A fuller understanding of eukaryotic DNA polymerase reaction mechanisms and response to DNA damage is required to predict accurately the biologic consequences of cellular exposure to environmental mutagens. Studies in this Project will focus on replicative DNA polymerases delta, alpha and epsilon. For DNA polymerase delta (pol delta), several aims will be pursued. A model will be tested for the ordered sequential interaction of mammalian pol delta, proliferating cell nuclear antigen (PCNA) and synthetic oligonucleotide template-primers. The interaction of mammalian pol delta and PCNA with base-mismatch and adduct-containing template-primers will be studied. Mammalian and Saccharomyces cerevisiae proteins will be compared. Effects of PCNA on the pol delta-associated exonuclease will be investigated. Sites of interaction between pol delta and PCNA as well as between pol delta/PCNA and both normal and damaged template-primers will be mapped. For DNA polymerase alpha (pol alpha)-holoenzyme, mobility shift assays similar to those optimized for pol delta/PCNA will be developed. Interactions of pol alpha-holoenzyme with template-primers containing 8- oxo-dG as well as other base-adducts will be examined. For DNA polymerase epsilon (pol epsilon), the molecular mechanism of substrate recognition and binding by purified pol epsilon will be elucidated. Interactions of pol epsilon with synthetic template-primers containing either primer region mismatches or base adducts in both template and primer regions will be characterized. Following the mechanistic characterization and comparison of the responses of pol delta, pol alpha-holoenzyme and pol epsilon to different forms of DNA damage, explicit biological hypotheses regarding leading and lagging strand mutagenesis will be formulated and tested.
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