DNA Polymerases η and ζ Combine to Bypass O2-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens

A. S. Prakasha Gowda, Thomas E. Spratt

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4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) are important human carcinogens in tobacco products. They are metabolized to produce a variety 4-(3-pyridyl)-4-oxobutyl (POB) DNA adducts including O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dT), the most abundant POB adduct in NNK- and NNN-treated rodents. To evaluate the mutagenic properties of O2-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past O2-POB-dT and O2-Me-dT by purified human DNA polymerases η, κ, ι, and yeast polymerase ζ in vitro. Under conditions of polymerase in excess, polymerase η was most effective at the insertion of dNTPs opposite O2-alkyl-dTs. The time courses were biphasic suggesting the formation of inactive DNA-polymerase complexes. The kpol parameter was reduced approximately 100-fold in the presence of the adduct for pol η, κ, and ι. Pol η was the most reactive polymerase for the adducts due to a higher burst amplitude. For all three polymerases, the nucleotide preference was dATP > dTTP 蠑 dGTP and dCTP. Yeast pol ζ was most effective in bypassing the adducts; the kcat/Km values were reduced only 3-fold in the presence of the adducts. The identity of the nucleotide opposite the O2-alkyl-dT did not significantly affect the ability of pol ζ to bypass the adducts. The data support a model in which pol η inserts ATP or dTTP opposite O2-POB-dT, and then, pol ζ extends past the adduct.

Original languageEnglish (US)
Pages (from-to)303-316
Number of pages14
JournalChemical research in toxicology
Issue number3
StatePublished - Mar 21 2016

All Science Journal Classification (ASJC) codes

  • Toxicology


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