Translesional synthesis on a DNA template containing a single stereoisomer of dG-(+)- or dG-(-)-anti-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene).
Oligodeoxynucleotides modified site-specifically with dG-(+)-trans- and dG-(+)-cis-anti-BPDE (7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) or dG-(-)-trans- and dG-(-)-cis-anti-BPDE were used as templates in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. The primer could be extended past the dG-(-)-trans-BPDE adduct with small amounts of dAMP incorporated opposite the lesion. A small amount of base deletions was also observed while, with the dG-(-)-cis-BPDE adduct, one- and two-base deletions predominated. When templates containing dG-(+)-trans-BPDE were used, small amounts of products containing one-base deletions were observed; with dG-(+)-cis-BPDE, substitution of dAMP opposite the lesion was also detected. The frequency of nucleotide insertion for dAMP opposite dG-(-)-trans-BPDE and the frequency of extension from the primer terminus containing the dA:dG-(-)-trans-BPDE pair were much higher than those observed with the other, stereochemically different BPDE adducts. Kinetic studies were in agreement with the results of the primer extension study. When the base flanking the 5' side of dG-BPDE was changed from dC to dT, the frequency of one-base deletions increased. We conclude that the trans- or cis-addition product of dG-(-)-anti-BPDE has a higher miscoding potential than dG-(+)-anti-BPDE in our model system and that G-->T transversions and deletions predominate. These observations are consistent with the types of mutations observed in vivo.
Shibutani
S
S
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651.
Margulis
L A
LA
Geacintov
N E
NE
Grollman
A P
AP
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004249:DNA Damage; D004256:DNA Polymerase I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D017353:Gene Deletion; D007700:Kinetics; D008969:Molecular Sequence Data; D013237:Stereoisomerism; D013698:Templates, Genetic
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA; D004256:DNA Polymerase I