Fidelity of DNA polymerases is predominantly governed by an induced fit mechanism in which the incoming dNTP in the ternary complex fits tightly into a binding pocket whose geometry is determined by the nature of the templating base. However, modification of the template with a bulky carcinogen may alter the dNTP binding pocket and thereby the polymerase incorporation fidelity. High fidelity DNA polymerases, such as bacteriophage T7 DNA polymerase, are predominantly blocked by bulky chemical lesions on the template strand during DNA replication. However, some mutagenic bypass can occur, which may lead to carcinogenesis. Experimental studies have shown that a DNA covalent adduct derived from (+)-anti-BPDE [(+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene], a carcinogenic metabolite of benzo[a]pyrene (BP), primarily blocks Sequenase 2.0, an exo- T7 DNA polymerase; however, a mismatched dATP can be preferentially inserted opposite the damaged adenine templating base within the active site of the polymerase [Chary, P., and Lloyd, R. S. (1995) Nucleic Acids Res. 23, 1398-1405]. The goal of this work is to elucidate structural features that contribute to DNA polymerase incorporation fidelity in the presence of this bulky covalent adduct and to interpret the experimental findings on a molecular level. We have carried out molecular modeling and molecular dynamics simulations with AMBER 6.0, investigating a T7 DNA polymerase primer-template closed ternary complex containing this 10S (+)-trans-anti-[BP]-N6-dA adduct in the templating position within the polymerase active site. All four incoming dNTPs were studied. The simulations show that the BP ring system fits well into an open pocket on the major groove side of the modified template adenine with anti glycosidic bond conformation, without disturbing critical polymerase-DNA interactions. However, steric hindrance between the BP ring system and the primer-template DNA causes displacement of the modified template adenine, so that the dNTP base binding pocket is enlarged. This alteration can explain the experimentally observed preference for incorporation of dATP opposite this lesion. These studies also rationalize the observed lower probabilities of incorporation of the other three nucleotides. Our results suggest that the differences in incorporation of dGTP, dCTP, and dTTP are due to the effects of imperfect geometric complementarity. Thus, the simulations suggest that altered DNA polymerase incorporation fidelity can result from adduct-induced changes in the dNTP base binding pocket geometry. Furthermore, plausible structural explanations for the observed effects of [BP]-N6-dA adduct stereochemistry on the observed stalling patterns are proposed.
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