Benzo[<i>a</i>]pyrene−DNA Adducts Inhibit the DNA Helicase Activity of the Bacteriophage T7 Gene 4 Protein

Abstract

The gene 4 protein of bacteriophage T7 provides the essential helicase and primase activities for the replication of the T7 genome. In addition, it also displays a DNA-dependent deoxyribonucleoside triphosphatase activity, the preferred substrate of which is dTTP. Previous investigations have demonstrated that the translocation of the gene 4 protein along single-stranded DNA is blocked by the presence of benzo[a]pyrene-DNA adducts and that the gene 4 protein is likely to be sequestered at the sites of these adducts. In the present study, we directly show that the helicase activity of the gene 4 protein is also profoundly inhibited by the benzo[a]pyrene-DNA adducts. The inhibitory effects of these adducts are strand-specific in that they block the DNA helicase activity of the gene 4 protein only when they are located in the DNA strand where the gene 4 protein translocates when it unwinds double-stranded DNA. Consistent with the hypothesis that the gene 4 protein is sequestered at the adduct site, we also show that the complexes formed by the gene 4 protein and benzo[a]pyrene-modified DNA are far more stable than those formed by the gene 4 protein and unmodified DNA.