Cross-Linking of the DNA Repair Protein O6-Alkylguanine DNA Alkyltransferase to DNA in the Presence of Antitumor Nitrogen Mustards (original) (raw)
2008, Chemical Research in Toxicology
The antitumor activity of chemotherapeutic nitrogen mustards including chlorambucil, cyclophosphamide, and melphalan, is commonly attributed to their ability to induce DNA-DNA cross-links by consecutive alkylation of two nucleophilic sites within the DNA duplex. DNA-protein cross-linking by nitrogen mustards is not well characterized, probably because of its inherent complexity and the insufficient sensitivity of previous methodologies. If formed, DNA-protein conjugates are likely to contribute to both target and off-target cytotoxicity of nitrogen mustard drugs. Here we show that the DNA repair protein, O 6-alkylguanine DNA alkyltransferase (AGT), can be readily cross-linked to DNA in the presence of nitrogen mustards. Both chlorambucil and mechlorethamine induced the formation of covalent conjugates between 32 P-labeled double-stranded oligodeoxynucleotides and recombinant human AGT protein, which were detected by SDS-PAGE. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT that had been treated with guanine half mustards of chlorambucil or mechlorethamine revealed the ability of the protein to form either one or two cross-links to guanine. C145A AGT-a variant containing a single point mutation in the protein's active site-was found capable of forming a single guanine conjugate, while cross-linking was virtually abolished upon treatment of the C145A/C150S AGT double mutant with the guanine half mustards. HPLC-ESI +-MS/MS sequencing of the tryptic peptides obtained from the wild type AGT protein that had been treated with nitrogen mustards in the presence of DNA confirmed that the cross-linking took place between the N7 position of guanine in DNA and two active site residues within the AGT protein (Cys 145 and Cys 150). The exact chemical structures of AGT-DNA cross-links induced by chlorambucil and mechlorethamine were identified as N-(2-[Scysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)-p-aminophenylbuyric acid and N-(2-[Scysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)methylamine, respectively, based upon HPLC-MS/MS analysis of protein hydrolysates in parallel with the corresponding amino acid conjugates prepared synthetically.
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