title: DNA Polymerases η and ζ Combine to Bypass O(2)-[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 O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT), the most abundant POB adduct in NNK- and NNN-treated rodents. To evaluate the mutagenic properties of O(2)-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past O(2)-POB-dT and O(2)-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 O(2)-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 ≫ 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 O(2)-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 O(2)-POB-dT, and then, pol ζ extends past the adduct.
authors: Department of Biochemistry and Molecular Biology Penn State Hershey Cancer Institute, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine , Hershey, Pennsylvania 17033, United States.
authors: A S Prakasha
authors: AS
authors: Gowda
authors: Department of Biochemistry and Molecular Biology Penn State Hershey Cancer Institute, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine , Hershey, Pennsylvania 17033, United States.
authors: Thomas E
authors: TE
authors: Spratt
mesh_terms: D002273:Carcinogens / Q000737:chemistry*; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism*; D013941:Thymine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D014026:Nicotiana / Q000737:chemistry*
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; C000613692:O(2)-(4-(3-pyridyl)-4-oxobutyl)thymine; D011725:Pyridines; D004259:DNA-Directed DNA Polymerase; D013941:Thymine