The repair of the tobacco specific nitrosamine derived adduct O-6-[4-oxo-4-(3-pyridyl)butyl]guanine by O-6-alkylguanine-DNA alkyltransferase variants

The tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent pulmonary carcinogen, both methylates and pyridyloxobutylates DNA. Both reaction pathways generate promutagenic O-6-alkylguanine adducts. These adducts, O-6-methylguanine (O-6-mG) and O-6- [4-oxo-4-(3-pyridyl)butyl] guanine (O-6-pobG), are repaired by O-6-alkylguanineDNA alkyltransferase (AGT). In this report, we demonstrate that pyridyloxobutyl DNA adducts are repaired by AGT in a reaction that results in pyridyloxobutyl transfer to the active site cysteine. Because minor changes within the binding pocket of AGT can alter the ability of this protein to repair bulky O-6-alkylguanine adducts relative to O-6-mG, we explored the ability of AGTs from different species as well as several human AGT variants and mutants to discriminate between O-6-mG or O-6-pobG adducts. We incubated proteins with equal molar amounts of oligodeoxynucleotides containing site specifically incorporated O-6-mG or O-6-pobG and measured repair. Bacterial AGTs poorly repaired O-6-pobG. Mouse and rat AGT repaired both adducts at comparable rates. Wild-type human AGT, variant I143V/K178R, and mutant N157H repaired O-6-mG approximately twice as fast as O-6-pobG. Human variant G160R and mutants P140K, Y158H, G156A, and E166G did not repair O-6-pobG until all of the O-6-mG was removed. To understand the role of adduct structure on relative repair rates, the competition experiments were repeated with two other bulky O-6-alkylguanine adducts, O-6-butylguanine (O-6-buG) and O-6-benzylguanine (O-6-bzG). The proteins displayed similar repair preference of O-6-mG relative to O-6-buG as observed with O-6-pobG. In contrast, all of the mammalian proteins, except the mutant P140K, preferentially repaired O-6-bzG. These studies indicate that the rate of repair of O-6-pobG is highly dependent on protein structure. Inefficient repair of O-6-pobG by bacterial AGT explains the high mutagenic activity of this adduct in bacterial systems. In addition, differences observed in the repair of this adduct by mammalian proteins may translate into differences in sensitivity to the mutagenic and carcinogenic effects of NNK or other pyridyloxobutylating nitrosamines

See More