ROLE OF MISMATCH REPAIR IN MAINTAINING GENOME STABILITY

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This project aims to understand the actions of eukaryotic mismatch repair (MMR) proteins in correcting DNA replication errors and in processing recombination events. In organisms ranging from E. coli to humans, MMR plays an important role in correcting misincorporation errors and slippage events that occur during DNA replication. In E. coli, MutS and MutL play critical roles in MMR; MutS binds to mispairs and MutL interacts with MutS-mispair complexes. Homologs of MutS (Msh) and MutL (Mlh) have been identified in yeast and higher eukaryotes. Studies in bacteria, yeast and humans have also shown that MMR plays an important role in maintaining chromosome stability by preventing recombination between homologous DNA sequences. The molecular mechanisms by which MMR proteins recognize mismatch substrates and transduce this information to downstream repair factors during DNA replication and genetic recombination are not well understood in eukaryotes. These issues will be addressed in the yeast S. cerevisiae through the following approaches: 1. A goal of the 1994 submission was to identify conditional msh2 mutations as they can reveal intermediate steps in a pathway and provide critical reagents for a second site suppressor screen. Genetic suppression analysis will be performed with six well characterized msh2 and four mlh1 conditional MMR mutations to identify interactions between MSH2 and MLH1 and new downstream MMR factors. 2. Biochemical approaches will be used to examine interactions between the Msh2p-Msh6p (mutS homolog) mismatch recognition complex and other DNA repair and replication factors. This analysis will take advantage of a large collection of dominant negative, conditional, and site specific msh2 and msh6 mutations that we isolated. 3. mlh1 mutants show distinct defects in MMR and in crossing over. To test the idea that MLH1 plays unique and distinct roles in each of these processes, we will mutagenize MLH1 with the goal of identifying mlh1 mutations that affect one but not both processes. 4. To understand how MMR factors are recruited to recombination intermediates, we are investigating the association of Msh2p with DNA using ChIP (chromatin crosslinking, immunoprecipitation and PCR). This procedure will be performed in wild type and recombination defective mutants using both plasmid and chromosomal recombination assays.