The broad objective of Project 3 is to study the mechanisms responsible for the fidelity of DNA synthesis andthus to address the most fundamental questions concerning mutagenesis, a root cause of cancer. Ourspecific approach is to investigate the fidelity of DNA polymerase beta, a key repair polymerase. Varients ofpol beta are associated with genome instability and human cancer. The unique aspect of Project 3 is that byclosely integrating its specific aims with those proposed for the structural characterization of Pol beta inProject 1 and the the theoretical computational study in Project 2, we can test quantitative predictions forhow active site amino acids govern the choice between incorporating right and wrong deoxynucleotidesubstrates. By providing a stringent test of theoretical-computational and structural predictions, the data willplay a key role in refining the theoretical models. Project 3 investigates dNTP substrate transition stateanalogs to provide new mechanistic information concerning the source of free energy available to enablepolymerases to distinguish right from wrong. The main experimental approach involves the use offluorescence and rapid quench presteady state kinetic techniques to measure overall fidelity as well asindividual fidelity base substitution and frameshift fidelity components. Project 3 will investigate geneticinstability more generally by constructing model in vitro systems to study the effects of strand displacementsynthesis on the expansion of mono- and dinucleotide repeat sequences yielding frameshift mutation thatcause cancer. The Program Project generally, and Experiment 3 more specifically, are timely given theresurgence of interest in the role of DNA polymerases in causing cancer. The studies in Experiment 3 ontransition state analogs, taken in conjunction with the structural and computational projects, should providepractical payoffs in pharmaceutical anticancer drug design, and offer a logical framework in which to designdrug intervention and prevention strategies to inhibit cancer progression. Project 3 features a newconsortium collaborator, Joann Sweasy, Yale University, who will indentify and characterize human tumorassociatedpol beta variants in Experiment 5.
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