Polymerase blockage and misincorporation of dNTPs opposite the ethylene dibromide-derived DNA adducts S-[2-(N7-guanyl)ethyl]glutathione, S-[2-(N2-guanyl)ethyl]glutathione, and S-[2-(O6-guanyl)ethyl]glutathione.
The carcinogen ethylene dibromide (EDB) has been shown to cause glutathione (GSH)-dependent base-substitution mutations, especially GC to AT transitions, in a variety of bacterial and eukaryotic systems. The known DNA adducts S-[2-(N7-guanyl)ethyl]GSH, S-[2-(N2-guanyl)ethyl]GSH, and S-[2-(O6-guanyl)ethyl]GSH were individually placed at a site in a single oligonucleotide. Polymerase extension studies were carried out using Escherichia coli polymerase I exo- (Klenow fragment, Kf-) and polymerase II exo- (pol II-), bacteriophage T7 polymerase exo-, and human immunodeficiency virus-1 reverse transcriptase in order to characterize misincorporation events. Even though extension was not as efficient as with the nonadducted template, some fully extended primers were observed with the template containing S-[2-(N7-guanyl)ethyl]GSH using all of these polymerases. dCTP was the most preferred nucleotide incorporated opposite S-[2-(N7-guanyl)ethyl]GSH by most of polymerases examined; however, dTTP incorporation was observed opposite S-[2-(N7-guanyl)ethyl]GSH with pol II-. Both S-[2-(N2-guanyl)ethyl]GSH and S-[2-(O6-guanyl)ethyl]GSH strongly blocked replication by all polymerases. Only dATP and dGTP were incorporated opposite S-[2-(N2-guanyl)ethyl]GSH by both Kf- and pol II-. S-[2-(O6-Guanyl)ethyl]GSH was shown to strongly code for dATP incorporation by Kf-. With pol II-, dTTP was incorporated opposite S-[2-(O6-guanyl)ethyl]GSH. In conclusion, all three GSH-guanyl adducts derived from the carcinogen EDB blocked the polymerases and were capable of miscoding.
Kim
M S
MS
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Guengerich
F P
FP
D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D015946:Ethylene Dibromide / Q000378:metabolism*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism
D018736:DNA Adducts; C112926:S-(2-(N(2)-guanyl)ethyl)glutathione; C112925:S-(2-(O(6)-guanyl)ethyl)glutathione; D015946:Ethylene Dibromide; C038886:S-(2-(N(7)-guanyl)ethyl)glutathione; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005978:Glutathione