Role of DNA polymerase 3'----5' exonuclease activity in the bypass of aminofluorene lesions in DNA

Carcinogenesis. 1990 Dec;11(12):2103-9. doi: 10.1093/carcin/11.12.2103.


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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetoxyacetylaminofluorene / pharmacology*
  • Chromatography, High Pressure Liquid
  • DNA Damage*
  • DNA-Directed DNA Polymerase / pharmacology
  • Deoxyguanosine / analogs & derivatives*
  • Deoxyguanosine / pharmacology
  • Dose-Response Relationship, Drug
  • Electrophoresis, Agar Gel
  • Fluorenes / pharmacology*
  • Hydrogen-Ion Concentration
  • In Vitro Techniques
  • RNA-Directed DNA Polymerase / pharmacology
  • Time Factors
  • Transfection


  • Fluorenes
  • N-(deoxyguanosin-8-yl)-2-aminofluorene
  • Acetoxyacetylaminofluorene
  • bacteriophage T7 induced DNA polymerase
  • RNA-Directed DNA Polymerase
  • DNA-Directed DNA Polymerase
  • Deoxyguanosine