Extending the understanding of mutagenicity: Structural insights into primer-extension past a benzo[a]pyrene diol epoxide-DNA adduct

Rebecca A. Perlow, Suse Broyde

Research output: Contribution to journalArticlepeer-review


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]-N2-dG adduct. Bulky adducts such as (+)-trans-anti-[BP]-N2-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]-N2-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]-N2-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]-N2-dG, we carried out molecular modeling and 1.25ns 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]-N2-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]-N2-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]-N2-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]-N2-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.

Original languageEnglish (US)
Pages (from-to)797-818
Number of pages22
JournalJournal of Molecular Biology
Issue number4
StatePublished - Apr 4 2003


  • Benzo[a]pyrene
  • Carcinogen-DNA adducts
  • DNA polymerase
  • Molecular dynamics
  • Mutagenicity

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


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