Role of DNA polymerase 3'----5' exonuclease activity in the bypass of aminofluorene lesions in DNA.
N-(Deoxyguanosin-8-yl)-2-(acetylamino)fluorene (AAF-G) adducts in the DNA of bacteriophage M13 can be converted to N-(deoxyguanosin-8-yl)-2-aminofluorene (AF-G) adducts in situ by treatment with 1.0 M NaOH for 45 min at room temperature. The conversion is accompanied by a dramatic increase in the transfection activity of the samples which is correlated with the measured deacetylation of the acetylaminofluorene adduct. The pair of substrates (AAF-G/AF-G) with adducts at identical places in the DNA has been used to study bypass synthesis catalyzed by T7 DNA polymerase, an altered T7 DNA polymerase from which the 3'----5' exonuclease has been genetically removed by an 84 nucleotide deletion (Sequenase 2), T4 DNA polymerase and Escherichia coli DNA polymerase I. All polymerases appear blocked at acetylaminofluorene lesions. Sequenase 2 is apparently able to add nucleotides opposite the acetylaminofluorene lesion but is unable to catalyze further elongation. T7 DNA polymerase, including thioredoxin and with an active 3'----5' exonuclease, is unable to bypass aminofluorene adducts, whereas Sequenase 2 bypasses the lesions readily. The data support the view that the elongation step is rate limiting in synthesis past lesions and that low 3'----5' exonuclease activity allows the priming nucleotide opposite the altered template site to remain in position long enough for elongation past particular adducts.
Department of Molecular Genetics and Cell Biology, University of Chicago, IL 60637.
B S
BS
Strauss
J
J
Wang
D000099:Acetoxyacetylaminofluorene / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000494:pharmacology; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D005449:Fluorenes / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D012194:RNA-Directed DNA Polymerase / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D005449:Fluorenes; C031838:N-(deoxyguanosin-8-yl)-2-aminofluorene; D000099:Acetoxyacetylaminofluorene; C034970:bacteriophage T7 induced DNA polymerase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine