of Work: The goal of this project is to understand how DNA polymerase selectivity and exonucleolytic proofreading contribute to DNA replication fidelity in human cells. We previously determined the fidelity of undamaged DNA replication by the multiprotein replication apparatus in extracts of normal human cells and tumor cells. Although the overall fidelity of replication is similar on the leading and lagging strands, base substitution and frameshift error rates do differ at some sites for leading and lagging strand replication aproteins. In order to better understand the effects of known mutagens and carcinogens on the fidelity of DNA synthesis, we have performed studies with DNA molecules containing several different types of DNA adducts. This year, the probability of termination versus bypass and the extent of mutatgenic bypass has been determined for psoralen monoadducts. In an attempt to understand the instabilty of microsatellite sequences in certain tumors and the instability in triplet repeat sequences in several hereditary human diseases, we extended earlier studies of the fideilty of replication of homopolymeric sequences to the copying of dinucleotide and triplet repeat sequences. Both polymerase selectivity and exonucleolytic proofreading are diminished during replication of these repetitive sequences. This may place an increased relative burden on post- replication repair processes to reduce rates of addition and deletion mutations in organisms whose genomes contain abundant simple repeat DNA sequences. We have also presented a novel model that can explain certain features of triplet repeat expansion, based on aberrant processing of Okazaki fragments during DNA replication. These studies are important for understanding the molecular genetic basis for the initiating events in diseases and the risk posed to individuals in the population by exposure to DNA damaging agents.
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