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DNA-protein cross-linking by 1,2,3,4-diepoxybutane.

ABSTRACT: 1,2,3,4-diepoxybutane (DEB) is a strongly genotoxic diepoxide hypothesized to be the ultimate carcinogenic metabolite of the common industrial chemical and environmental carcinogen 1,3-butadiene. DEB is a bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs), which are thought to play a central role in its biological activity. Previous studies with recombinant proteins have shown that the biological outcomes of DEB-induced DPCs are strongly influenced by protein identities. The present work combines affinity capture methodology with mass spectrometry-based proteomics and immunological detection to identify the proteins that form DPCs in nuclear extracts from human cervical carcinoma (HeLa) cells. We identified 39 human proteins that form covalent DPCs in the presence of DEB. DNA-protein cross-linking efficiency following treatment with 25 mM DEB was 2-12%, depending on protein identity. High-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI+-MS/MS) analysis of the total proteolytic digests of cross-linked proteins revealed the presence of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol conjugates, suggesting that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA.

SUBMITTER: Michaelson-Richie ED

PROVIDER: S-EPMC2956319 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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DNA-protein cross-linking by 1,2,3,4-diepoxybutane.

Michaelson-Richie Erin D ED Loeber Rachel L RL Codreanu Simona G SG Ming Xun X Liebler Daniel C DC Campbell Colin C Tretyakova Natalia Y NY

Journal of proteome research 20100901 9

1,2,3,4-diepoxybutane (DEB) is a strongly genotoxic diepoxide hypothesized to be the ultimate carcinogenic metabolite of the common industrial chemical and environmental carcinogen 1,3-butadiene. DEB is a bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs), which are thought to play a central role in its biological activity. Previous studies with recombinant proteins have shown that the biological outcomes of DEB-induced DPCs are str ...[more]

PMID: 20666492

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Project description:1,2,3,4-Diepoxybutane (DEB) is a key carcinogenic metabolite of the important industrial chemical 1,3-butadiene. DEB is a bifunctional alkylating agent capable of reacting with DNA and proteins. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine monoadducts, which can react with another nucleophilic site to form cross-linked adducts. A recent report revealed a strong correlation 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-DNA cross-links (Valadez, J. G., et al. (2004) Activation of bis-electrophiles to mutagenic conjugates by human O6-alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 17, 972-982). The purpose of our study was to analyze the formation and structures of DEB-induced AGT-DNA conjugates and to identify specific amino acid residues within the protein involved in cross-linking. DNA-protein cross-link formation was detected by SDS-PAGE when 32P-labeled double-stranded oligodeoxynucleotides were exposed 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 with N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-deoxyguanosine (dG monoepoxide) revealed the ability of the protein to form either one or two butanediol-dG cross-links, corresponding to mass shifts of +353 and +706 Da, 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 a neighboring active site residue, Cys150. The same two amino acid residues of hAGT became covalently cross-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 represents the active site tryptic fragment of hAGT (C = Cys145). The replacement of the catalytic cysteine residue with alanine in the C145A hAGT mutant abolished DEB-induced cross-linking at this site, while the formation 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/MS analysis of the amino acids resulting from the total digestion of modified proteins analyzed in parallel with an authentic standard. AGT-DNA cross-linking is a likely mechanism of DEB-mediated cytotoxicity in cells expressing this important repair protein.

Project description:1,2,3,4-Diepoxybutane (DEB) is a carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is considered the ultimate carcinogenic species of BD because of its potent genotoxicity and mutagenicity attributed to its ability to form DNA-DNA cross-links and exocyclic nucleoside adducts. Mutagenesis studies suggest that DEB adducts formed at adenine bases may be critically important, as it induces large numbers of A ? T transversions. We have recently identified three types of exocyclic DEB-dA lesions: N?,N?-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N?,N?-DHB-dA), 1,N?-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N?-?-HMHP-dA), and 1,N?-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2'-deoxyadenosine (1,N?-?-HMHP-dA) [Seneviratne, U., et al. (2010) Chem. Res. Toxicol. 23, 118-133]. In the work presented here, a postsynthetic methodology for preparing DNA oligomers containing stereospecific and site-specific N?,N?-DHB-dA and 1,N?-?-HMHP-dA adducts was developed. DNA oligomers containing site-specific 6-chloropurine were coupled with optically pure 1-amino-2-hydroxy-3,4-epoxybutanes to generate oligomers containing N?-(2-hydroxy-3,4-epoxybut-1-yl)-2'-deoxyadenosine adducts, followed by their spontaneous cyclization to 1,N?-?-HMHP-dA lesions. N?,N?-DHB-dA containing strands were prepared analogously by coupling 6-chloropurine containing DNA with (3S,4S)- or (3R,4R)-pyrrolidine-3,4-diols. Oligodeoxynucleotide structures were confirmed by ESI-MS, exonuclease ladder sequencing, and HPLC-MS/MS of enzymatic digests. UV melting and CD spectroscopy studies of DNA duplexes containing N?,N?-DHB-dA and 1,N?-?-HMHP-dA revealed that both lesions lower the thermodynamic stability of DNA. Interestingly, structurally modified DNA duplexes were more thermodynamically stable when an adenine residue was placed opposite 1,N?-?-HMHP-dA instead of thymine, suggesting that these adducts may preferentially pair with dA.

Project description:Nitrogen mustards are antitumor agents used clinically for the treatment of a variety of neoplastic conditions. The biological activity of these compounds is typically attributed to their ability to induce DNA-DNA cross-links. However, nitrogen mustards are able to produce a variety of other lesions, including DNA-protein cross-links (DPCs). DPCs induced by nitrogen mustards are not well-characterized because of their structural complexity and the insufficient specificity and sensitivity of previously available experimental methodologies. In the present work, affinity capture methodology in combination with mass spectrometry-based proteomics was employed to identify mammalian proteins that form covalent cross-links to DNA in the presence of a simple nitrogen mustard, mechlorethamine. Following incubation of 5'-biotinylated DNA duplexes with nuclear protein extracts, DPCs were isolated by affinity capture on streptavidin beads, and the cross-linked proteins were identified by high-performance liquid chromatography-electrospray tandem mass spectrometry of tryptic peptides. Mechlorethamine treatment resulted in the formation of DPCs with nuclear proteins involved in chromatin regulation, DNA replication and repair, cell cycle control, transcriptional regulation, and cell architecture. Western blot analysis was employed to confirm protein identification and to quantify the extent of drug-mediated cross-linking. Mass spectrometry of amino acid-nucleobase conjugates found in total proteolytic digests revealed that mechlorethamine-induced DPCs are formed via alkylation of the N7 position of guanine in duplex DNA and cysteine thiols within the proteins to give N-[2-[S-cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]methylamine lesions. The results described herein suggest that cellular exposure to nitrogen mustards leads to cross-linking of a large spectrum of nuclear proteins to chromosomal DNA, potentially contributing to the cytotoxic and mutagenic effects of these drugs.

Dataset Information

DNA-protein cross-linking by 1,2,3,4-diepoxybutane.

Publications

DNA-protein cross-linking by 1,2,3,4-diepoxybutane.

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