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DNA−Protein Cross-Linking via Guanine Oxidation: Dependence upon Protein and Photosensitizer

2003; American Chemical Society; Volume: 42; Issue: 34 Linguagem: Inglês

10.1021/bi020713p

1943-295X

Kristina Kurbanyan, Kim L. Nguyen, Phuong To, Eunice V. Rivas, Alexis M. K. Lueras, Cynthia Kosinski, Mary Steryo, Arcelia González, Daisy Ann Mah, Eric D. A. Stemp,

Metal-Catalyzed Oxygenation Mechanisms

DNA−protein cross-links form when guanine undergoes a 1-electron oxidation in a flash−quench experiment, and the importance of reactive oxygen species, protein, and photosensitizer is examined here. In these experiments, a strong oxidant produced by oxidative quenching of a DNA-bound photosensitizer generates an oxidized guanine base that reacts with protein to form the covalent adduct. These cross-links are cleaved by hot piperidine and are not the result of reactive oxygen species, since neither a hydroxyl radical scavenger (mannitol) nor oxygen affects the yield of DNA−histone cross-linking, as determined via a chloroform extraction assay. The cross-linking yield depends on protein, decreasing as histone > cytochrome c > bovine serum albumin. The yield does not depend on the cytochrome oxidation state, suggesting that reduction of the guanine radical by ferrocytochrome c does not compete effectively with cross-linking. The photosensitizer strongly influences the cross-linking yield, which decreases in the order Ru(phen)2dppz2+ [phen = 1,10-phenanthroline; dppz = dipyridophenazine] > Ru(bpy)32+ [bpy = 2,2'-bipyridine] > acridine orange > ethidium, in accordance with measured oxidation potentials. A long-lived transient absorption signal for ethidium dication in poly(dG-dC) confirms that guanine oxidation is inefficient for this photosensitizer. From a polyacrylamide sequencing gel of a 32P-labeled 40-mer, all of these photosensitizers are shown to damage guanines preferentially at the 5' G of 5'-GG-3' steps, consistent with a 1-electron oxidation. Additional examination of ethidium shows that it can generate cross-links between histone and plasmid DNA (pUC19) and that the yield depends on the quencher. Altogether, these results illustrate the versatility of the flash−quench technique as a way to generate physiologically relevant DNA−protein adducts via the oxidation of guanine and expand the scope of such cross-linking reactions to include proteins that may associate only transiently with DNA.