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

The production of guanine radicals in DNA via the flash−quench technique is shown to cause the formation of covalent adducts between DNA and histone protein. In the flash−quench experiment, the DNA-bound intercalator Ru(phen) 2 dppz 2+ (phen = 1,10-phenanthroline, dppz = dipyridophenazine) is excited with 442 nm light and quenched oxidatively by Co(NH 3 ) 5 Cl 2+, methyl viologen (MV 2+ ), or Ru(NH 3 ) 6 3+ to produce Ru(phen) 2 dppz 3+, a strong oxidant (+1.6 V) that can oxidize a nearby guanine base (+1.3 V). The guanine radical thus produced is vulnerable to nucleophilic attack and can react with amino acid side chains to form DNA−protein cross-links. Evidence for DNA−protein cross-linking was provided by the chloroform extraction assay, a filter binding assay, and gel electrophoretic analysis. After flash−quench treatment, pUC19 plasmid DNA undergoes a dramatic decrease in mobility that is reversed upon digestion with proteinase K, as seen by agarose gel electrophoresis. In polyacrylamide gel electrophoresis (SDS-PAGE) experiments, the histone protein shows similar mobility shifts. Cross-linking is observed with poly(dG-dC) and mixed sequence DNA, but not with poly(dA-dT), indicating that the reaction requires guanine bases. Measurements of emission quenching indicate 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 amount of quenching and decreases in the order Co(NH 3 ) 5 Cl 2+ > MV 2+ > Ru(NH 3 ) 6 3+ . This trend in cross-linking correlates instead with the lifetime of the guanine radical measured by transient absorption spectroscopy, and suggests that the cross-linking reaction requires > 100 μs. These results demonstrate that the flash−quench technique is an effective approach for the study of covalent adducts between DNA and protein formed as a result of guanine oxidation, and suggest one possible fate for oxidatively damaged DNA in vivo.