Characterization of DNA−Protein Cross-Links Induced by Oxanine:  Cellular Damage Derived from Nitric Oxide and Nitrous Acid

• Published in: Biochemistry (Easton) Vol. 46; no. 13; pp. 3952 - 3965
• Main Authors: Chen, Hauh-Jyun Candy, Hsieh, Chia-Jong, Shen, Li-Ching, Chang, Chia-Ming
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 03.04.2007
• Subjects: Chromatography, High Pressure Liquid; Cross-Linking Reagents - chemistry; Deoxyribonucleosides - chemistry; DNA - chemistry; Glutathione - chemistry; Glutathione Disulfide - chemistry; Nitric Oxide - chemistry; Nitrous Acid - chemistry; Nuclear Magnetic Resonance, Biomolecular; Proteins - chemistry; Purine Nucleosides - chemistry; Reactive Nitrogen Species - chemistry; Tandem Mass Spectrometry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0620398

Reactive nitrogen species are implicated in inflammatory diseases and cancers. Oxanine (Oxa) is a DNA lesion derived from the guanine base with nitric oxide, nitrous acid, or N-nitrosoindoles. It was shown by gel electrophoresis that oxanine mediated the formation of DNA−protein cross-links (DPCs) with DNA-binding proteins and in the cell extract. Although 2‘-deoxyoxanosine was shown to react with amines including the N-terminal amino group of glycine, the structures of DNA−protein cross-links induced by oxanine have not been characterized. In this study, we find that the thiol group of the amino acid side chain is reactive toward oxanine, forming a thioester. Two reaction products of oxanine, namely, the thioester and the amide adducts, with the endogenous tripeptide glutathione (GSH) as a model protein were characterized on the basis of their UV, NMR (1H- and 13C-), and mass spectra. Interestingly, the disulfide GSSG also reacts with oxanine, forming the thioester adduct. The thioester and the amide adducts are generated when GSH and GSSG react with oxanine-containing calf thymus DNA, and they might be possible forms of cellular DPCs. Because the repair mechanism of DPCs is not extensively investigated, the characterization of oxanine-derived DPC structures should shed light on their detection in vivo and on their biological consequences.