Summary of Work: Rare replication errors are corrected by the cell's spellchecking machinery, post-replication DNA mismatch repair (MMR). The goal of this project is to understand the biochemistry and genetics of MMR in normal and mutant eukaryotic cells. This year we had three main accomplishments. To test the hypothesis that MSH3 is important for MMR and for reducing cancer incidence, we studied mice deficient in MSH3 alone or in combination MSH6. The (-/-) mice were found to have an increased incidence of cancer and extracts of cells derived from these mice are deficient in MMR activity. To test the hypothesis that polymorphisms in the MSH2 coding sequence may be functionally important, we studied three different polymorphisms that are reported at 1-8% allele frequency. We provided evidence that all three MSH2 gene sequence changes partially reduced MMR, suggesting that people harboring these polymorphisms may be at increased risk of developing cancer. To investigate the function of the Mlh1oPms1 heterodimer in MMR, we are developing a procedure to purify this complex. As part of this effort, we used mass spectroscopy to identify protein contaminants in the partially purified protein preparation. This permitted redesign of the purification procedure to remove the contaminants, thus providing highly purified MutL alpha heterodimer for ongoing biochemical characterizations. These studies are important for understanding the genetics and biochemistry of DNA mismatch repair, the functions of mismatch repair genes, the molecular genetic basis for the initiating events in cancer and its subsequent treatment, and the risk posed to individuals in the population by exposure to DNA damaging agents.