The long-term objectives of this project are to understand the molecular mechanism of human mismatch repair and its impact on human cancer. DNA mismatch repair plays a crucial role in maintaining genomic stability by correcting mismatches generated from DNA biosynthetic errors and DNA recombination. Defects in human mismatch repair are the primary cause of both hereditary colorectal cancer and sporadic colorectal cancers that display microsatellite instability. Microsatellite instability, which correlates with mismatch repair deficiency, has also been demonstrated in a substantial fraction of many types of sporadic cancer, including bladder cancers. Recently, sporadic bladder cancers have been shown to display a higher rate of microsatellite instability than other sporadic cancers. The goals of this application are to determine if mismatch repair deficiency is associated with sporadic bladder cancers and to isolate and characterize novel mismatch repair components/genes. Experiments will be developed in the following three specific areas. 1) The mismatch repair proficiency of bladder cell lines with microsatellite instability will be determined using an in vitro biochemical mismatch repair assay. 2) Novel mismatch repair activities will be first characterized by complementation experiments using the known mismatch repair proteins and/or mutant cell lines, and then be purified from HeLa nuclear extracts by virtue of their ability to restore mismatch repair to the novel mutant cell lines. 3) The gene(s) encoding the novel protein(s) will be cloned by the """"""""reverse genetic"""""""" approach. Peptide sequences will be obtained from the novel protein(s) and used for designing degenerate primers to amplify DNA fragments of interest, which are in turn used to identify full length cDNAs. This study will not only provide insight into the etiology of sporadic bladder cancers, but also lead to the identification of novel mismatch repair components.
