title: Kinetics of O(6)-methyl-2'-deoxyguanosine repair by O(6)-alkylguanine DNA alkyltransferase within K-ras gene-derived DNA sequences.
abstract: 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'-G1TA G2TT G3G4A G5CT G6G7T G8G9C G10TA G11G12C AAG13 AG14T-3', where G5, G6, G7, G8, G9, G10, or G11 was replaced with O(6)-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. 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 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 O(6)-Me-dG adducts located at the first and second positions of the K-ras codon 12 (5'-G6G7T-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.
authors.affiliation: Department of Medicinal Chemistry and the Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Guza
authors.forename: Mathur
authors.initials: M
authors.lastname: Rajesh
authors.forename: Qingming
authors.initials: Q
authors.lastname: Fang
authors.forename: Anthony E
authors.initials: AE
authors.lastname: Pegg
authors.forename: Natalia
authors.initials: N
authors.lastname: Tretyakova
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D004260:DNA Repair; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000235:genetics; D011905:Genes, ras; D006801:Humans; D007700:Kinetics; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D017931:DNA Primers; D004247:DNA; C015558:O(6)-methyl-2'-deoxyguanosine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003849:Deoxyguanosine