The broad objective of Project 3 is to study the mechanisms responsible for the fidelity of DNA synthesis and thus to address the most fundamental questions concerning mutagenesis, a root cause of cancer. Our specific approach is to investigate the fidelity of DNA polymerase beta, a key repair polymerase. Varients of pol beta are associated with genome instability and human cancer. The unique aspect of Project 3 is that by closely integrating its specific aims with those proposed for the structural characterization of Pol beta in Project 1 and the the theoretical computational study in Project 2, we can test quantitative predictions for how active site amino acids govern the choice between incorporating right and wrong deoxynucleotide substrates. By providing a stringent test of theoretical-computational and structural predictions, the data will play a key role in refining the theoretical models. Project 3 investigates dNTP substrate transition state analogs to provide new mechanistic information concerning the source of free energy available to enable polymerases to distinguish right from wrong. The main experimental approach involves the use of fluorescence and rapid quench presteady state kinetic techniques to measure overall fidelity as well as individual fidelity base substitution and frameshift fidelity components. Project 3 will investigate genetic instability more generally by constructing model in vitro systems to study the effects of strand displacement synthesis on the expansion of mono- and dinucleotide repeat sequences yielding frameshift mutation that cause cancer. The Program Project generally, and Experiment 3 more specifically, are timely given the resurgence of interest in the role of DNA polymerases in causing cancer. The studies in Experiment 3 on transition state analogs, taken in conjunction with the structural and computational projects, should provide practical payoffs in pharmaceutical anticancer drug design, and offer a logical framework in which to design drug intervention and prevention strategies to inhibit cancer progression. Project 3 features a new consortium collaborator, Joann Sweasy, Yale University, who will indentify and characterize human tumorassociated pol beta variants in Experiment 5.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Program--Cooperative Agreements (U19)
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Special Emphasis Panel (ZCA1-GRB-S)
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University of Southern California
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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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