HUMAN MISMATCH REPAIR IN CHEMICAL CARCINOGENESIS

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DESCRIPTION: Dr. Li's long term goal is to understand the molecular mechanism of mismatch repair (MMR) and its role in cancer avoidance. MMR is a mutation avoidance system and plays an important role in maintaining genetic stability. It has been shown that defects in the human system confer a strong cancer predisposition including hereditary polyposis colorectal cancer. Environmental carcinogens covalently modify DNA to form carcinogen-DNA adducts, which induce mutations that initiate carcinogenesis. Recently, MMR components have been shown to recognize certain forms of carcinogen-DNA adducts that are previously thought to be only processed by nucleotide excision repair (NER). It is also documented that MMR-proficient cells are much more sensitive to the cytotoxic effects of N-methyl-N'-nitro-nitrosoguanidine (MNNG) compared to MMR-deficient cells. Given these findings, we hypothesize that MMR components either directly participate in repair of carcinogen-DNA adducts or function as a sensor to activate programmed cell death. To test this hypothesis, four lines of work are proposed in this application. First, using an electrophoretic mobility shift assay, purified hMutSa, a human mismatch recognition protein, will be tested for its ability to bind oligonucleotide duplexes containing site-specific adducts of acetylaminofluorene, benzo[a]pyrene diol epoxide, and MNNG, the three most critical chemical carcinogens. Second, cell extracts derived from tumor cells with different MMR backgrounds will be examined to process circular plasmid DNA containing site-specific adducts of the carcinogens listed above. Third, to investigate the physiological importance of the recognition and processing of carcinogen-DNA adducts by MMR, MMR-normal and mutant cells will be treated with carcinogens listed above or transfected with carcinogen-modified pZ189 plasmid, an SV40-based shuttle vector, and analyzed for apoptosis. Since MNNG-induced cell death of MMR-proficient cells has been attributed to their futile attempts to remove MMNG adducts in the template DNA strand, it is anticipated that replication of carcinogen-modified genomic or plasmid DNA will induce apoptosis in MMR-proficient cells. Finally, to determine if carcinogen adducts can be remove in vivo by MMRcriti, genomic DNA from MMR-proficient and MMR-deficient cells that are treated with carcinogens will be digested into mononucleosides, and followed by adduct detection and quantitation.