Kinetics of O6-Methyl-2‘-deoxyguanosine Repair by O6-Alkylguanine DNA Alkyltransferase within K-ras Gene-Derived DNA Sequences

Abstract

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 G₃G₄A G₅CT G₆G₇T G₈G₉C 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 O⁶-Me-G remaining in DNA. The relative extent of demethylation for O⁶-Me-dG adducts located at G₅, G₆, G₇, 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 × 10⁶ 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.