Mechanistic studies of DNA repair and damage response

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The overall goal of this proposal is to elucidate the structural and dynamic properties of MutS¿- DNA complexes that signal initiation of DNA mismatch repair (MMR) versus initiation of DNA damage-induced apoptosis. MMR is the mechanism by which DNA synthesis errors are corrected post-replicatively, and it is central to the maintenance to the integrity of the genome. MMR proteins are also involved in several other DNA transactions, including DNA damage sensing. In addition to dramatically increasing the frequency of mutations, inactivation of MMR genes decreases apoptosis, increases cell survival, and results in resistance to chemotherapy. In humans, mutations in MMR genes are directly linked to hereditary non-polyposis colorectal cancer and are associated with several sporadic cancers. MMR is initiated by MutS homologs binding to a mismatch. Subsequently, MutL homologs interact with the MutS homologs in an ATP dependent manner and coordinate protein-protein interactions that signal excision and resynthesis of the newly synthesized DNA strand containing the incorrect nucleotide. Initiation of DNA damage- induced apoptosis in eukaryotes follows a very similar initiation pathway. The MutS homolog MSH2-MSH6 (MutS¿) binds preferentially to a DNA lesion and subsequently interacts with the MutL homolog MLH1-PMS2 (MLH1-PMS1 in yeast; MutL¿), but instead of initiating DNA mismatch repair, these interactions activate cell-cycle checkpoints and signal apoptosis. The central goal of this grant is to understand how interaction of MutS¿ with damaged DNA signals a cell-cycle checkpoint response and apoptosis, while interaction of MutS¿ with mismatches signals MMR. We seek to uncover the conformational and dynamic properties of MutS¿-DNA complexes that elicit different cellular fates depending on whether MutS¿ is bound to mismatch or to a DNA lesion. We propose a systematic series of experiments in which we use a combination of bulk and single-molecule fluorescence techniques in conjunction with atomic force microscopy to characterize the binding and dynamic conformational properties of MutS¿- DNA complexes that signal repair versus apoptosis. We will examine the interactions of wildtype and a mutant of MutS¿, which exhibits a separation of function for MMR and DNA damage response,with damaged DNA and with mismatches that exhibit different efficiencies of repair, in the presence and absence of nucleotide cofactors.