DNA-Protein Cross-Linking from Oxidation of Guanine via the Flash-Quench Technique

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• 作者：Kim L. Nguyen ; Mary Steryo ; Kristina Kurbanyan ; Kristina M. Nowitzki ; Sara M. Butterfield ; Suzie R. Ward ; and Eric D. A. Stemp
• 刊名：Journal of the American Chemical Society
• 出版年：2000
• 出版时间：April 19, 2000
• 年：2000
• 卷：122
• 期：15
• 页码：3585 - 3594
• 全文大小：293K
• 年卷期：v.122,no.15(April 19, 2000)
• ISSN：1520-5126

文摘

The production of guanine radicals in DNA via the flash-quench technique is shown to cause theformation of covalent adducts between DNA and histone protein. In the flash-quench experiment, the DNA-bound intercalator Ru(phen)₂dppz²⁺ (phen = 1,10-phenanthroline, dppz = dipyridophenazine) is excited with442 nm light and quenched oxidatively by Co(NH₃)₅Cl²⁺, methyl viologen (MV²⁺), or Ru(NH₃)₆³⁺ to produceRu(phen)₂dppz³⁺, a strong oxidant (+1.6 V) that can oxidize a nearby guanine base (+1.3 V). The guanineradical thus produced is vulnerable to nucleophilic attack and can react with amino acid side chains to formDNA-protein cross-links. Evidence for DNA-protein cross-linking was provided by the chloroform extractionassay, a filter binding assay, and gel electrophoretic analysis. After flash-quench treatment, pUC19 plasmidDNA undergoes a dramatic decrease in mobility that is reversed upon digestion with proteinase K, as seen byagarose gel electrophoresis. In polyacrylamide gel electrophoresis (SDS-PAGE) experiments, the histone proteinshows similar mobility shifts. Cross-linking is observed with poly(dG-dC) and mixed sequence DNA, but notwith poly(dA-dT), indicating that the reaction requires guanine bases. Measurements of emission quenchingindicate that for a given quencher, the amount of cross-linking is correlated to the amount of quenching.When comparing different quenchers, however, the amount of cross-linking is inversely related to the amountof quenching and decreases in the order Co(NH₃)₅Cl²⁺ > MV²⁺ > Ru(NH₃)₆³⁺. This trend in cross-linkingcorrelates instead with the lifetime of the guanine radical measured by transient absorption spectroscopy, andsuggests that the cross-linking reaction requires > 100 ages/entities/mgr.gif">s. These results demonstrate that the flash-quenchtechnique is an effective approach for the study of covalent adducts between DNA and protein formed as aresult of guanine oxidation, and suggest one possible fate for oxidatively damaged DNA in vivo.