Elucidating the factors that control the replication fidelity of DNA polymerases is a goal of great fundamental and practical importance. In order to advance in this direction we will generate realistic atomic-level simulations of the catalysis and replication fidelity of wild-type and mutant DNA polymerases, focusing on DNA polymerase (pol), which is a key player In human base excision repair and has been Implicated in the incidence of cancer. The main strength of our laboratories has been our combined expertise in state-of- the art simulations of enzyme catalysis and catalytic landscapes, with a deep understanding of the available experimental information on the structural and mechanistic underpinnings of the fidelity of DNA polymerases. Our theoretical toolbox includes the empirical valence bond (EVB), the paradynamics that provides effective way of obtaining ab initio quantum mechanical /molecular mechanics (QM/MM) free energy profiles using the EVB as a reference potential, as well as coarse grained (CG) renormalizatlon approaches for exploring long time coupling between the conformational and chemical coordinates. We have also develop and refined effective sampling methods that should allow us to increase the accuracy of the calculated free energies. These computational studies will be applied in concert with the biochemical studies of Project 3 of kinetic effects of mutations of pol and of changes in its substrates. Our simulations will reproduce and/or predict the functional effects of these changes and analyze their origin. At the same time, we will rely on the important structural information from Project 1. The atomic level understanding Of the structure of the transition state for the insertion of a new nucleotide by pol and the malleability of this structure by modifications of the active site will greatly increase the possibilities of effective design of a potent and selective inhibitor of pol .

Public Health Relevance

Our strategies will allow us to explore the effect of mutations (including distant mutations) and realistically simulate the coupling between the conformational changes induced by the binding of dNTP substrate and the formation of the new PO bond by DNA polymerases. We will also explore the change of this coupling and the resulting rate-limiting transition states due for the right and wrong dNTP substrates

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program--Cooperative Agreements (U19)
Project #
5U19CA177547-02
Application #
8754982
Study Section
Special Emphasis Panel (ZCA1)
Project Start
Project End
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Southern California
Department
Type
DUNS #
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
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Maximoff, Sergey N; Kamerlin, Shina Caroline Lynn; Florián, Jan (2017) DNA Polymerase ? Active Site Favors a Mutagenic Mispair between the Enol Form of Deoxyguanosine Triphosphate Substrate and the Keto Form of Thymidine Template: A Free Energy Perturbation Study. J Phys Chem B 121:7813-7822
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Vorobyov, Igor; Kim, Ilsoo; Chu, Zhen T et al. (2016) Refining the treatment of membrane proteins by coarse-grained models. Proteins 84:92-117
Hwang, Candy S; Xu, Liang; Wang, Wei et al. (2016) Functional interplay between NTP leaving group and base pair recognition during RNA polymerase II nucleotide incorporation revealed by methylene substitution. Nucleic Acids Res 44:3820-8

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