| Abstract
| - The tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), apotent pulmonary carcinogen, both methylates and pyridyloxobutylates DNA. Both reactionpathways generate promutagenic O6-alkylguanine adducts. These adducts, O6-methylguanine(O6-mG) and O6-[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 adductsare repaired by AGT in a reaction that results in pyridyloxobutyl transfer to the active sitecysteine. Because minor changes within the binding pocket of AGT can alter the ability of thisprotein to repair bulky O6-alkylguanine adducts relative to O6-mG, we explored the ability ofAGTs from different species as well as several human AGT variants and mutants todiscriminate between O6-mG or O6-pobG adducts. We incubated proteins with equal molaramounts of oligodeoxynucleotides containing site specifically incorporated O6-mG or O6-pobGand measured repair. Bacterial AGTs poorly repaired O6-pobG. Mouse and rat AGT repairedboth adducts at comparable rates. Wild-type human AGT, variant I143V/K178R, and mutantN157H repaired O6-mG approximately twice as fast as O6-pobG. Human variant G160R andmutants P140K, Y158H, G156A, and E166G did not repair O6-pobG until all of the O6-mGwas removed. To understand the role of adduct structure on relative repair rates, thecompetition experiments were repeated with two other bulky O6-alkylguanine adducts, O6-butylguanine (O6-buG) and O6-benzylguanine (O6-bzG). The proteins displayed similar repairpreference of O6-mG relative to O6-buG as observed with O6-pobG. In contrast, all of themammalian proteins, except the mutant P140K, preferentially repaired O6-bzG. These studiesindicate that the rate of repair of O6-pobG is highly dependent on protein structure. Inefficientrepair of O6-pobG by bacterial AGT explains the high mutagenic activity of this adduct inbacterial systems. In addition, differences observed in the repair of this adduct by mammalianproteins may translate into differences in sensitivity to the mutagenic and carcinogenic effectsof NNK or other pyridyloxobutylating nitrosamines.
|
