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

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

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
Volume29
Issue number3
DOIs
StatePublished - Mar 21 2016

Fingerprint

N'-nitrosonornicotine
Thymine
Tobacco
DNA Adducts
DNA-Directed DNA Polymerase
Carcinogens
Yeast
Nucleotides
Yeasts
Tobacco Products
Rodentia
Adenosine Triphosphate
thymidine 5'-triphosphate

All Science Journal Classification (ASJC) codes

  • Toxicology

Cite this

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title = "DNA Polymerases η and ζ Combine to Bypass O2-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens",
abstract = "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.",
author = "{Prakasha Gowda}, {A. S.} and Spratt, {Thomas E.}",
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DNA Polymerases η and ζ Combine to Bypass O2-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens. / Prakasha Gowda, A. S.; Spratt, Thomas E.

In: Chemical research in toxicology, Vol. 29, No. 3, 21.03.2016, p. 303-316.

Research output: Contribution to journalArticle

TY - JOUR

T1 - DNA Polymerases η and ζ Combine to Bypass O2-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens

AU - Prakasha Gowda, A. S.

AU - Spratt, Thomas E.

PY - 2016/3/21

Y1 - 2016/3/21

N2 - 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.

AB - 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.

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