This PPG integrates multiple disciplines to apply X-ray structural studies, presteady state kinetic and theoretical computational analyses and novel chemical probes to elucidate the molecular basis of DNA polymerase catalysis incorporating base-pair discrimination, a fundamental issue in mutagenesis relevant to cancer. The Program Project contains three research projects, structural (Project 1), theoretical computational (Project 2), and kinetics coupled with an approach toward translational paths (Project 3). Our success at synthesizing dNTP substrate analogs, by replacing one or both phosphate bridging oxygen molecules with a large variety of halo-methylene derivatives containing widely differing electrostatic charge and steric properties, allows us to probe fidelity from a transitions state (T) perspective. The use of these substrate analogs is a uniquely powerful aspect of our PPG, and will allow us for the first time to investigate TS effects using stereoisomeric probes, while offering a feasible approach for targeted inhibition of Pol p, on a path toward cancer cell inhibition (Project 3). The objective of Project 1 is to obtain high-resolution structural data for normal and aberrant forms of Pol , using the dNTP analogs designed in Project 3 and synthesized in Core B. The goal of Project 2 is the application of theoretical and computer modeling to perform structure/function analyses of catalytic mechanisms that govern base selection both in the ground-state and TS. The computations are aimed at calculating free energies, which are used to predict individual contributions of amino acid side chains to fidelity, including substrate binding and catalysis in the pol active site. Central to our PPG is that the theory (Project 2) serves as the intellectual framework with which to marry structural analysis (Project 1) with kinetic mechanistic analysis (Project 3). It is atypical for the experimentalist t test a priori computational predictions. A defining aspect of this PPG is its bidirectional interply, where structural data serve as a starting point for computational predictions, which are tested experimentally, and where the experimental data are used to refine the theory.

Public Health Relevance

Our primary goals are focused on understanding the principles of polymerase fidelity defined by the atomic and molecular interactions between specific amino acid side chains, primer/template bases and dNTP substrates at the pol active site. The target enzyme DNA pol ?plays a key role in base excision repair, which is central to oncogenesis.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program--Cooperative Agreements (U19)
Project #
5U19CA177547-03
Application #
8918543
Study Section
Special Emphasis Panel (ZCA1)
Program Officer
Pelroy, Richard
Project Start
2013-09-03
Project End
2016-08-31
Budget Start
2015-09-01
Budget End
2016-08-31
Support Year
3
Fiscal Year
2015
Total Cost
Indirect Cost
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
90032
Oertell, Keriann; Kashemirov, Boris A; Negahbani, Amirsoheil et al. (2018) Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State. Biochemistry 57:3925-3933
Alnajjar, Khadijeh S; Garcia-Barboza, Beatriz; Negahbani, Amirsoheil et al. (2017) A Change in the Rate-Determining Step of Polymerization by the K289M DNA Polymerase ? Cancer-Associated Variant. Biochemistry 56:2096-2105
Alnajjar, Khadijeh S; Negahbani, Amirsoheil; Nakhjiri, Maryam et al. (2017) DNA Polymerase ? Cancer-Associated Variant I260M Exhibits Nonspecific Selectivity toward the ?-? Bridging Group of the Incoming dNTP. Biochemistry 56:5449-5456
Ni, Feng; Kung, Alvin; Duan, Yankun et al. (2017) Remarkably Stereospecific Utilization of ATP ?,?-Halomethylene Analogues by Protein Kinases. J Am Chem Soc 139:7701-7704
Petruska, John; Goodman, Myron F (2017) Relating DNA base-pairing in aqueous media to DNA polymerase fidelity. Nat Rev Chem 1:
Yoon, Hanwool; Warshel, Arieh (2017) Simulating the fidelity and the three Mg mechanism of pol ? and clarifying the validity of transition state theory in enzyme catalysis. Proteins 85:1446-1453
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
Klva?a, Martin; Bren, Urban; Florián, Jan (2016) Uniform Free-Energy Profiles of the P-O Bond Formation and Cleavage Reactions Catalyzed by DNA Polymerases ? and ?. J Phys Chem B 120:13017-13030
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 ?. Proteins 84:1644-1657
Kim, Taejin; Freudenthal, Bret D; Beard, William A et al. (2016) Insertion of oxidized nucleotide triggers rapid DNA polymerase opening. Nucleic Acids Res 44:4409-24

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