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

A. S.Prakasha Gowda, Krishne Gowda, Zucai Suo, Shantu Amin, Thomas Spratt

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15 Scopus citations

Abstract

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 O2-[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 O2-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past both O 2-POB-dT and O2-methylthymidine (O2-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 kcat/Km for the Kf catalyzed incorporation opposite and extension past the adducts was reduced ∼106-fold when compared to undamaged DNA. Dpo4 catalyzed the incorporation opposite and extension past the adducts approximately 103-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 O2-Me-dT and dT the preferred base pair partner for O2-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.

Original languageEnglish (US)
Pages (from-to)1195-1202
Number of pages8
JournalChemical Research in Toxicology
Volume25
Issue number6
DOIs
StatePublished - Jun 18 2012

All Science Journal Classification (ASJC) codes

  • Toxicology

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