Date of Award
Doctor of Philosophy
Molecular Biology, Microbiology and Biochemistry
Genome integrity is constantly challenged by ionizing radiation, UV light, hydrolysis, DNA-damaging chemicals, and DNA replication errors. If DNA damage and replication errors are left unrepaired, they cause the formation of mutations that can alter the cell’s phenotype and lead to numerous diseases including cancer. DNA repair is essential for maintaining genome integrity. The mismatch repair (MMR) system is a major DNA repair system. Most of the functions of the MMR system promote genome stability, but some of its functions trigger the formation of mutations at certain genomic loci. MMR functions are initiated by recognition of a mismatch by MutSα (MSH2-MSH6 complex) or MutSβ (MSH2-MSH3 complex). MutSα recognizes base-base mismatches and small insertion/deletion loops whereas MutSβ recognizes small insertion/deletion loops. Once the mismatch is recognized, MutSα or MutSβ undergoes an ATP-dependent conformational change and recruits a MutL complex. The recruitment of the MutL complex MutLα (MLH1-PMS2 complex in humans and Mlh1-Pms1 complex in budding yeast) leads to the activation of its endonuclease activity that cleaves the daughter strand in a reaction that requires MutSα/MutSβ, RFC-loaded PCNA, and ATP. Even though several MMR factors have been identified, the precise functions of certain factors and their interactions with other cellular processes remain elusive. Previous research showed that deletion of MLH2 results in a weak mutator phenotype, which is particularly under conditions when another MMR component is limiting. Our data revealed that Mlh2 may contribute to DNA ribonucleotide removal. We also analyzed genetic interactions between the MMR system and RAD27. This analysis provided evidence that MutSβ and the Rad27 endonuclease act in overlapping pathways that suppress the formation of small deletions and certain duplications. Intriguingly, we observed template switch events in msh3 rad27 double mutant cells, but not in msh3 or rad27 single mutant cells. This observation suggests that MutSβ and Rad27 function in overlapping pathways that defend against mutations that arise because of DNA template switching. We also studied the contribution of the MMR system to the cellular response to Sn1-type alkylating agents. Our data suggest that the MMR system preferentially suppresses MNNG- induced mutations on the transcribed strand of the CAN1 gene. Additionally, we investigated several pms1 mutants that mimic human pms2 variants of uncertain significance. The data revealed that the pms1 mutations do not significantly affect MutLα functions in MMR and the cellular response to MNNG, suggesting that the conserved Pms1/PMS2 residues are important for a different MutLα function. Finally, we evaluated the contribution of the MMR system to the cellular response to cisplatin treatment. Our data showed that deletion of MSH3 decreases the survival of cells treated with cisplatin. Collectively, our findings shed light on the role of MLH2, the interaction of the MMR system with RAD27, the mechanism of removal of MNNG-induced DNA damage by the MMR system, the effects of several conserved pms1 residues on MutLα function, and the contribution of the MMR system to the cellular response to cisplatin, thereby expanding our understanding of the intricate mechanisms underlying genome maintenance and repair processes.
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