Genomic stability in mammalian cells requires proper functioning of multiple DNA replication, repair and recombination processes. One such process, DNA mismatch repair (MMR), plays roles in: 1) correcting replication- and damage-induced DNA mismatches, 2) correcting mispairs in heteroduplex DNA associated with genetic recombination, and 3) suppressing genetic recombination between similar but non-identical sequences. Not surprisingly, MMR deficiency contributes to both spontaneous and/or hereditary cancer in humans and mice. Our primary objective is to elucidate the mechanism and roles of mammalian MMR by studying the MutL homologs Mlh1p and Pms2p, comprising the heterodimer MutL alpha, which helps to couple mismatch binding to downstream repair events. Also, we will study Exo1p, which is involved in the excision reaction of MMR-mediated mutation avoidance. To enhance our mammalian MMR studies, we will continue molecular genetic studies of MMR in budding yeast focusing on Mlh1p and Pms1p, and Exo1p. Conservation between mammalian and yeast MMR and the """"""""synergy"""""""" between the two for studying DNA repair pathways serve to justify yeast investigations as an efficient supplemental means to reach our primary goals. As one goal, we will continue structure/function analyses of MutL alpha using molecular genetic strategies. For human Mlh1p and Pms2p, we will use in vivo complementation relying on stable expression of wild type and various mutant forms of the proteins in mouse embryonic fibroblast (MEF) cells. In addition, we will use in vitro MMR assays with extracts of MEF and human cells expressing """"""""mutated"""""""" forms of Mlh1p and/or Pms2p. For certain studies, we will use wild type and mutant MutL alpha, purified with the baculovirus expression system. The mutant alleles of human and yeast MutL homologs to be studied are based on mutations observed in cancer families, mutations that affect conserved motifs of the MutL protein family and mutations from screens in yeast designed to detect separation of function (SOF) and dominant mutations. These studies are intended to define better the roles of human MutL alpha and """"""""interacting"""""""" proteins, e.g. exonucleases, in mismatch repair-mediated processes, including mutation avoidance, responses to certain DNA damaging agents and genetic recombination. In turn, the studies should provide insight into which MMR-related functions are most important for cancer prevention. Finally, we anticipate identifying additional proteins involved in MMR-related processes, which may have functions in preventing human disease such as cancer.
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