Defects in the human mismatch repair system are the cause of hereditary nonpolyposis colon cancer and familial colon cancer, and have also been implicated in etiology of sporadic tumors. Inactivation of this pathway renders cells genetically unstable due to DNA replication errors, illegitimate recombination events, and failure to respond normally to certain DNA damaging agents, including several that are used as anti-tumor drugs. Despite the importance of this genetic stabilization system, our understanding of its molecular nature is limited. Although seven activities have been implicated in human strand-specific mismatch repair (MutS, MutS, MutLo, PCNA, RPA, EXOI, and DNA polymerase), these are not sufficient to reconstitute the reaction. A major goal for the requested extension of this project is the identification and isolation of other required activities. MutSo and MutL play key roles in the initiation of mismatch repair, but the molecular modes by which they interact with DNA and the roles of their nucleotide hydrolytic functions are not well defined and have been the subject of controversy.
The second aim of this application addresses these questions, with emphasis on the protein oligomerization states involved in the interaction with DNA, and further clarification of the function of their ATPase centers as modulators of DNA-protein interaction. The nature of multi-protein and multi-protein-DNA assemblies involved in early stages of the mismatch repair reaction will also be examined. These studies will emphasize interactions of MutSo with MutLo, MutS with PCNA, and EXOI with MutSo and MutLa, as well as modulation of activated EXOI by RPA. The fourth line of proposed work addresses the role of the mismatch repair system and the BLM helicase in recombination fidelity. Illegitimate recombination events are elevated in mismatch repair-deficient and in BLM-deficient cells, and MutSo has been shown to modulate the activity of the BLM helicase. Consequently, the investigators will ask whether these two systems function in a coordinated manner to suppress illegitimate recombination events.
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