Low fidelity bypass of O2-(3-pyridyl)-4-oxobutylthymine, the most persistent bulky adduct produced by the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by model polymerases.


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 <italic>O</italic><sup>2</sup>-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (<italic>O</italic><sup>2</sup>-POB-dT) because it is the most abundant POB adduct in NNK-treated rodents. To evaluate the mutagenic properties of <italic>O</italic><sup>2</sup>-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past both <italic>O</italic><sup>2</sup>-POB-dT and <italic>O</italic><sup>2</sup>-methylthymidine (<italic>O</italic><sup>2</sup>-Me-dT) by two model polymerases, <italic>E. coli</italic> DNA polymerase I (Klenow fragment) with the proofreading exonuclease activity inactivated (Kf) and <italic>Sulfolobus solfataricus</italic> DNA polymerase IV (Dpo4). We found that the size of the alkyl chain only marginally affect 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 ~10<sup>6</sup>-fold when compared to undamaged DNA. Dpo4 catalyzed the incorporation opposite and extension past the adducts approximately 1000-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 <italic>O</italic><sup>2</sup>-Me-dT and dT the preferred base pair partner for <italic>O</italic><sup>2</sup>-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.



Nucleotide Analogs / Template Lesions


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