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 > or = C) differed from that when the adduct and partner were the nascent base-pair (A > T > or = 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.
Perlow
Rebecca A
RA
Department of Biology, New York University, 100 Washington Square East, New York, NY 10003, USA.
Broyde
Suse
S
D017123:Bacteriophage T7 / Q000201:enzymology; D020137:Base Pair Mismatch / Q000235:genetics; D020029:Base Pairing; D001564:Benzo(a)pyrene / Q000378:metabolism* / Q000494:pharmacology; D002274:Carcinogens, Environmental / Q000378:metabolism / Q000494:pharmacology; D003198:Computer Simulation; D018736:DNA Adducts / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004852:Epoxy Compounds / Q000737:chemistry*; D006147:Guanine / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D012997:Solvents / Q000737:chemistry; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
D002274:Carcinogens, Environmental; D018736:DNA Adducts; D017931:DNA Primers; D004852:Epoxy Compounds; D009711:Nucleotides; D012997:Solvents; D001564:Benzo(a)pyrene; D006147:Guanine; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium