title: Characterization of a mutagenic DNA adduct formed from 1,2-dibromoethane by O6-alkylguanine-DNA alkyltransferase.
abstract: It has been proposed that the DNA repair protein O6-alkylguanine-DNA alkyltransferase increases the mutagenicity of 1,2-dibromoethane by reacting with it at its cysteine acceptor site to form a highly reactive half-mustard, which can then react with DNA (Liu, L., Pegg, A. E., Williams, K. M., and Guengerich, F. P. (2002) J. Biol. Chem. 277, 37920-37928). Incubation of Escherichia coli-expressed human alkyltransferase with 1,2-dibromoethane and single-stranded oligodeoxyribonucleotides led to the formation of covalent transferaseoligo complexes. The order of reaction determined was Gua>Thy>Cyt>Ade. Mass spectrometry analysis of the tryptic digest of the reaction product indicated that some of the adducts led to depurination with the release of the Gly136-Arg147 peptide cross-linked to a Gua at the N7 position, with the site of reaction being the active site Cys145 as established by chromatographic retention time and the fragmentation pattern determined by tandem mass spectrometry of a synthetic peptide adduct. The alkyltransferase-mediated mutations produced by 1,2-dibromoethane were predominantly Gua to Ade transitions but, in the spectrum of such rifampicin-resistant mutations in the RpoB gene, 20% were Gua to Thy transversions. The latter are likely to have arisen from the apurinic site generated from the Gua-N7 adduct. Support exists for an additional adduct/mutagenic pathway because evidence was obtained for DNA adducts other than at the Gua N7 atom and for mutations other than those attributable to depurination. Thus, chemical and biological evidence supports the existence of at least two alkyltransferase-dependent pathways for 1,2-dibromoethane-induced mutagenicity, one involving Gua N7-alkylation by alkyltransferase-S-CH2CH2Br and depurination, plus another as yet uncharacterized system(s).
authors.affiliation: Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
authors.forename: Liping
authors.initials: L
authors.lastname: Liu
authors.forename: David L
authors.initials: DL
authors.lastname: Hachey
authors.forename: Gerardo
authors.initials: G
authors.lastname: Valadez
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Williams
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Loktionova
authors.forename: Sreenivas
authors.initials: S
authors.lastname: Kanugula
authors.forename: Anthony E
authors.initials: AE
authors.lastname: Pegg
mesh_terms: D000818:Animals; D002417:Cattle; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D015946:Ethylene Dibromide / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D006801:Humans; D066298:In Vitro Techniques; D015394:Molecular Structure; D009153:Mutagens / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D018736:DNA Adducts; D009153:Mutagens; D011994:Recombinant Proteins; D015946:Ethylene Dibromide; D019853:O(6)-Methylguanine-DNA Methyltransferase