DNA Replication Fidelity

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Summary of Work: The long-term goal of this project is to understand the fidelity of DNA synthesis by multiprotein DNA replication and repair complexes. This year, progress was made in three areas. We established an in vivo system in yeast cells to determine the fidelity of DNA replication by the leading and lagging strand DNA replication machinery and used the system to demonstrate that yeast origins establish a strand bias for replicational mutagenesis induced by two base analogs, one of which (8-oxo-guanine) is a major DNA lesion generated by oxidative stress. We investigated the roles of translesion synthesis polymerases in yeast upon exposure to simulated sunlight. These studies are more relevant to the true physiological roles of these polymerases in suppressing or perhaps promoting skin cancer than are all previous studies of cytotoxicity and mutagenesis induced by irradiation with 254 nm UV light, because the spectrum of lesions generated by sunlight are significantly different (e.g., much more oxidative stress). This year, we also described an assay to measure the fidelity of BER reconstituted with purified enzymes, and used it to determine the fidelity of a four-protein single-nucleotide BER reaction involving Pol beta. The base substitution fidelity of Pol beta dependent BER was found to be 3 to 8-fold higher than is single nucleotide gap filling by Pol beta alone, suggesting that other proteins in the BER reaction may enhance the base substitution fidelity of Pol beta during single-nucleotide BER