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.

Differential inactivation of polymorphic variants of human O6-alkylguanine-DNA alkyltransferase

“…The I143V/ K178R hAGT was active in the repair of O 6 -n-butylguanine, O 6 -[4-oxo-4-(3-pyridyl)butyl] guanine or BG when these were contained in oligodeoxynucleotides [39]. However, the repair of O 6 -[4-oxo-4-(3-pyridyl)butyl]guanine by this variant was less sensitive to sequence context than wild type or the L84F form [40]. These results indicate that the I143V substitution alters the geometry of the hAGT substrate-binding pocket to permit efficient repair of bulky O 6 -[4-oxo-4-(3-pyridyl)butyl]guanine even when it is in conformations that may be poorly repaired by wild type hAGT.…”

“…The purified L84F recombinant protein does not differ in activity from wild type hAGT in the repair of O 6 -methylguanine in vivo or in vitro or in the relative repair in vitro of O 6 -[4-oxo-4-(3-pyridyl)butyl]guanine, an adduct formed by the tobacco specific nitrosamines [40,43]. The lack of alteration in DNA repair ability for L84F is consistent with the fact that this is a conservative change of one hydrophobic amino acid side-chain for another at a position in the N-domain of hAGT, a significant distance away from the active site.…”

“…Plasmids for the production of C-terminal His 6 -tagged hAGT [37] and for the production of N-terminal His 6 -tagged hAGT [38], and the mutants L84F, I143V and I143V/K178R were prepared as described previously [39,40]. Plasmids for the production of N-terminal His 6 -tagged W65C and K178R of hAGT were constructed from the template plasmids of pQE30-hAGT [38] with Quick Change Site-directed Mutagenesis Kit used according to the manufacturer's instructions.…”

DNA repair systems

“…Unlike most mechanisms of direct repair, O6-methylguanine DNA methyltransferase (MGMT, EC 2.1.1.63) repairs damage inflicted by alkyla on (usually, methyla on) of bases. The enzyme catalyses the restora on of the original nucleo de by transferring the alkyl radical onto its own molecule (self-alkyla on) [42]. More details about the exact process may be found in Chapter VI -Mechanisms of DNA repair.…”

“…Spore photoproduct photolyase uses the energy of visible light to break the C-C bond between two thymine residues and restore the ini al thymines. The reac on requires the presence of S-adenosylmethionine as a cofactor [203].О6-methylguanine DNA methyltransferase(О6-alkylguanine DNA alkyltransferase)О6-methylguanine DNA methyltransferase works by transferring methyl groups from O6-methylguanine and other alkyl groups from various alkylated substrates to a designated cysteine residue in the ac ve centre of the enzyme molecule [42,204]. The methylated protein is subsequently degraded via the ubiqui n-dependent mechanism.…”