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

Abstract

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.

Cross-Linking of the DNA Repair Protein O6-Alkylguanine DNA Alkyltransferase to DNA in the Presence of Antitumor Nitrogen Mustards

“…These same two sites were also modified when the recombinant protein was treated with chlorambucil or mechlorethamine in the absence of DNA (Supporting Information S-13 through S-18), suggesting selective reactivity of these residues towards nitrogen mustards. These results are consistent with our previous results for AGT-DNA cross-linking by diepoxybutane (6).…”

Structure of the 1,4-Bis(2‘-deoxyadenosin-N6-yl)-2S,3S-butanediol Intrastrand DNA Cross-Link Arising from Butadiene Diepoxide in the Human N-ras Codon 61 Sequence

“…The genotoxicity is probably due to its potential to form DNA-DNA (43)(44)(45)(46) and DNA-protein cross-links (47,48), the latter having been observed in mice (49,50). Mice possess greater sensitivity to butadiene exposure than rats, and this is attributed to their efficient oxidation of BD to BDO 2 (51,52), presumably facilitating DNA cross-linking.…”

Formation and repair of DNA-protein crosslink damage

“…DPCs can be induced upon exposure to ionizing radiation, ultraviolet light, and various transition metal ions, including chromium and nickel [Swenberg et al, 2011]. A number of other carcinogens are also known to induce DPCs, including bifunctional alkylating agents such as 1,3-butadiene, diepoxybutane, acrolein, and crotonaldehyde [Costa et al, 1997; Kurtz and Lloyd, 2003; Loeber et al, 2006; Minko et al, 2008]. …”

“…O 6 -methylguanine-DNA methyltransferase (MGMT), an enzyme responsible for removing alkyl adducts from DNA, readily forms DPCs when cells are treated with nitrogen mustard or the carcinogen 1,2,3,4-diepoxybutane [Loeber et al, 2006; Loeber et al, 2008; Cheng et al, 2016]. Poly(ADP-ribose) polymerase-1 (PARP-1), which is implicated in several DNA repair, DNA damage detection, and chromatin remodeling processes, has been shown to covalently bond with abasic sites derived during BER, forming a DPC [Prasad et al, 2014].…”