Role of DNA polymerase 3′→ 5′ exonuclease activity in the bypass of aminofluorene lesions in DNA

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Abstract
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 mm 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 ideiitical 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 elonga tion. T7 DNA polymerase, including thioredoxin and with an active 3′ → 5′ exonudease, 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.

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