DNA mismatch repair plays a prominent role in genomic stability in mammalian cells by correcting mispairs that form during replication, by chemical damage of DNA and by homologous recombination. Data indicates that mutation in any one of five human mismatch repair gene homologs, MSH2, MSH6, MLH1, PMS2 and PMS2 (sic) contributes to spontaneous and hereditary cancers. As a primary example, HNPCC is frequently associated with germline mutations in the MMR genes and accounts for approximately 5% of the total colorectal cancer burden. Biochemical studies indicate the MutS and MutAL mammalian homologs each function as heterodimers to ensure efficient repair. The MutS heterodimers and the """"""""MutL"""""""" heterodimers appear to form a higher order complex, most likely in conjunction with other proteins, that is important for the initial steps in the repair process. Although much has been learned about the biochemical activities of the MSH gene products, little functional information is available for the major MutL homologs, MLH1 and pMS2. One significant unanswered question is """"""""What determines 'strand-choice' in correction of replicative errors, i.e., the preferential repair of the newly synthesized strand? To help answer such questions, we will perform a structure/function analysis of MLH1 and PMS2 using both in vivo and in vitro approaches. We will develop complementation systems using expression vector transfections into cultured fibroblasts for both MLH1 and PMS2. We will use cell-based and cell extract-based assays with transfected mutant forms of MLH1 or PMS2 for the analyses. We will focus the assays on mutation avoidance functions and on responses to DNA damaging agents. We will use certain mutant forms MLH1 and/or PMS2 to identify additional """"""""Mut-L"""""""" interacting proteins. Such interacting proteins will represent candidates for other mismatch repair proteins, or, alternatively, proteins that function in processes that """"""""cooperate"""""""" with mismatch repair. The elucidation of the roles of MLH1 and PMS2 proteins, and isolation of additional interacting genes, is likely to contribute not only to our understanding of mismatch repair, but also to the identification of genes involved in human disease, in particular in tumorigenesis.
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