Molecular dynamics of a food carcinogen-DNA adduct in a replicative DNA polymerase suggest hindered nucleotide incorporation and extension.

Abstract:

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant of the carcinogenic heterocyclic aromatic amines in the human diet, and the major mutagenic effect of dietary PhIP is G-->T transversions. The major PhIP-derived DNA adduct is to C8 of guanine. We have investigated this adduct in a PhIP-induced mutational hotspot 5'-GGGA-3' of the Apc tumor suppressor gene, frequently mutated in mammalian colon tumors. We have carried out a molecular dynamics study to elucidate on a structural level nucleotide incorporation and extension opposite this major adduct during replication. The PhIP adduct was modeled into the ternary complex closed conformation of DNA polymerase RB69, at incorporation and extension positions, with normal cytosine or mismatched partner adenine. RB69 polymerase is a member of the B family as are most replicative eukaryotic DNA polymerases such as DNA polymerase alpha. These systems were subjected to molecular dynamics simulations with AMBER. Our results show that the adduct can reside on the major groove side of the modified DNA template opposite an incoming dCTP or dATP. In the case of the normal partner, disturbance to the active site is observed at the incorporation step, but there is less perturbance in the extension simulation. In the case of the mismatched partner, a less disturbed active site is observed during the incorporation step, but extension appears to be more difficult. Disturbances include adverse impacts on Watson-Crick hydrogen bonding in the nascent base pair, on the distance between the alpha-phosphate of the incoming dNTP and the primer terminus 3'-OH, and on critical protein interactions with the dNTP. However, in all of these cases, a near reaction ready distance (within 3.5 angstroms) between the 3'-terminal oxygen of the primer and the Palpha of the incoming nucleotide triphosphate is sampled occasionally (0.4-23.5% of the time). Thus, error-free bypass or the induction of a G-->T transversion mutation could occur at times and contribute to an extent to the mutagenic effect of PhIP. Polymerase stalling would be the more common outcome and in vivo could lead to switch to an error-prone bypass polymerase.

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