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

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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. .

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Program--Cooperative Agreements (U19)
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Special Emphasis Panel (ZCA1-RPRB-B (M2))
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University of Southern California
Los Angeles
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