The purpose of this study is to examine the role of the mismatch repair proteins MSH3 and MSH6 in cancer susceptibility. Mutations in the hMSH2 gene lead to HNPCC. Since MSH3 and MSH6 are thought to form functional complexes with MSH2, the prediction is that knockout mice in these genes will be cancer prone. Most of our understanding of the properties of the mammalian DNA mismatch repair system is derived from biochemical studies of the Escherichia coli MutHLS system. In bacteria the repair of DNA mismatches, which are the result of DNA replication errors, is initiated by the binding of the MutS protein to the mismatched bases. In eukaryotes the recognition of DNA mismatches is more complex and requires subsets of three different MutS homologs: MSH2, MSH3 and MSH6. Studies in yeast have indicated the DNA mismatch repair is initiated by two different complexes: A complex between MSH2-MSH6 for the recognition of single base mismatches and a complex between MSH2-MSH3 for the recognition of two to four base pair insertion/deletions. Because germline mutations in hMSH2 lead to HNPCC it is likely that hMSH3 and hMSH6, which interact with hMSH2, are also involved in the development of neoplasms. Their role in these events, however, remains unclear. In this proposal we plan to test the hypothesis that the mammalian homologs of yeast MSH3 and MSH6 share the same function in mitotic mismatch repair and to analyze the cancer susceptibility of mice carrying targeted mutations in these genes. The 3 specific aims of this proposal are: 1. To generate mice that are deficient in MSH3 and MSH6 and to study the cancer susceptibility of these mouse lines. Dr. Edelmann will study the consequences of loss of MSH3 and MSH6 for cancer susceptibility with special emphasis on gastrointestinal carcinogenesis. 2. To test the hypothesis that the mammalian msh2, msh3 and msh 6 genes are genetically equivalent to their yeast homologs. Experiments in yeast suggest the MSH2-MSH3 and MSH2-MSH6 are involved in the repair of different types of DNA mismatches. We will use msh2, msh3 and msh6 mutant mouse lines and double mutant msh3/msh6 mouse lines as a source for mouse embryonic fibroblast cell lines to analyze microsatellite instability and to measure heteroduplex mismatch repair in cell extracts. 3. To examine the effect of MSH2, MSH3 and MSH6 deficiency of the Apc tumor suppressor gene. Colorectal cancer cell lines with mutations in the hMSH2 gene accumulate mutations in the tumor suppressor gene APC. We will generate mouse lines that are mutant for msh3 or msh6 and heterozygous for the A1638 allele. The analysis of the tumor spectrum and the onset and progression of tumor formation will indicate the significance of MSH2, MSH3 and MSH6 for gastrointestinal tumorigenesis and provide a detailed analysis of the spectrum of mutations that results from deficiency in each of these repair genes.
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