trans-lesion synthesis past bulky benzo[a]pyrene diol epoxide N2-dG and N6-dA lesions catalyzed by DNA bypass polymerases

The effectiveness of in vitro primer elongation reactions catalyzed by human bypass DNA polymerases kappa (hDinB1), pol eta (hRad30A), pol iota (hRad30B), and yeast pol zeta (Rev3 and Rev7) in site-specifically modified template oligonucleotide strands were studied in vitro. The templates contained single bulky lesions derived from the trans-addition of the mutagenic (+)- or (-)-enantiomers of r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (a metabolite of the environmental carcinogen benzo[a]pyrene), to the exocyclic amino groups of guanine or adenine in oligonucleotide templates 33, or more, bases long. In "running start" primer extension reactions, pol kappa effectively bypassed both the stereoisomeric (+)- and (-)-trans-guanine adducts but not the analogous adenine adducts. In sharp contrast, pol eta, which exhibits considerable sequence homology with pol kappa (both belong to the group of Y family polymerases), is partially blocked by the guanine adducts and the (-)-trans-adenine adduct, although the stereoisomeric (+)-trans-adenine adduct is more successfully bypassed. Neither pol iota nor pol zeta, either alone or in combination, were effective in trans-lesion synthesis past the same adducts. In all cases, the fidelity of insertion is dependent on adduct stereochemistry and structure. Generally, error-free nucleotide insertion opposite the lesions tends to depend more on adduct stereochemistry than error-prone insertion. None of the polymerases tested are a universal bypass polymerase for the stereoisomeric bulky polycyclic aromatic hydrocarbon-DNA adducts derived from anti-BPDE.