Benzo[a]pyrene diol epoxide-deoxyguanosine adducts are accurately bypassed by yeast DNA polymerase zeta in vitro.
The possible role of bypass DNA polymerase zeta 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 10S-BP DE-dG and 10R-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 zeta (complex of Gst-Rev3p and Rev7p) formed extension products (total of all lengths) of 71, 74 and 88% of a primer annealed to the 10S-BP DE-dG, 10R-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 10S-BP DE-dG and 10R-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 zeta in translesion synthesis past these BP DB-dG lesions in vitro.
Simhadri
Srilatha
S
Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA.
Kramata
Pavel
P
Zajc
Barbara
B
Sayer
Jane M
JM
Jerina
Donald M
DM
Hinkle
David C
DC
Wei
Caroline S-J
CS
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D003849:Deoxyguanosine / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D013698:Templates, Genetic; D015003:Yeasts / Q000201:enzymology*; D000099270:Y-Family DNA Polymerases
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D018736:DNA Adducts; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D005656:Fungal Proteins; D007041:Hypoxanthine Phosphoribosyltransferase; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases