DNA Sequence Context Affects Repair of the Tobacco-Specific Adduct O6-[4-Oxo-4-(3-pyridyl)butyl]guanine by Human O6-Alkylguanine-DNA Alkyltransferases

Abstract

The repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) protects cells from the mutagenic and carcinogenic effects of alkylating agents by removing O(6)-alkylguanine adducts from DNA. Recently, we established that AGT protects against the mutagenic effects of pyridyloxobutylation resulting from the metabolic activation of the tobacco-specific nitrosamines (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosonornicotine by repairing O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine (O(6)-pobG). There have been several epidemiologic studies examining the association between the I143V/K178R AGT genotype and lung cancer risk. Two studies have found positive associations, suggesting that AGT proteins differ in their repair of DNA damage caused by TSNA. However, it is not known how this genotype alters the biochemical activity of AGT. We proposed that AGT proteins may differ in their ability to remove large O(6)-alkylguanine adducts, such as O(6)-pobG, from DNA. Therefore, we examined the repair of O(6)-pobG by wild-type (WT) human, I143V/K178R, and L84F AGT proteins when contained in multiple sequence contexts, including the twelfth codon of H-ras, a mutational hotspot within this oncogene. The AGT-mediated repair of O(6)-pobG was more profoundly influenced by sequence context than that of O(6)-methylguanine. These differences are not the result of secondary structure (hairpin) formation in DNA. In addition, the I143V/K178R variant seems less sensitive to the effects of sequence context than the WT or L84F proteins. These studies indicate that the sequence dependence of O(6)-pobG repair by human AGT (hAGT) varies with subtle changes in protein structure. These data establish a novel functional difference between the I143V/K178R protein and other hAGTs in the repair of a toxicologically relevant substrate, O(6)-pobG.