This Program Project is designed to address fundamental issues in mutagenesis relevant to the root causes of cancer, in accordance with the mission of NCI. We propose to investigate the molecular basis of DNA polymerase accuracy, relating theory to experiment and vice versa, using human DNA polymerase beta as a model system. Pol beta plays a key role in the avoidance of cancer, because its loss of regulation or disruption by mutation induces chromosome instability and tumorigenesis. Our primary goals are focused on understanding the principles of polymerase fidelity 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 Synthetic and 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 ? using a new class of nucleotide analogs designed in Project 3 and synthesized in Core B. These analogs will be used in drug design and delivery strategies to establish their potential use as anticancer agents in mouse and cultured cell model systems, in a translational approach to target bone tumors. A unique and timely aspect of the PPG is the application of theoretical and computer-modeling approaches to structure/function analysis of catalytic efficiencies in polymerase active sites, as proposed in Project 2. The modeling analysis calculates free energies, which are 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 analyses described in Project 3. 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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program--Cooperative Agreements (U19)
Project #
5U19CA105010-09
Application #
8306988
Study Section
Special Emphasis Panel (ZCA1-GRB-S (J1))
Program Officer
Pelroy, Richard
Project Start
2004-08-16
Project End
2013-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
9
Fiscal Year
2012
Total Cost
$1,030,757
Indirect Cost
$287,830
Name
University of Southern California
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Yoon, Hanwool; Warshel, Arieh (2016) The control of the discrimination between dNTP and rNTP in DNA and RNA polymerase. Proteins 84:1616-1624
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
Matute, Ricardo A; Yoon, Hanwool; Warshel, Arieh (2016) Exploring the mechanism of DNA polymerases by analyzing the effect of mutations of active site acidic groups in Polymerase β. Proteins 84:1644-1657
Batra, Vinod K; Beard, William A; Pedersen, Lars C et al. (2016) Structures of DNA Polymerase Mispaired DNA Termini Transitioning to Pre-catalytic Complexes Support an Induced-Fit Fidelity Mechanism. Structure 24:1863-1875
Warshel, Arieh; Bora, Ram Prasad (2016) Perspective: Defining and quantifying the role of dynamics in enzyme catalysis. J Chem Phys 144:180901
Vorobyov, Igor; Kim, Ilsoo; Chu, Zhen T et al. (2016) Refining the treatment of membrane proteins by coarse-grained models. Proteins 84:92-117
Mukherjee, Shayantani; Bora, Ram Prasad; Warshel, Arieh (2015) Torque, chemistry and efficiency in molecular motors: a study of the rotary-chemical coupling in F1-ATPase. Q Rev Biophys 48:395-403
Perera, Lalith; Freudenthal, Bret D; Beard, William A et al. (2015) Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse. Proc Natl Acad Sci U S A 112:E5228-36
Freudenthal, Bret D; Beard, William A; Perera, Lalith et al. (2015) Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide. Nature 517:635-9
Frushicheva, Maria P; Mills, Matthew J L; Schopf, Patrick et al. (2014) Computer aided enzyme design and catalytic concepts. Curr Opin Chem Biol 21:56-62

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