Hereditary nonpolyposis colorectal cancer (HNPCC; OMIM 120435-6) is an autosomal dominant inherited syndrome caused by germline mutations in DNA mismatch repair (MMR) genes such as hMSH2 and hMLH1. With the loss of both alleles, DNA mismatch repair activity is completely lost and mutation rates increase significantly in specific regions of the genome called microsatellites. This phenomenon, referred to as microsatellite instability (MSI), leads to accelerated accumulation of frameshift mutations in specific tumor suppressors and other genes. Inactivation of these tumor suppressor genes contributes to stepwise neoplastic progression. This proposal is focused on two hypotheses regarding MSI and its relationship to DNA repair genes and HNPCC. The first hypothesis is that different DNA replication and repair genes influence microsatellite instability and mutation rates in human tumor suppressors and other cancer-related genes. While the effects of the MMR genes, MSH2 and MLH1, on MSI and mutation rates have been studied, there are other genes which contribute to MSI but are not as well characterized. The second hypothesis is that there are other human genes, as yet unidentified, which are susceptible to MSI mutations, and thus may be important contributors to colon epithelial neoplastic development in HNPCC. Although a number of genes vulnerable to MSI have been discovered, undoubtedly there are others that contribute to tumorigenesis. In this proposal, the principal investigator describes a strategy using S. cerevisiae as a model organism system to test these hypotheses and expand the genetic studies of MSI-related mutations in human tumor genes. Using isogenic S. cerevisiae strains with deletion mutations for a variety of DNA repair and replication (such as polymerases) genes I will test the effects these mutants have on human gene microsatellite instability. This quantitative data will be used to discern different DNA repair and replication genes' contribution to MSI in human genes. In addition, I will measure the mutation rates of selected tumor suppressor microsatellites in MMR-deficient mammalian cell lines to study the conservation of MSI between yeast and higher eukaryotes. I will identify hypermutable microsatellites in the coding region of new human genes that participate in neoplastic development in HNPCC-related malignancies. To accomplish this goal, I will screen a cDNA library derived from normal colon tissue for new genes which are susceptible to microsatellite instability mutations.
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