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 O⁶-alkylguanine adducts. These adducts, O ⁶-methylguanine (O⁶-mG) and O ⁶-[4-oxo-4-(3-pyridyl)butyl]guanine (O⁶-pobG), are repaired by O⁶-alkylguanine-DNA 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 O⁶-alkylguanine adducts relative to O⁶-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 O⁶-pobG adducts. We incubated proteins with equal molar amounts of oligodeoxynucleotides containing site specifically incorporated O⁶-mG or O⁶-pobG and measured repair. Bacterial AGTs poorly repaired O⁶-pobG. Mouse and rat AGT repaired both adducts at comparable rates. Wild-type human AGT, variant I143V/K178R, and mutant N157H repaired O⁶-mG approximately twice as fast as O ⁶-pobG. Human variant G160R and mutants P140K, Y158H, G156A, and E166G did not repair O⁶-pobG until all of the O⁶-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, O⁶-butylguanine (O ⁶-buG) and O⁶-benzylguanine (O⁶-bzG). The proteins displayed similar repair preference of O⁶-mG relative to O⁶-buG as observed with O⁶-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 O⁶-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.