Poster abstracts

Poster number 62 submitted by Andrew Reed

Insights into DNA Polymerase β Nucleotide Stereoselectivity and Post-chemistry Events

Andrew J. Reed (OSBP), Rajan Vyas (Chem and Biochem), Austin T. Raper (OSBP), Walter J. Zahurancik (OSBP), Petra C. Wallenmeyer (Chem and Biochem)

Abstract:
Antivirals with L-stereochemistry are an effective treatment for viral infections presumably due to the strong D-stereoselectivity exhibited by human DNA polymerases relative to viral reverse transcriptases. However, the D-stereoselectivity of DNA polymerases has only recently been explored structurally with all polymerases studied exhibiting unique selectivity strategies. To further understand toxicities associated with these treatments we have solved structures of human DNA polymerase β (hPolβ), in complex with DNA and the antiviral drugs lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP) and performed pre-steady-state kinetic analysis to determine the D-stereoselectivity mechanism of hPolβ. Overall, we have determined that hPolβ discriminates against L-stereochemistry through accumulation of several active site rearrangements that lead to a decreased nucleotide binding affinity and incorporation rate. In addition, while the mechanism of nucleotide incorporation by DNA polymerases has been extensively studied, key steps following phosphodiester bond formation remain structurally uncharacterized. For these steps, it is thought that the release of pyrophosphate (PPi) triggers reverse conformational changes in the polymerase in order to complete a full catalytic cycle and allow subsequent incorporation events. Fortuitously, by using these L-nucleotides, we have structurally revealed the correct sequence of post-chemistry steps during nucleotide incorporation by hPolβ and provide a structural basis for PPi release. These post-catalytic structures reveal hPolβ in an open conformation with PPi bound, thereby strongly suggesting that the reverse conformational changes occur prior to PPi release. Furthermore, our structures suggest the recently discovered third metal ion does not participate in product release but rather the next correct nucleotide stimulates PPi dissociation

Keywords: DNA Polymerase, Antiviral Drugs, Structure