Inhibition of Rad3 DNA helicase activity by DNA adducts and abasic sites: implications for the role of a DNA helicase in damage-specific incision of DNA.
The yeast nucleotide excision repair gene RAD3 is absolutely required for damage-specific incision of DNA. Rad3 protein is a DNA helicase, and previous studies have shown that its catalytic activity is inhibited by ultraviolet (UV) radiation damage. This inhibition is observed when base damage is confined to the DNA strand on which Rad3 protein binds and translocates, and not when damage is present exclusively on the complementary strand. In the present study, we show that Rad3 DNA helicase activity is inhibited in an identical strand-specific fashion by bulky base adducts formed by treating DNA with the antineoplastic agent cisplatin or the antibiotic compound CC-1065, which alter the secondary structure of DNA in different ways. In addition, Rad3 helicase activity is inhibited by small adducts generated by treatment of DNA with diethyl sulfate and by the presence of sites at which pyrimidines have been lost (abasic sites). No inhibition of Rad3 helicase activity was detected when DNA was methylated at various base positions. Cisplatin-modified single-stranded DNA and poly(deoxyuridylic acid) containing abasic sites are more effective competitors for Rad3 helicase activity than their undamaged counterparts, suggesting that Rad3 protein is sequestered at such lesions, resulting in the formation of stable Rad3 protein-DNA complexes. The observations of strand-specific inhibition of Rad3 helicase activity and the formation of stable complexes with the covalently modified strand suggest a general mechanism by which the RAD3 gene product may be involved in nucleotide excision repair in yeast.
Department of Pathology, University of Texas Southwestern Medical Center, Dallas 75235.
H
H
Naegeli
L
L
Bardwell
E C
EC
Friedberg
D000225:Adenine / Q000031:analogs & derivatives; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D002945:Cisplatin; D004247:DNA / Q000737:chemistry / Q000187:drug effects; D018736:DNA Adducts; D004249:DNA Damage; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004260:DNA Repair; D004054:Diethylstilbestrol / Q000737:chemistry; D000080890:Duocarmycins; D007211:Indoles; D007933:Leucomycins; D008745:Methylation; D009690:Nucleic Acid Conformation; D029701:Saccharomyces cerevisiae Proteins
D000903:Antibiotics, Antineoplastic; C055641:CC-1065-(N(3)-adenine)-DNA; D018736:DNA Adducts; D000080890:Duocarmycins; D007211:Indoles; D007933:Leucomycins; D029701:Saccharomyces cerevisiae Proteins; C057270:cisplatin-DNA adduct; D004054:Diethylstilbestrol; D004247:DNA; D000251:Adenosine Triphosphatases; C061897:Rad3 protein, S cerevisiae; D004265:DNA Helicases; D000225:Adenine; D002945:Cisplatin