PROJECT SUMMARY DNA mismatch repair (MMR) plays critical roles in eukaryotic cells including: 1) suppressing mutations that result from misincorportation errors during DNA replication that escape DNA polymerase proofreading; 2) suppressing mutations that result from misincorporation events that occur due to chemical modification of DNA or DNA precursors; 3) preventing genome rearrangements due to recombination between divergent DNA sequences; 4) correcting mispaired bases in recombination intermediates; and 5) detecting DNA damage and activating signaling pathways linked to cellular responses, including cell cycle control and cell death. Consequently, MMR defects cause increased rates of accumulating mutations and genome rearrangements resulting in a characteristic genome instability signature and resistance to killing by some DNA damaging agents. In humans, MMR defects underlie both inherited and sporadic cancers, cause tumors to become resistant to some chemotherapy agents and appear to cause quite striking sensitivity of cancers to immunotherapy. Thus, a better understanding of MMR pathways and the consequences of MMR defects will impact human health by: 1) informing our understanding of MMR status; and 2) guiding improvements in the development and use of therapies for MMR-deficient cancers. The proposed studies use Saccharomyces cerevisiae as a model system to study the mechanisms of the conserved eukaryotic MMR pathways. The following lines of investigation will be carried out: 1) genetic approaches will be used to study Msh2- and Mlh1-interacting proteins, identify new MMR proteins and study the activation of the Mlh1-Pms1 endonuclease; 2) reconstitution approaches will be used to study the MMR pathways in which mispair excision is mediated by either Exo1 or the Rad27 endonuclease focusing on mispair excision mechanisms, the recruitment of Exo1 and Rad27 to MMR reactions and whether DNA pol ε can act in Rad27-mediated MMR; 3) reconstitution approaches will be used to study MMR pathways that are dependent on the Mlh1-Pms1 endonuclease including pathways where Mlh1-Pms1 initiates mispair excision by Exo1 and Rad27 and a novel, newly reconstituted pathway where mispair excision is mediated only by Mlh1-Pms1; and, 4) individual steps in MMR reactions will be studied primarily by investigating the protein-protein interactions and higher order complexes of proteins that drive MMR using Surface Plasmon Resonance and single molecule biochemistry methods. The long-term goal of these studies is to develop a detailed understanding of the biochemical and molecular mechanisms of MMR and how cells utilize MMR to prevent mutations and genome rearrangements. Because MMR is highly conserved, the results from studies of S. cerevisiae MMR will provide insights into the mechanisms of MMR in human cells. Consequently, this project will provide insights that can be applied to understanding the genetics of human cancers and the biology of MMR defects in human ...