The long-term goal of this project is to define the fine structure of DNA replication fidelity. Replication of the human genome involves a number of complex reactions including initiation of synthesis at origins, elongation on the leading strand and lagging strands and replacement of RNA primers with DNA. Current models suggest that more than one DNA polymerase is required for replication, that the proteins that start or finish chains may be different than those that perform the bulk of chain elongation, and that the proteins that replicate the leading and lagging strands may be different. To determine whether these differences result in different replication error rates, we are examining the fidelity of DNA synthesis catalyzed by eukaryotic DNA polymerases alpha, beta, delta, epsilon and gamma and the fidelity of bidirectional DNA replication by the multiprotein replication apparatus in extracts of human HeLa cells. We have found that the DNA polymerases have distinctly different error rates and specificities, which have implications for their roles in the various stages of DNA replication. Also, 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. Finally, 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 defined lesions, including psoralen monoadducts, UV photoproducts and AAF adducts. We intend to continue these studies to understand the fidelity of each of the polymerization reactions required for complete replication of the human genome.
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