O6-Methyl-2′-deoxyguanosine (O6-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). O6-Me-dG is directly repaired by the specialized DNA repair protein, O6-alkylguanine DNA alkyltransferase (AGT), which transfers the O6-alkyl group from the modified guanine to a cysteine thiol within the active site of the protein. Previous investigations suggested that AGT repair of O6-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 O6-Me-dG adducts placed at different sites within the double-stranded DNA sequence representing codons 8-17 of the K-ras protooncogene, 5′-G1TA G2TT G 3G4A G5CT G6G7T G 8G9C G10TA G11G12C AAG13 AG14T-3′, where G5, G6, G7, G8, G9, G10, or G11 was replaced with O6-Me-dG. The second guanine of K-ras codon 12 (G7 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. O6-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 C6-Me-G remaining in DNA. The relative extent of demethylation for O6-Me-dG adducts located at G5, G6, G 7, G8, G9, G10, or G11 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 O6-Me-dG adducts located at the first and second positions of the K-ras codon 12 (5′-G6G7T-3′) were similar (1.4 × 107 M-1 s-1 vs 7.4 × 10 6 M-1 s-1, respectively), suggesting that O6-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.
All Science Journal Classification (ASJC) codes
- Drug Discovery
- Organic Chemistry
- Health, Toxicology and Mutagenesis