Formation of 8-oxo-7,8-dihydroguanine-radicals in -irradiated DNA by multiple one-electron oxidations
Journal - Nucleic Acids Research
Received August 10, 2004; Revised November 2, 2004; Accepted November 23, 2004
Electron spin resonance (ESR) studies of radicals formed byradiation-induced multiple one-electron oxidations of guaninemoieties in DNA are reported in this work. Annealing of gamma-irradiatedDNA from 77 to 235 K results in the hydration of one electronoxidized guanine (G•+) to form the 8-hydroxy-7,8-dihydroguanin-7-yl-radical(•GOH) having one ß-proton coupling of 17–28G and an anisotropic nitrogen coupling, A||, of 20 G, A = 0with g|| = 2.0026 and g = 2.0037. Further annealing to 258 Kresults in the formation of a sharp singlet at g = 2.0048 withline-width of 5.3 G that is identified as the 8-oxo-7,8-dihydroguanineone-electron-oxidized radical (8-oxo-G•+). This speciesis formed via two one-electron oxidations of •GOH. Thesetwo one-electron oxidation steps leading to the formation of8-oxo-G•+ from •GOH in DNA, are in accordance withthe expected ease of oxidation of •GOH and 8-oxo-G. Theincorporation of oxygen from water in G•+ leading to •GOHand to 8-oxo-G•+ is verified by ESR studies employing 17Oisotopically enriched water, which provide unambiguous evidencefor the formation of both radicals. ESR analysis of irradiated-DNAin the presence of the electron scavenger, Tl3+, demonstratesthat the cationic pathway leads to the formation of the 8-oxo-G•+.In irradiated DNA–Tl3+ samples, Tl3+ captures electrons.Tl2+ thus produced is a strong oxidant (2.2 V), which is metastableat 77 K and is observed to increase the formation of G•+and subsequently of 8-oxo-G•+ upon annealing. We find thatin the absence of the electron scavenger the yield of 8-oxo-G•+is substantially reduced as a result of electron recombinationswith G•+ and possible reaction with •GOH.
Ensemble and single-molecule fluorescence spectroscopic study of the binding modes of the bis-benzimidazole derivative Hoechst 33258 with DNA
Journal - Nucleic Acids Research
Ensemble and single-molecule fluorescence measurements of 2'-(4-hydroxyphenyl)-5-[5-(4-methylpiperazine-1-yl)benzimidazo-2-yl]-benzimidazole (H-258)– calf thymus (CT)DNA complexes at various [H-258]/[DNA bp] ratios were performedto elucidate the binding of H-258 with DNA. Upon binding todouble-stranded CT DNA (CT ds DNA) at a [H-258]/[DNA bp] ratioof 0.05 the relative fluorescence quantum yield, f, of H-258increases from 0.02 to 0.58. The fluorescence decay can be fittedalmost by a mono-exponential model with a lifetime of 3.6 ns.This indicates that H-258 binds almost quantitatively in theminor groove of DNA at low [H-258]/[DNA bp] ratios. With increasing[H-258]/[DNA bp] ratios, e.g. 0.15 and 0.20, the fluorescencequantum yield of H-258 decreases to 0.28 and 0.19, respectively.Fitting of the fluorescence decays measured for higher [H-258]/[DNAbp] ratios reveals the presence of additional shorter fluorescencelifetime components in the range of 0.5–2.0 ns. Our resultssuggest that H-258 partially intercalates in G:C sequences athigher [H-258]/[DNA bp] ratios reflected by a lifetime componentof 1.5–2 ns. In addition, stacking or adsorption of H-258molecules on DNA occurs at higher [H-258]/[DNA bp] ratios. Thesemolecules exhibit a short fluorescence lifetime of 500 ps andare more exposed to the aqueous environment. Fluorescence transientsof the intensity and lifetime of single H-258 CT ds DNA demonstratethat weakly (unspecific) bound H-258 molecules exhibit a shorterfluorescence lifetime and a strongly reduced photostability.