Cross-Linking of the Human DNA Repair Protein O6-Alkylguanine DNA Alkyltransferase to DNA in the Presence of 1,2,3,4-Diepoxybutane

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• 作者：Rachel Loeber ; Mathur Rajesh ; Qingming Fang ; Anthony E. Pegg ; and Natalia Tretyakova
• 刊名：Chemical Research in Toxicology
• 出版年：2006
• 出版时间：May 2006
• 年：2006
• 卷：19
• 期：5
• 页码：645 - 654
• 全文大小：253K
• 年卷期：v.19,no.5(May 2006)
• ISSN：1520-5010

文摘

1,2,3,4-Diepoxybutane (DEB) is a key carcinogenic metabolite of the important industrial chemical1,3-butadiene. DEB is a bifunctional alkylating agent capable of reacting with DNA and proteins. InitialDNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine monoadducts, whichcan react with another nucleophilic site to form cross-linked adducts. A recent report revealed a strongcorrelation between cellular expression of the DNA repair protein O⁶-alkylguanine DNA alkyltransferase(AGT) and the cytotoxic and mutagenic activity of DEB, suggesting that DEB induces AGT-DNAcross-links (Valadez, J. G., et al. (2004) Activation of bis-electrophiles to mutagenic conjugates by humanO⁶-alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 17, 972-982). The purpose of our studywas to analyze the formation and structures of DEB-induced AGT-DNA conjugates and to identifyspecific amino acid residues within the protein involved in cross-linking. DNA-protein cross-linkformation was detected by SDS-PAGE when ³²P-labeled double-stranded oligodeoxynucleotides wereexposed to DEB in the presence of either wild-type hAGT or a C145A hAGT mutant. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of hAGT that had been treated withN7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-deoxyguanosine (dG monoepoxide) revealed the ability of the proteinto form either one or two butanediol-dG cross-links, corresponding to mass shifts of +353 and +706Da, respectively. HPLC-ESI⁺-MS/MS sequencing of the tryptic peptides obtained from dG monoepoxide-treated protein indicated that the two cross-linking sites were the alkyl acceptor site, Cys¹⁴⁵, and aneighboring active site residue, Cys¹⁵⁰. The same two amino acid residues of hAGT became covalentlycross-linked to DNA following DEB treatment. Modification of Cys¹⁴⁵ was further confirmed by HPLC-ESI⁺-MS/MS analysis of dG monoepoxide-treated synthetic peptide GNPVPILIPCHR which representsthe active site tryptic fragment of hAGT (C = Cys¹⁴⁵). The replacement of the catalytic cysteine residuewith alanine in the C145A hAGT mutant abolished DEB-induced cross-linking at this site, while theformation of conjugates via neighboring Cys¹⁵⁰ was retained. The exact chemical structure of the cross-linked lesion was established as 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol by HPLC-ESI⁺-MS/MSanalysis of the amino acids resulting from the total digestion of modified proteins analyzed in parallelwith an authentic standard. AGT-DNA cross-linking is a likely mechanism of DEB-mediated cytotoxicityin cells expressing this important repair protein.