key regions of the Ubc13-Mms2 heterodimer; the Ubc13 active-site, the Ubc13-E3 (Ub ligating enzyme) interface, the Mms2-Ub interface, and the Ubc13-Mms2 interface. <p>Underscoring the importance of the Ub conjugation by Ubc13-Mms2, a Ubc13-C87S active-site mutation was created that could bind to Mms2 but was unable to function in DDT. Regarding the Ubc13-E3 interface, a single Ubc13-M64A point mutation had a potent effect on disrupting Ubc13 function in DDT, as well as its physical interaction with Rad5, TRAF6, and CHFR. The results suggest that different RING finger E3s use the same Ubc13 surface to sequester the Ub conjugation activity of Ubc13-Mms2. Two human Mms2 mutations at Ser32 and Ile62, which are contained within the Mms2-Ub interface, were found to reduce the ability of Mms2 to bind Ub. When the corresponding yeast mutations are combined, a synergistic loss in DDT function is observed. The relative orientation of Ser32 and Ile62 suggests that the Mms2 and Tsg101 Uev families use different Uev surfaces to physically interact with Ub. A 200 ìM dissociation constant for the wild-type Mms2-Ub interaction was also determined. The systematic mutagenesis and testing of 14 Ubc13-Mms2 interface residues led to mutants with partial or complete disruption of binding and function. Using this data, a model involving the insertion of a specific Mms2-Phe residue into a unique Ubc13 hydrophobic pocket was created to explain the specificity of Mms2 for Ubc13, and not other E2s. In addition, the dissociation constant for the wild-type Ubc13-Mms2 heterodimer was determined to be approximately 50 nM. <p>The structural and functional studies strongly support the notion that Ubc13-Mms2 complex has the unique ability to conjugate Lys63-linked Ub chains. However, several reported instances of Lys63-linked Ub chains in vivo have not yet been attributed to Ubc13 or Mms2. To address the disparity I was able to demonstrate and map a physical interaction between Mms2 and Rsp5, an E3 implicated in Lys63-linked Ub conjugation. Surprisingly, it was found that MMS2 is not responsible for the RSP5-dependent Lys63-linked Ub conjugation of a plasma membrane protein. A possible explanation for the apparent paradox is presented.

Ubiquitylation is an indispensable post-translational modification system in eukaryotic cells that leads to the covalent attachment of a small ubiquitin (Ub) protein onto a target. The traditional and best-characterized role for ubiquitylation is a fundamental regulatory mechanism whereby target proteins are tagged with a characteristic Lys48-linked Ub chain that signals for their elimination through proteasomal degradation. Challenging this conventional wisdom is the finding that some ubiquitylated proteins are modified by Ub chains linked through Lys63, providing a molecular signal that is thought to be structurally and functionally distinct from Lys48-linked Ub chains. Of further interest and significance is that the Lys63-linked Ub chains are apparently synthesized through a novel biochemical mechanism employing a unique complex formed between a true Ub conjugating enzyme (E2), Ubc13, and an E2-variant (Uev), Mms2 (or Uev1A). The goal of this thesis was to employ structural and functional approaches in order to better characterize the Ubc13-Mms2 Lys63-linked Ub conjugation complex. <p>Error-free DNA damage tolerance (DDT) in the budding yeast is dependent on Lys63-linked Ub chains synthesized by Ubc13-Mms2 and thus provided the opportunity to experimentally test the function of the human UBC13 and MMS2 genes in a simple model organism. Human UBC13 and MMS2 were each shown to function in place of their yeast counterparts and in accordance, human Ubc13 was shown to physically interact with yeast Mms2, and vice versa. Two human MMS2 homologs were also tested and it was determined that UEV1A but not UEV1B can function in place of mms2 in yeast DDT. Physical interactions were observed between Ubc13 and Uev1A, but not between Ubc13 and Uev1B, suggesting that Ubc13-Uev complex formation is required for function. <p>In collaboration with a research group at the University of Alberta, crystal structure and NMR data were used to develop a mechanistic model for the conjugation of Lys63-linked Ub chains by the Ubc13-Mms2 heterodimer, whereby the special orientation of two Ub molecules facilitates a specific Ub-Ub linkage via Lys63. In order to help support the in vitro model and to determine how the Ubc13-Mms2 structure relates to biological function, I used a structure-based approach to direct the creation of point mutations within four key regions of the Ubc13-Mms2 heterodimer; the Ubc13 active-site, the Ubc13-E3 (Ub ligating enzyme) interface, the Mms2-Ub interface, and the Ubc13-Mms2 interface. <p>Underscoring the importance of the Ub conjugation by Ubc13-Mms2, a Ubc13-C87S active-site mutation was created that could bind to Mms2 but was unable to function in DDT. Regarding the Ubc13-E3 interface, a single Ubc13-M64A point mutation had a potent effect on disrupting Ubc13 function in DDT, as well as its physical interaction with Rad5, TRAF6, and CHFR. The results suggest that different RING finger E3s use the same Ubc13 surface to sequester the Ub conjugation activity of Ubc13-Mms2. Two human Mms2 mutations at Ser32 and Ile62, which are contained within the Mms2-Ub interface, were found to reduce the ability of Mms2 to bind Ub. When the corresponding yeast mutations are combined, a synergistic loss in DDT function is observed. The relative orientation of Ser32 and Ile62 suggests that the Mms2 and Tsg101 Uev families use different Uev surfaces to physically interact with Ub. A 200 ìM dissociation constant for the wild-type Mms2-Ub interaction was also determined. The systematic mutagenesis and testing of 14 Ubc13-Mms2 interface residues led to mutants with partial or complete disruption of binding and function. Using this data, a model involving the insertion of a specific Mms2-Phe residue into a unique Ubc13 hydrophobic pocket was created to explain the specificity of Mms2 for Ubc13, and not other E2s. In addition, the dissociation constant for the wild-type Ubc13-Mms2 heterodimer was determined to be approximately 50 nM. <p>The structural and functional studies strongly support the notion that Ubc13-Mms2 complex has the unique ability to conjugate Lys63-linked Ub chains. However, several reported instances of Lys63-linked Ub chains in vivo have not yet been attributed to Ubc13 or Mms2. To address the disparity I was able to demonstrate and map a physical interaction between Mms2 and Rsp5, an E3 implicated in Lys63-linked Ub conjugation. Surprisingly, it was found that MMS2 is not responsible for the RSP5-dependent Lys63-linked Ub conjugation of a plasma membrane protein. A possible explanation for the apparent paradox is presented.

No description available.