DNA mismatch repair (MMR) plays critical roles in eukaryotic cells including: 1) suppression of mutations that result from misincorportation events caused by errors in DNA replication and by chemically damaged DNA and DNA precursors; 2) prevention of genome rearrangements due to non-allelic homologous recombination; 3) repair of mispaired bases in heteroduplex recombination intermediates; and 4) DNA damage signaling linked to cellular responses, including cell cycle control and cell death. As a consequence, MMR defects cause increased rates of accumulating mutations and altered recombination events resulting in a characteristic genome instability signature as well as increased resistance to killig by some DNA damaging agents. In humans, MMR defects can underlie both inherited and sporadic cancers and can cause tumors to become resistance to chemotherapy. Thus, a better understanding of MMR pathways and of the genetic consequences of MMR defects will impact human health by: 1) informing the development and improvement of clinical tests for the MMR status of patients and tumors; and 2) guiding improvements in therapies for MMR-deficient cancers or cancers that have acquired MMR defects as a result of developing resistance to chemotherapy. The goal of this proposal is to use Saccharomyces cerevisiae to study the conserved biochemical and genetic mechanisms of the eukaryotic MutS and MutL homologue-dependent MMR pathways. The following lines of investigation will be carried out: 1) MMR genes and proteins that function in overlapping MMR sub- pathways will be identified using genetic approaches, and the mutations identified in these studies will be used in biochemical studies of MMR mechanisms; 2) the biochemical activities of individual purified MMR proteins will be characterized in detail to provide insights into the roles that each protein plays in MMR; 3) MMR reactions, including those coupled to DNA replication, will be reconstituted in vitro using purified proteins to study the mechanisms of MMR; and 4) individual steps in the multi-protein reactions that function in MMR will be studied in detail by characterizing the protein-protein interactions that target individual proteins to specific steps in MMR and by using electron microscopy and single molecule biochemistry methods to visualize features of MMR. The ultimate goal of these experiments is to understand the biochemical mechanisms of MMR and how cells utilize MMR to prevent mutations and genome rearrangements. Because MMR is highly conserved, a key feature of the proposed studies is that studies of S. cerevisiae MMR will provide insights into the mechanisms of MMR in human cells. As a consequence, the proposed studies will provide insights into the genetics of human cancer susceptibility and the biology of MMR defects in human cancers in addition to providing a basic understanding of MMR mechanisms.