The general significance of this Program Project is that it is designed to address fundamental issues in mutagenesis and in so doing will have important relevance to the root causes of cancer, in accordance with the mission of NCI. We propose to investigate the molecular and atomic basis for DNA polymerase accuracy, relating theory to experiment and vice versa, using normal and mutant DNA polymerases including error-prone polymerases. Our primary goals are focused on understanding the principles of polymerase fidelity, the relationship of dynamics to thermodynamics, the source of free energy enabling the polymerase to distinguish right from wrong, and active site geometrical constraints as defined by the detailed interactions between specific amino acid side chains, primer/template bases and dNTP substrates at the Pol active site. The Program Project contains three research projects, structural (Project 1), theoretical computational (Project 2), kinetics (Project 3) and three core facilities, a Biochemical Analysis Core (Core B), a Computational Core (Core C) and an Administrative Core (Core A). The goal of Project 1 is to obtain high-resolution structural data for normal and mutant forms of Pol beta to investigate the mechanism of specificity and catalytic efficiency of this critically important human repair polymerase, using information that will be evaluated theoretically in Project 2 and experimentally in Project 3. A unique and timely aspect of the PPG is the application of theoretical and compute modeling approaches in structure/function analysis of catalytic efficiencies in polymerase active sites, as proposed in Project 2. The modeling analysis calculates free energies used to predict individual contributions of amino acid side chains to fidelity including substrate binding and catalysis in the polymerase active site. The theory serves as the intellectual framework with which to marry structural analysis with kinetic mechanistic analysis described in Project 3. The usual interplay between molecular computations and experiment is usually unidirectional - experimental data already in the literature are used to """"""""fit"""""""" the theory, either for better or for worse. It is usually atypical for the experimentalist to test a priori computational predictions. Thus, a defining aspect of this PPG is its bidirectional interplay, where computational predictions are tested experimentally and new experimental data are used to refine the theory.
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