Simulating Large-Scale Conformational Rearrangements and Reaction Kinetics Profiles in DNA Polymerase Beta to Interpret DNA Synthesis Fidelity Mechanisms

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Studying large-scale, long-time biological processes such as enzyme catalysis, protein folding, and macromolecular assembly is a challenging task in computational biophysics. Since these processes occur over microseconds to seconds, much beyond the scope of traditional dynamics simulations, new techniques are needed to provide insights into detailed, local motions to supplement experiments. In this project, funded jointly by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences and the Computational Math Program in the Division of Mathematical Sciences, the PI will develop, compare, and apply two rigorous and complementary path-generation methods, Elber's stochastic path approach (SPA) and Chandler's transition path sampling (TPS), to study the conformational transitions between closed and open states for human DNA polymerase beta (pol beta) complexed with DNA template/primer. This millisecond process is thought to be key in maintaining DNA synthesis fidelity. With these new tools, the PI will pursue several fundamental biological questions related to DNA synthesis fidelity, including the identification of slow conformational steps that steer the enzyme toward the chemistry-competent state and determination of rate-limiting steps in the enzyme's pathway. Atomic-level mechanistic insights, as well as associated free-energy barriers, will be delineated and related to enzyme function. The methodology developed is widely applicable to many other fundamental processes in molecular biophysics, and the biological findings will provide atomic-level interpretations to puzzling experimental variations in catalytic rates and error frequencies. Thus, the biological findings will help interpret fundamental fidelity mechanisms employed by DNA polymerases to replicate and repair DNA faithfully from one generation to the next.DNA polymerases maintain genomic integrity in the cell by replicating DNA and repairing damages in the genome from generation to generation. The goal of this project is to dissect the conformational aspects of the selectivity and fidelity of DNA repair process. Fidelity refers to the ability of DNA polymerases to discriminate among the various nucleotide building blocks as each base unit is synthesized and choose the correct base (parent strand's partner) for insertion and extension. The PI will employ modeling and simulation by novel path-generation schemes that can capture large-scale long-time processes to study these polymerase mechanisms to explain fidelity. Such information has important ramifications to our understanding the fundamental DNA synthesis and repair fidelity processes. This project represents a collaboration with theoreticians and experimentalists with expertise in nucleic-acid structure, polymerase mechanisms, and simulation methodology, and relies on solid groundwork in both methodology for biomolecular modeling. The tools developed are also widely applicable to many other important problems in biology. Involving undergraduate and graduate students and postdoctoral fellows, the project offers important multidisciplinary educational and training opportunities to young scientists, including women and minorities, in molecular modeling and computational biology, fields of growing importance to science and society.