DNA polymerase mechanisms of base selection and catalysis are explored using a tool-kit of dNTP analogs that have bisphosphonates in place of the B,Y-bridge oxygen. These analogs are Pol B substrates that have leaving groups with widely dispirate electronic properties, pKa4 values ranging from 7.8 to 12.3, enabling us to use presteady state kinetic measurements to determine the selection of right and wrong deoxynucleotides occurring at the chemical transition state. Especially important members of the toolkit include all four individually synthesized (R)- and (S)-B,Y-CHF and B,Y-CHCI diastereomers. Our recent observation of a pronounced stereoselection for (R)-CHF in Pol B involving an electrostatic interaction of F with Arg183, unique to family X pols such as Pol B, serves as the impetus for a

Public Health Relevance

The proposed research will apply innovative experimental strategies to elucidate the mechanisms of fidelity occurring in the active site of DNA polymerase. We will focus on studies of human DNA polymerase p, an exceptionally important repair enzyme. Mutants of Pol B have been associated with numerous different human cancers. We have devised a logical strategy using a new class of polymerase substrate analogs to selectively inhibit Pol B in cell free systems and in cultured cancer cells. .

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program--Cooperative Agreements (U19)
Project #
1U19CA177547-01
Application #
8591712
Study Section
Special Emphasis Panel (ZCA1-RPRB-B (M2))
Project Start
2013-09-03
Project End
2018-08-31
Budget Start
2013-09-03
Budget End
2014-08-31
Support Year
1
Fiscal Year
2013
Total Cost
$580,534
Indirect Cost
$175,736
Name
University of Southern California
Department
Type
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
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
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
Oertell, Keriann; Harcourt, Emily M; Mohsen, Michael G et al. (2016) Kinetic selection vs. free energy of DNA base pairing in control of polymerase fidelity. Proc Natl Acad Sci U S A 113:E2277-85
Jeřábek, Petr; Florián, Jan; Martínek, Václav (2016) Lipid molecules can induce an opening of membrane-facing tunnels in cytochrome P450 1A2. Phys Chem Chem Phys 18:30344-30356
Hwang, Candy S; Kung, Alvin; Kashemirov, Boris A et al. (2015) 5'-β,γ-CHF-ATP diastereomers: synthesis and fluorine-mediated selective binding by c-Src protein kinase. Org Lett 17:1624-7
Kadina, Anastasia P; Kashemirov, Boris A; Oertell, Keriann et al. (2015) Two Scaffolds from Two Flips: (α,β)/(β,γ) CH2/NH ""Met-Im"" Analogues of dTTP. Org Lett 17:2586-9
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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