Low fidelity bypass of O(2)-(3-pyridyl)-4-oxobutylthymine, the most persistent bulky adduct produced by the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by model DNA polymerases

Chem Res Toxicol. 2012 Jun 18;25(6):1195-202. doi: 10.1021/tx200483g. Epub 2012 May 1.


4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most important human carcinogens. It is metabolized to produce a variety of methyl and 4-(3-pyridyl)-4-oxo-butyl (POB) DNA adducts. A potentially important POB adduct is O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT) because it is the most abundant POB adduct in NNK-treated rodents. To evaluate the mutagenic properties of O(2)-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past both O(2)-POB-dT and O(2)-methylthymidine (O(2)-Me-dT) by two model polymerases, E. coli DNA polymerase I (Klenow fragment) with the proofreading exonuclease activity inactivated (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4). We found that the size of the alkyl chain only marginally affected the reactivity and that the specificity of adduct bypass was very low. The k(cat)/K(m) for the Kf catalyzed incorporation opposite and extension past the adducts was reduced ∼10(6)-fold when compared to undamaged DNA. Dpo4 catalyzed the incorporation opposite and extension past the adducts approximately 10(3)-fold more slowly than undamaged DNA. The dNTP specificity was less for Dpo4 than for Kf. In general, dA was the preferred base pair partner for O(2)-Me-dT and dT the preferred base pair partner for O(2)-POB-dT. With enzyme in excess over DNA, the time courses of the reactions showed a biphasic kinetics that indicates the formation inactive binary and ternary complexes.

Publication types

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

MeSH terms

  • DNA / chemistry
  • DNA / metabolism
  • DNA Polymerase I / chemistry
  • DNA Polymerase I / metabolism*
  • DNA Polymerase beta / chemistry
  • DNA Polymerase beta / metabolism*
  • Escherichia coli / enzymology
  • Models, Biological*
  • Molecular Structure
  • Nitrosamines / chemistry
  • Nitrosamines / metabolism*
  • Sulfolobus solfataricus / enzymology
  • Thymine / analogs & derivatives*
  • Thymine / chemistry
  • Thymine / metabolism
  • Tobacco / chemistry*


  • Nitrosamines
  • O(2)-(3-pyridyl)-4-oxobutylthymine
  • 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone
  • DNA
  • DNA Polymerase I
  • DNA Polymerase beta
  • Thymine