Toward Understanding the Mutagenicity of an Environmental Carcinogen: Structural Insights into Nucleotide Incorporation Preferences

Bulky carcinogen–DNA adducts, including (+)- trans- anti-[BP]- N 2-dG derived from the reaction of (+)- anti-benzo[ a]pyrene diol epoxide with guanine, often block the progression of DNA polymerases. However, when rare bypass of the lesions does occur, they may be misreplicated. Experimental results have shown that nucleotides are inserted opposite the (+)- trans- anti-[BP]- N 2-dG adduct by bacteriophage T7 DNA polymerase with the order of preference A>T≥G>C. To gain structural insights into the effects of the bulky adduct on nucleotide incorporation within the polymerase active site, molecular modeling and molecular dynamics simulations were carried out using T7 DNA polymerase to permit the relation of function to structure. We modeled the (+)- trans- anti-[BP]- N 2-dG adduct opposite incoming dGTP, dTTP and dCTP nucleotides, as well as unmodified guanine opposite its normal partner dCTP as a control, to compare with our previous simulation with dATP opposite the adduct. The modeling required that the (+)- trans- anti-[BP]- N 2-dG adduct adopt the syn conformation in each case to avoid deranging essential protein–DNA interactions. While the dATP: (+)- trans- anti-[BP]- N 2-dG pair was well accommodated within the active site of T7 DNA polymerase, dCTP fit poorly opposite the adduct, adopting an orientation perpendicular to the plane of the syn modified guanine during the simulation. Rotation about the glycosidic bond of the dCTP residue to this abnormal position was allowed because only one hydrogen bond between dCTP and the (+)- trans- anti-[BP]- N 2-dG residue evolved during the simulation, and this hydrogen bond was directly across from the dCTP glycosidic bond. The dTTP and dGTP nucleotides, incorporated with an intermediate preference opposite (+)- trans- anti-[BP]- N 2-dG, were accommodated reasonably well, but not as stably as the dATP nucleotide, due to a skewed primer-template alignment and more exposed BP moiety, respectively. In addition, the extent of stabilizing interactions between the nascent base-pair in each simulation was correlated positively with the incorporation preference of that particular nucleotide. The dATP nucleotide is accommodated most stably opposite the adduct, with protein–DNA hydrogen bonding interactions and an active-site pocket size that do not deviate significantly from those of the control simulation. The simulations of dTTP and dGTP opposite (+)- trans- anti-[BP]- N 2-dG exhibited more instability in interactions b