ACCURACY OF DNA REPLICATION IN VITRO

点击翻译按钮获取中文摘要

The goal of this project is to understand the three processes reponsible for DNA replication fidelity in human cells: DNA polymerase base selectivity, exonucleolytic proofreading and post-replication mismatch repair. We have determined the fidelity of DNA synthesis catalyzed by the five classes of eukaryotic DNA polymerases. The DNA polymerases have distinctly different error rates and specificities, which has implications for their roles in the various stages of DNA replication. We have also determined the fidelity of bidirectional 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 two strands, base substitution and frameshift error rates do differ at some sites for the leading and lagging strand replication apparatus. In order to better understand the effects of known mutagens and carcinogens on the fidelity of DNA synthesis, we are performing studies of replication fidelity with DNA molecules containing DNA adducts. Emphasis is now on uniquely-placed AAF adducts, defining the probability of termination versus bypass and the extent of mutatgenic bypass. We have shown that the multiprotein replication apparatus in extracts of human HeLa cells is indeed capable of mutagenic translesion bypass of three very different types of adducts, which has implications for their possible roles in the etiology of cancer. 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 have recently begun to examine the fideilty of replication of simple repetitive di- and tri-nucleotide repeat sequences. Lastly, we are examining the ability of normal and mutant human cell extracts to correct DNA substrates containing mispaired or unpaired nucleotides. These studies are important for understanding the molecular genetic basis for the initiating events in carcinogenesis and the risk posed to individuals in the population by exposure to DNA damaging agents.