The long term goals are to define the process of mismatch repair in eukaryotes. Because of the high degree of conservation of mismatch repair genes and proteins between yeast and humans, we will use the yeast Saccharomyces cerevisae as a model for these studies. We will examine the roles of the Msh2-Msh3, Msh2-Msh6, and Mlh1-Pms1 complexes in mismatch recognition and in subsequent steps of mismatch repair and will determine at which step(s) the ATP binding/hydrolysis activity of Msh2-Msh3 and Msh2-Msh6 is utilized in mismatch repair. Mismatch binding by the Msh2-Msh3 and Msh2-Msh6 complexes will be examined by Dnase I and hydroxyl radical footprinting, and the effect of Mlh1-Pms1 on mismatch binding by Msh complexes determined. Genetic and biochemical studies of mutant msh2, msh3, and msh6 genes and proteins harboring a lysine to alanine alteration in the GKS nucleotide binding motif will examine the role of ATP binding/hydrolysis by the Msh protein complexes in mismatch recognition, in ternary complex formation with Mlh1-Pms1 and mismatched DNA, and in the looping of mismatched DNA. The other components of mismatch repair, including the 5' yields 3' and 3' yields 5' exonucleases and DNA helicases, will be identified by genetic and biochemical studies; their biochemical activities will be further defined, and their manner of interaction with the other mismatch repair proteins determined. Mismatch repair will be examined in vitro in yeast extracts, and the involvement of different nucleases, helicases, and of other enzymes in mismatch repair will be determined. Mismatch repair will be reconstituted with the highly purified yeast proteins, setting the stage for in-depth analyses of the intricacies of this repair process. Mismatch repair maintains the integrity of genomic DNA by correcting replication errors. Defects in mismatch repair lead to enhanced mutability and microsatellite instability, and mutations in human mismatch repair genes result in hereditary non-polyposis colorectal cancer (HNPCC) and other types of cancers. Because of the remarkable conservation of the mismatch repair machinery between yeast and humans, these studies should provide important and novel insights into the mechanisms of this repair process in humans.
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