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

Abstract

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)2dppz2+ (phen = 1,10-phenanthroline, dppz = dipyridophenazine) is excited with 442 nm light and quenched oxidatively by Co(NH3)5Cl2+, methyl viologen (MV2+), or Ru(NH3)6 3+ to produce Ru(phen)2dppz3+, 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(NH3)5Cl2+ > MV2+ > Ru(NH3)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.

DNA-Mediated Charge Transport in Redox Sensing and Signaling

“…Cytochrome c, which is not natively a DNA-binding protein but has extensive lysine content, also undergoes crosslinking with DNA, demonstrating the generality of this decomposition. The rate of crosslinking is slower than 10 4 s -1 ,94 comparable to that which has been reported for degradation of guanine radical in the presence of in situ generated superoxide. In the absence of protein, superoxide or other diffusing reductants, guanine radical survives for seconds 96.…”

“…The guanine radical that is generated upon DNA photooxidation degrades to 8-oxo-7,8-dihydroguanine (oxoG), among other products, following nucleophilic attack by water at the C8 carbon 26,88. After it was demonstrated that the tripeptides KYK and KWK are oxidized by DNA with the radical localized on the tyrosine and tryptophan respectively (Figure 4),89-91 several groups explored reactions of DNA-binding peptides that lack a stable radical acceptor but have nucleophilic groups that might attack the guanine radical 92-94. Consistent with the oxidative decomposition products of covalent guanosine-lysine conjugates,95 trilysine for ms crosslinks with oxidized DNA.…”

Control and utilization of ruthenium and rhodium metal complex excited states for photoactivated cancer therapy

“…This O 2 -independent cleavage occurs via guanine oxidation by the long-lived photogenerated Ru(III) center in the excited state species. This effect is similar to Ru(II) tris-polypyridyl complexes in the presence of an electron acceptor [67,68]. …”

One-electron oxidation reactions of purine and pyrimidine bases in cellular DNA

“…This may be rationalized in terms of the initial generation of guanine radical cations since 6-thioguanine has been shown to act as a type I photosensitizer. Further support is gained by previous model studies that have involved several one-electron oxidants (Nguyen et al 2000, Kurbanyan et al 2003, Madison et al 2012) including high intensity UVC laser irradiation (Angelov et al 2003) as generators of DPC. A detailed mechanistic study describing the covalent attachment of TGT trinucleotide to trilysine peptide (KKK) is now available (Perrier et al 2006).…”