Evading the proofreading machinery of a replicative DNA polymerase: Induction of a mutation by an environmental carcinogen

DNA replication fidelity is dictated by DNA polymerase enzymes and associat ed proteins. When the template DNA is damaged by a carcinogen, the fidelity of DNA replication is sometimes compromized, allowing mispaired bases to p ersist and be incorporated into the DNA, resulting in a mutation. A key que stion in chemical carcinogenesis by metabolically activated polycyclic arom atic hydrocarbons (PAHs) is the nature of the interactions between the carc inogen-damaged DNA and the replicating polymerase protein that permits the mutagenic misincorporation to occur. PAHs are environmental carcinogens tha t, upon metabolic activation, can react with DNA to form bulky covalently l inked combination molecules known as carcinogen-DNA adducts. Benzo[a]pyrene (BP) is a common PAH found in a wide range of material ingested by humans, including cigarette smoke, car exhaust, broiled meats and fish, and as a c ontaminant in other foods. BP is metabolically activated into several highl y reactive intermediates, including the highly tumorigenic (+)-antibenzo[a] pyrene diol epoxide (BPDE). The primary product of the reaction of (+)-anti -BPDE with DNA, the (+)-trans-anti-benzo[a]pyrene diol epoxide-N-2-dG ((+)- ta-[BP]G) adduct, is the most mutagenic BP adduct in mammalian systems and primarily causes G-to-T transversion mutations, resulting from the mismatch of adenine with BP-damaged guanine during replication. In order to elucida te the structural characteristics and interactions between the DNA polymera se and carcinogen-damaged DNA that allow a misincorporation opposite a DNA lesion, we have modeled a (+)-ta-[BP]G adduct at a primer-template junction within the replicative phage T7 DNA polymerase containing an incoming dATP , the nucleotide most commonly mismatched with the (+)-ta-[BP]G adduct duri ng replication. A one nanosecond molecular dynamics simulation, using AMBER 5.0, has been carried out, and the resultant trajectory analyzed. The mode ling and simulation have revealed that a (+)-ta-[BP]G:A mismatch can be acc ommodated stably in the active site so that the fidelity mechanisms of the polymerase are evaded and the polymerase accepts the incoming mutagenic bas e. In this structure, the modified guanine base is in the syn conformation, with the BP moiety positioned in the major groove, without interfering wit h the normal protein-DNA interactions required for faithful polymerase func tion. This structure is stabilized by a hydrogen bond between the modified guanine base and dATP partner, hydrophobic interactions between the BP moie ty and the polymerase, a hydrogen bond between the modified guanine base an d the polymerase, and several hydrogen bonds between the BP moiety and poly merase side-chains. Moreover, the G:A mismatch in this system closely resem bles the size and shape of a normal Watson-Crick pair. These features revea l how the polymerase proofreading machinery may be evaded in the presence o f a mutagenic carcinogen-damaged DNA, so that a mismatch can be accommodate d readily, allowing bypass of the adduct by the replicative T7 DNA polymera se. (C) 2001 Academic Press.