SUNY at Albany
June 19-23, 2001
The Making of a Mutation: "Tricking" T7 DNA Polymerase into Accepting a Mismatched dATP Opposite a Carcinogen Damaged Guanine in the Active Site
DNA polymerases govern the fidelity of DNA replication, and replicative DNA polymerases in different organisms share common structural motifs. Certain residues within polymerases are involved in maintaining replication fidelity. When the template DNA is damaged by a carcinogen, the fidelity of DNA replication is sometimes compromised, allowing mispaired bases to persist and be incorporated into the DNA, resulting in a mutation. A key question in chemical carcinogenesis by activated polycyclic aromatic hydrocarbons (PAHs) is the nature of the interaction between the DNA adduct and replicating polymerase that permits the mutagenic misincorporation to occur. The environmental PAH, benzo[a]pyrene (BP) is metabolized to a highly reactive, tumorigenic derivative, (+)-anti-benzo[a]pyrene diol epoxide (BPDE). (+)-anti-BPDE attacks DNA and forms a major covalent adduct at the N2 position of guanine by trans epoxide opening, the (+)-trans-anti-N2-[BP]-dG adduct ((+)-ta-BP-dG). In order to elucidate the structural characteristics of the DNA polymerase/DNA adduct interactions that allow a misincorporation opposite a DNA lesion, we have modeled the (+)-ta-BP-dG adduct at a model replication fork within the replicative T7 DNA polymerase containing a mismatched incoming dATP, the nucleotide most commonly mismatched with these adducts. A one nanosecond molecular dynamics simulation, using AMBER 5.0, has been completed, and the resultant trajectory analyzed. The modeling and simulation have revealed that a (+)-ta-BP-dG:A mismatch can be stably accommodated in the active site so that the fidelity mechanisms of the polymerase can be "tricked" into accepting the mutagenic partner. In this structure, the modified guanine is in the syn conformation, with the BP moiety positioned in the major groove, without interfering with the normal protein-DNA interactions required for faithful polymerase function. During the simulation, several stabilizing interactions develop, including, a hydrogen bond between the modified guanine and dATP partner, hydrophobic interactions between the BP moiety and the polymerase, and a hydrogen bond between the modified guanine and the polymerase.
Rebecca Perlow and Suse Broyde
Department of Biology, New York University,