The main objectives of this proposal are; (i) To determine if base selection by RB69 pol is governed by a conformational change before chemistry or by the nucleotidyl transfer step itself; (ii) To understand how the triple mutant, that we have produced, can have dramatically reduced base selectivity and, at the same time, exhibit pre-steady state kpol and Kd values that are very similar to the wild-type enzyme for the incorporation of complementary dNMPs. (iii) To determine the mechanism used by RB69 pol, and several of its mutants, to generate deletions and to carry out lesion bypass synthesis. To address these problems we will determine rates of conformational changes by stopped-flow fluorescence and compare the results with rates obtained from chemical quench in the bulk phase. We then plan to use single molecular fluorescence (SMF) and single molecule fluorescence resonance energy transfer (SMFRET) to investigate the dynamics of the nucleotide addition cycle with the aim of capturing transients in the reaction pathway. We will employ single molecule fluorescence to investigate extension and excision of mispaired nucleotide residues at the primer terminus. To complement these kinetic studies we will perform in vitro and in vivo biological assays, '^collaboration with the Karam and Drake labs, to determine the type and frequency of mutations caused by RB69 pol """"""""fidelity"""""""" mutants. By studying the kinetic behavior of these mutants with pulse-chase, chemical- quench and stopped-flow fluorescence, as well as by single molecule techniques, we hope to gain insights into mechanisms that control fidelity. In conjunction with this work, structural studies on RB69 pol """"""""fidelity"""""""" mutants will be carried out by C. Kisker and S. Doublie as our studies draw heavily on knowledge of the crystal structures of RB69 pol complexes. Since RB69 pol is a member of the B family (which includes two human replicative DNA polymerases, pol alpha and pol delta); information obtained from RB69 pol and its mutants should be relevant to these DNA pols as well. Taken together, the results from these collaborative efforts will contribute to a better understanding of diseases that involve accumulated alterations in DNA caused by cellular or viral polymerases such as HIV-RT. They will also provide a structural basis for understanding mutagenic mechanisms that compromise the ability of replicative and repair polymerases to produce faithful replicas of their DNA substrates. ? ? ?
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