Kinetics of O6-methyl-2′-deoxyguanosine repair by O6-alkylguanine DNA alkyltransferase within K-ras gene-derived DNA sequences

O-6-Methyl-2'-deoxyguanosine (O-6-Me-dG) is a potent mutagenic DNA adduct that can be induced by a variety of methylating agents, including tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). O-6-Me-dG is directly repaired by the specialized DNA repair protein, O-6-alkylguanine DNA alkyltransferase (AGT), which transfers the O-6-alkyl group from the modified guanine to a cysteine thiol within the active site of the protein. Previous investigations suggested that AGT repair of O-6-alkylguanines may be sequence-dependent as a result of flanking nucleobase effects on DNA conformation and energetics. In the present work, a novel high-performance/pressure liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI+-MS/MS)-based approach was developed to analyze the kinetics of AGT-mediated repair of O-6-Me-dG adducts placed at different sites within the double-stranded DNA sequence representing codons 8-17 of the K-ras protooncogene, 5'-G(1)TA G(2)TT G(3)G(4)A G(5)CT G(6)G(7)T G(8)G(9)C G(10)TA G(11)G(12)C AAG(13) AG(14)T-3', where G(5), G(6), G(7), G(8), G(9), G(10), or G(11) was replaced with O-6-Me-dG. The second guanine of K-ras codon 12 (G(7) in our numbering system) is a major mutational hotspot for G - A transitions observed in lung tumors of smokers and in neoplasms induced in laboratory animals by exposure to methylating agents. O-6-Me-dG-containing duplexes were incubated with human recombinant AGT protein, and the reactions were quenched at specific times. Following acid hydrolysis to release purines, isotope dilution HPLC-ESI-MS/MS was used to determine the amounts of O-6-Me-G remaining in DNA. The relative extent of demethylation for O-6-Me-dG adducts located at G(5), G(6), G(7), G(8), G(9), G(10), or G(II) following a 10 s incubation with AGT showed little variation as a function of sequence position. Furthermore, the second-order rate constants for the repair of O-6- Me-dG adducts located at the first and second positions of the K-ras codon 12 (5'-G(6)G(7)T-3') were similar (1.4 x 10(7) M-1 s(-1) vs 7.4 x 10(6) M-1 s(-1), respectively), suggesting that O-6-Me-dG repair by AGT is not the determining factor for K-ras codon 12 mutagenesis following exposure to methylating agents. The new HPLC-ESI-MS/MS assay developed in this work is a valuable tool which will be used to further explore the role of local sequence environment and endogenous DNA modifications in shaping mutational spectra of NNK and other methylating agents.