Biochemical investigations into the incorporation of 8-oxo-2′-deoxyguanosine-5′-triphosphate with two A-family polymerases

Abstract

8-Oxo-2′-deoxyguanosine-5′-triphosphate (OdGTP) is a promutagenic oxidatively damaged nucleotide that can base pair to both 2′-deoxycytidine (dC) and 2′-deoxyadenosine (dA) and may play a role in antibiotic initiated bacterial cell death. We evaluated the insertion of OdGTP, dGTP, and eight related analogs opposite dC and dA with two A-family bacterial replicative polymerases, nuclease deficient Polymerase I from E. coli (KF-exo) and Geobacillus Stearothermophilus (BF). Results from these studies demonstrate that KF-exo is much less likely than BF to incorporate OdGTP opposite either dC or dA as compared to incorporation of dGTP opposite dC. Our work also highlights similarities and differences in the active sites that can occur between two polymerases within the same family. For example, the C2-amine of OdGTP appears to have little impact during incorporation opposite dA with either enzyme. However, when inserting opposite dC, the C2-amine can form a stabilizing hydrogen bond to the template dC, and increase activity, especially when large atoms are present at the C8-position. KF-exo is more sensitive than BF to large substituents at the C8-position during incorporation opposite dC, while BF, but not KF-exo, likely forms a minor groove hydrogen bond to the C8‑oxygen during incorporation opposite dA. Overall, these findings provide mechanistic insights into how oxidative stress can contribute to genomic instability through polymerase-mediated misincorporation events.