DNA-protein crossing-linking by BIS-electrophiles

Loeber, Rachel Lea
2008-10

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DNA-protein crossing-linking by BIS-electrophiles

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2008-10

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A key carcinogenic metabolite of the important industrial chemical 1,3-butadiene (BD), DEB is a bifunctional alkylating agent capable of reacting with both DNA and proteins. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)- guanine monoadducts, which can then react with a second nucleophilic site to form crosslinked adducts. A recent report revealed a strong correlation between expression of the human DNA repair protein AGT in bacteria and the cytotoxic and mutagenic activity of DEB (J. G. Valadez et al., Chem. Res. Toxicol. 17 (2004) 972-982). As AGT expression appeared to enhance the toxic effects of this bis-electrophile, the authors proposed that DEB induces AGT-DNA cross-links. The purpose of our study was to structurally characterize DEB-induced AGT-DNA conjugates and to identify amino acid residues within the protein involved in cross-linking. DNA-protein cross-link formation was first detected by SDS-PAGE when 32P-labeled double-stranded oligodeoxynucleotides were exposed to DEB in the presence of both wild-type AGT or a C145A AGT mutant. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT 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 tryptic peptides obtained from dG monoepoxide-treated AGT indicated that the two cross linking sites were located at the alkyl acceptor site, Cys145 39 and a neighboring active site residue, Cys150. The same two active site cysteines became 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 AGT (C = Cys145 ). Replacement of the catalytic cysteine residue with alanine (C145A AGT) 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 total digestion of modified AGT analyzed in parallel with an authentic standard. Based upon these results, the formation of AGT-DNA cross-links is a likely mechanism to explain the enhanced cytotoxicity of DEB in cells expressing this important repair protein.

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University of Minnesota Ph.D. dissertation. October 2008. Major: Medicinal chemistry. Advisor: Dr. Natalia Tretyakova. 1 computer file (PDF); xiv, 239 pages, includes col. illustration.

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