Mutations in the DNA mismatch repair genes hMLH1, hPMS2 and hPMS1 are involved in human hereditary colon cancer. Lack of MMR activity, as determined by the presence of microsatellite instability, is also a common feature in a significant fraction of sporadic tumors from a variety of tissues, and is associated with the development of resistance to several chemotherapeutic agents. We have recently generated and characterized mice deficient for the Mlh1, Pms2 and Pms1 DNA mismatch repair gene. Our analysis has demonstrated that mice carrying mutations in these genes differ in tumor susceptibility, and that there is no obvious correlation between intestinal tumorigenesis and the frequency of DNA replication errors in the intestinal epithelium. These findings imply that individual DNA mismatch repair genes have unique biological roles, and that poorly characterized factors play a role in tumorigenesis associated with DNA mismatch repair deficiency. The broad goal of this proposal is to define the mechanism(s) of tumor formation due to defects in DNA mismatch repair. We hypothesize that different tumor susceptibility in Mlh1, Pms2 and Pms1 deficient mice is due to gene-specific roles in either spontaneous or chemically induced mutagenesis. In order to examine this hypothesis, we propose to construct isogenic cell lines carrying mutations in Mlh1, Pms2 and Pms1 through homologous recombination (Aim 1). These cell lines will be used in conjunction with previously generated Msh2-/-cell lines in a number of assays that will allow us to systematically determine the relative contribution of different MMR genes to spontaneous mutation, mutation spectra at defined loci, instability of simple-repeat tracts, and genetic recombination between diverged DNA elements (Aim 2). Additionally, we will determine if mismatch repair genes affect chemically induced mutagenesis, and if base modifying agents can affect tumor development in mismatch repair deficient mice (Aim 3). The proposed research program will define the role of different mismatch repair genes in several aspects of mutagenesis at the cellular level, and results will be applied in the study of tumor formation at the organismal level in DNA mismatch repair deficient mice. Elucidation of novel and specific roles of DNA mismatch repair genes will provide new strategies for cancer prevention in families carrying mismatch repair gene mutations, and will suggest new therapeutic avenues for treatment of DNA mismatch repair deficient tumors.