title: Kinetics of O(6)-pyridyloxobutyl-2'-deoxyguanosine repair by human O(6)-alkylguanine DNA alkyltransferase.
abstract: Tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN) are potent carcinogens believed to contribute to the development of lung tumors in smokers. NNK and NNN are metabolized to DNA-reactive species that form a range of nucleobase adducts, including bulky O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]deoxyguanosine (O(6)-POB-dG) lesions. If not repaired, O(6)-POB-dG adducts induce large numbers of G → A and G → T mutations. Previous studies have shown that O(6)-POB-dG can be directly repaired by O(6)-alkylguanine-DNA alkyltransferase (AGT), which transfers the pyridyloxobutyl group from O(6)-alkylguanines in DNA to an active site cysteine residue within the protein. In the present study, we investigated the influence of DNA sequence context and endogenous cytosine methylation on the kinetics of AGT-dependent repair of O(6)-POB-dG in duplex DNA. Synthetic oligodeoxynucleotide duplexes containing site-specific O(6)-POB-dG adducts within K-ras and p53 gene-derived DNA sequences were incubated with recombinant human AGT protein, and the kinetics of POB group transfer was monitored by isotope dilution HPLC-ESI(+)-MS/MS analysis of O(6)-POB-dG remaining in DNA over time. We found that the second-order rates of AGT-mediated repair were influenced by DNA sequence context (10-fold differences) but were only weakly affected by the methylation status of neighboring cytosines. Overall, AGT-mediated repair of O(6)-POB-dG was 2-7 times slower than that of O(6)-Me-dG adducts. To evaluate the contribution of AGT to O(6)-POB-dG repair in human lung, normal human bronchial epithelial cells (HBEC) were treated with model pyridyloxobutylating agent, and O(6)-POB-dG adduct repair over time was monitored by HPLC-ESI(+)-MS/MS. We found that HBEC cells were capable of removing O(6)-POB-dG lesions, and the repair rates were significantly reduced in the presence of an AGT inhibitor (O(6)-benzylguanine). Taken together, our results suggest that AGT plays an important role in protecting human lung against tobacco nitrosamine-mediated DNA damage and that inefficient AGT repair of O(6)-POB-dG at a specific sequences contributes to mutational spectra observed in smoking-induced lung cancer.
authors.affiliation: Department of Medicinal Chemistry and the Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Delshanee
authors.initials: D
authors.lastname: Kotandeniya
authors.forename: Daniel
authors.initials: D
authors.lastname: Murphy
authors.forename: Shuo
authors.initials: S
authors.lastname: Yan
authors.forename: Soobong
authors.initials: S
authors.lastname: Park
authors.forename: Uthpala
authors.initials: U
authors.lastname: Seneviratne
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Koopmeiners
authors.forename: Anthony
authors.initials: A
authors.lastname: Pegg
authors.forename: Sreenivas
authors.initials: S
authors.lastname: Kanugula
authors.forename: Fekadu
authors.initials: F
authors.lastname: Kassie
authors.forename: Natalia
authors.initials: N
authors.lastname: Tretyakova
mesh_terms: D001483:Base Sequence; D001980:Bronchi / Q000166:cytology; D002273:Carcinogens / Q000737:chemistry / Q000494:pharmacology; D002478:Cells, Cultured; D018736:DNA Adducts / Q000737:chemistry* / Q000378:metabolism; D019175:DNA Methylation; D004260:DNA Repair; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D006147:Guanine / Q000031:analogs & derivatives; D006801:Humans; D007700:Kinetics; D009602:Nitrosamines / Q000737:chemistry / Q000494:pharmacology; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011119:Polynucleotides / Q000737:chemistry; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D011725:Pyridines / Q000737:chemistry* / Q000378:metabolism; D020545:Respiratory Mucosa / Q000201:enzymology; D044366:Transition Temperature; D018631:ras Proteins / Q000235:genetics
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; C117307:KRAS protein, human; D009602:Nitrosamines; C473029:O(6)-(4-oxo-4-(3-pyridyl)butyl)guanine; D011119:Polynucleotides; D011518:Proto-Oncogene Proteins; D011725:Pyridines; D006147:Guanine; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D019853:O(6)-Methylguanine-DNA Methyltransferase; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins; D003849:Deoxyguanosine