title: DNA-protein cross-linking via guanine oxidation: dependence upon protein and photosensitizer.
abstract: 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)(2)dppz(2+) [phen = 1,10-phenanthroline; dppz = dipyridophenazine] > Ru(bpy)(3)(2+) [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 (32)P-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.
authors.affiliation: Department of Physical Sciences and Mathematics, Mount St. Mary's College, Los Angeles, California 90049, USA.
authors.forename: Kristina
authors.initials: K
authors.lastname: Kurbanyan
authors.forename: Kim L
authors.initials: KL
authors.lastname: Nguyen
authors.forename: Phuong
authors.initials: P
authors.lastname: To
authors.forename: Eunice V
authors.initials: EV
authors.lastname: Rivas
authors.forename: Alexis M K
authors.initials: AM
authors.lastname: Lueras
authors.forename: Cynthia
authors.initials: C
authors.lastname: Kosinski
authors.forename: Mary
authors.initials: M
authors.lastname: Steryo
authors.forename: Arcelia
authors.initials: A
authors.lastname: González
authors.forename: Daisy Ann
authors.initials: DA
authors.lastname: Mah
authors.forename: Eric D A
authors.initials: ED
authors.lastname: Stemp
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry; D003574:Cytochrome c Group / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D004563:Electrochemistry / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006147:Guanine / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006657:Histones / Q000737:chemistry*; D006736:Horses; D017665:Hydroxyl Radical / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010777:Photochemistry / Q000379:methods; D017319:Photosensitizing Agents / Q000032:analysis / Q000737:chemistry*; D010880:Piperidines / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry*
chemical_list: D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D006657:Histones; D017319:Photosensitizing Agents; D010880:Piperidines; D012710:Serum Albumin, Bovine; D017665:Hydroxyl Radical; D006418:Heme; D006147:Guanine; D004247:DNA; C427203:calf thymus DNA