Abstract
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- anti-[BP]- N 2-dG may pose a greater block to a high fidelity DNA polymerase than does nucleotide incorporation opposite the adduct because the presence of the modified base-pair in the +1 position is more disruptive to the polymerase–DNA interactions than it is within the active site itself. The dN:(+)- trans- anti-[BP]- N 2-dG base-pair is strained to shield the bulky aromatic BP moiety from contact with the solvent in the +1 position, causing disruption of protein–DNA interactions that would likely result in decreased extension of the base-pair. These studies reveal in molecular detail the kinds of specific structural interactions that determine the function of a processive DNA polymerase when challenged by a bulky DNA adduct.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.