The goal of this project is to understand the three processes responsible for DNA replication fidelity in human cells: DNA polymerase base selectivity, exonucleolytic proofreading and post-replication mismatch repair. Recently, we i) determined the fidelity of leading and lagging strand replication of undamaged DNA by the multiprotein replication apparatus in extracts of normal human cells and tumor cells, ii) studied replication fidelity with DNA molecules containing defined adducts of known carcinogens and iii) examined the fidelity of replication of simple repetitive sequences. Both polymerase selectivity and exonucleolytic proofreading are diminished during replication of 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. Lastly, we examined the ability of normal and mutant human cell extracts to correct DNA substrates containing mispaired or unpaired nucleotides. Mismatch repair activity is defective in cancer cell lines containing mutations in any of five different genes, MSH2, MSH3, MSH6, MLH1 and PMS2. Each of these lines has been corrected by transfer of a wild-type chromosome to the cell line and the resulting phenotypes are being examined. 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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