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.

Agency
National Institute of Health (NIH)
Type
Research Program--Cooperative Agreements (U19)
Project #
5U19CA177547-02
Application #
8729570
Study Section
Special Emphasis Panel (ZCA1)
Program Officer
Pelroy, Richard
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Southern California
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Kirby, Thomas W; Derose, Eugene F; Beard, William A et al. (2014) Substrate rescue of DNA polymerase ? containing a catastrophic L22P mutation. Biochemistry 53:2413-22
Wu, Sangwook; Beard, William A; Pedersen, Lee G et al. (2014) Structural comparison of DNA polymerase architecture suggests a nucleotide gateway to the polymerase active site. Chem Rev 114:2759-74
Prasad, Rajendra; Horton, Julie K; Chastain 2nd, Paul D et al. (2014) Suicidal cross-linking of PARP-1 to AP site intermediates in cells undergoing base excision repair. Nucleic Acids Res 42:6337-51
Freudenthal, Bret D; Beard, William A; Wilson, Samuel H (2014) Watching a DNA polymerase in action. Cell Cycle 13:691-2
Towle-Weicksel, Jamie B; Dalal, Shibani; Sohl, Christal D et al. (2014) Fluorescence resonance energy transfer studies of DNA polymerase ?: the critical role of fingers domain movements and a novel non-covalent step during nucleotide selection. J Biol Chem 289:16541-50
Beard, William A; Wilson, Samuel H (2014) Structure and mechanism of DNA polymerase ?. Biochemistry 53:2768-80
Oertell, Keriann; Chamberlain, Brian T; Wu, Yue et al. (2014) Transition state in DNA polymerase ? catalysis: rate-limiting chemistry altered by base-pair configuration. Biochemistry 53:1842-8
Hwang, Candy S; Kashemirov, Boris A; McKenna, Charles E (2014) On the observation of discrete fluorine NMR spectra for uridine 5'-?,?-fluoromethylenetriphosphate diastereomers at basic pH. J Org Chem 79:5315-9
Seamon, Kyle J; Hansen, Erik C; Kadina, Anastasia P et al. (2014) Small molecule inhibition of SAMHD1 dNTPase by tetramer destabilization. J Am Chem Soc 136:9822-5
Sassa, Akira; Ça?layan, Melike; Dyrkheeva, Nadezhda S et al. (2014) Base excision repair of tandem modifications in a methylated CpG dinucleotide. J Biol Chem 289:13996-4008