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.
Strauss
B S
BS
Department of Molecular Genetics and Cell Biology, University of Chicago, IL 60637.
Wang
J
J
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