Extending the Understanding of Mutagenicity: Structural Insights into Primer-extension Past a Benzo[ a]pyrene Diol Epoxide-DNA adduct

DNA polymerase enzymes employ a number of innate fidelity mechanisms to ensure the faithful replication of the genome. However, when confronted with DNA damage, their fidelity mechanisms can be evaded, resulting in a mutation that may contribute to the carcinogenic process. The environmental carcinogen benzo[ a]pyrene is metabolically activated to reactive intermediates, including the tumorigenic (+)- anti-benzo[ a]pyrene diol epoxide, which can attack DNA at the exocyclic amino group of guanine to form the major (+)- trans- anti-[BP]- N 2-dG adduct. Bulky adducts such as (+)- trans- anti-[BP]- N 2-dG primarily block DNA replication, but are occasionally bypassed and cause mutations if paired with an incorrect base. In vitro standing-start primer-extension assays show that the preferential insertion of A opposite (+)- trans- anti-[BP]- N 2-dG is independent of the sequence context, but the primer is extended preferentially when dT is positioned opposite the damaged base in a 5′-CG ∗T-3′ sequence context. Regardless of the base positioned opposite (+)- trans- anti-[BP]- N 2-dG, extension of the primer past the lesion site poses the greatest block to polymerase progression. In order to gain insight into primer-extension of each base opposite (+)- trans- anti-[BP]- N 2-dG, we carried out molecular modeling and 1.25 ns unrestrained molecular dynamics simulations of the adduct in the +1 position of the template within the replicative pol I family T7 DNA polymerase. Each of the four bases was modeled at the 3′ terminus of the primer, incorporated opposite the adduct, and the next-to-be replicated base was in the active site with its Watson–Crick partner as the incoming nucleotide. As in our studies of nucleotide incorporation, (+)- trans- anti-[BP]- N 2-dG was modeled in the syn conformation in the +1 position, with the BP moiety on the open major groove side of the primer–template duplex region, leaving critical protein–DNA interactions intact. The present work revealed that the efficiency of primer-extension past this bulky adduct opposite each of the four bases in the 5′-CG ∗T-3′ sequence can be rationalized by the stability of interactions between the polymerase protein, primer–template DNA and incoming nucleotide. However, the relative stabilization of each nucleotide opposite (+)- trans- anti-[BP]- N 2-dG in the +1 position (T>G>A≥C) differed from that when the adduct and partner were the nascent base-pair (A>T≥G>C). In addition, extension past (+)- trans-