Benzo[a]pyrene diol epoxide–deoxyguanosine adducts are accurately bypassed by yeast DNA polymerase ζ in vitro

Abstract The possible role of bypass DNA polymerase ζ in mutagenic translesion synthesis past benzo[ a ]pyrene (BP) 7,8-diol-9,10-epoxide (DE) N 2 -deoxyguanosine (dG) adducts has been examined. We prepared 59-mer DNA templates containing dG adducts derived from trans opening of enantiomers of BP DE-2, in which the 7-hydroxyl group and epoxide oxygen are trans . The 10 S -BP DE–dG and 10 R -BP DE–dG adducts derive from the (+)- and (−)-DE-2 enantiomers, respectively. The adducted dG is located at a site identified as a G→T mutational hotspot in random mutagenesis studies of (+)-BP DE-2 in Chinese hamster V-79 cells. Yeast pol ζ (complex of Gst-Rev3p and Rev7p) formed extension products (total of all lengths) of 71, 74 and 88% of a primer annealed to the 10 S -BP DE–dG, 10 R -BP DE–dG and non-adducted 59-mer templates, respectively. However, only 18 and 19% of the primer was extended to the full-length product on 10 S -BP DE–dG and 10 R -BP DE–dG adducted templates compared to 55% of the primer on the non-adducted template. A major 34-mer product corresponding to primer elongation up to and including the base before the adduct indicated that nucleotide incorporation opposite both adducts was strongly blocked. Full-length products were isolated from gels and subjected to PCR amplification and cloning. Sequence analysis of more than 300 clones of these full-length products on each template showed that only the correct dCMP was incorporated opposite both the adducted and non-adducted G-hotspot in the template. This corresponds to a probability of mutation lower than 0.3%, the limit of detection, and demonstrates the remarkable fidelity of yeast pol ζ in translesion synthesis past these BP DB–dG lesions in vitro.