Ruthenium-mediated DNA oxidation in condensed media

DNA oxidation is a process that has been implicated in mutagenesis and cancer. Previous studies to determine the pathways and products of DNA oxidation have been carried out in dilute aqueous solutions or organic solvents. These conditions may or may not be representative of the interior of a cell where there is a high concentration of biomolecules and DNA is in a highly condensed form. This work focuses on determining the role of DNA condensation on the mechanisms and products of DNA oxidation. DNA is condensed in reverse micelle (RM) media or in the presence of histone subunits. The DNA oxidation reactions are carried out using a flash/quench photochemical technique and ruthenium(II) polypyridyl complexes to produce ruthenium(III) oxidants or singlet oxygen.One-electron guanine oxidation in single- and double-stranded DNA has been investigated in anionic RMs. Tris(2-2'-bipyridine)ruthenium(II) chloride ([Ru(bpy)3]Cl2) is used to photochemically produce oxidatively generated damage to DNA that is quantified by high-resolution polyacrylamide gel electrophoresis. The steady state quenching efficiency of Ru(bpy)32+ with the oxidative quencher potassium ferricyanide (K3[Fe(CN)6]) is two-fold higher in buffer than in RMs. Consistent with the difference in quenching efficiencies in the two media, the amount of piperidine-labile oxidation products from double-stranded DNA is 1.5-fold higher in buffer than RMs. However, a 13-fold decrease is observed in RMs for single-stranded DNA. Circular dichroism spectra reveal that the single-stranded DNA undergoes a large structural change in the anionic RMs. This structural change may be due to cation-mediated adsorption of the DNA phosphates on the anionic headgroups of the surfactant, which protect the guanines from oxidation.DNA oxidation also has been investigated in the medium of cationic RMs. The oxidative chemistry is photochemically initiated using the DNA intercalator bis(bipyridine)dipyridophenazine ruthenium(II) chloride ([Ru(bpy)2dppz]Cl 2) bound to double-stranded DNA in the RMs. In buffer solution, the addition of the oxidative quencher [Fe(CN)6]3-leads to an increase in the amount of piperidine-labile guanine oxidation products generated via one-electron oxidation. In RMs, however, the yield of oxidatively-generated damage is decreased. With or without [Fe(CN)6]3- quencher in the RMs, the yield of oxidatively-generated products is approximately the same. Under anaerobic conditions, samples in RMs show decreased levels of piperidine-labile oxidation products, suggesting that the primary oxidant in RMs is singlet oxygen. Guanine oxidation is enhanced in D2O and deuterated heptane and diminished in the presence of sodium azide in RMs, also supporting 1O2 as the main guanine oxidant in RMs. Isotopic labeling experiments show that the O-atom in an oxidation product produced in RMs is not from water and likely coming from O2. The observed change in guanine oxidation mechanism from a one-electron process in buffer to mostly 1O2 in RMs illustrates the importance of both DNA structure and DNA environment on the chemistry of guanine oxidation.DNA oxidation in the presence of histone subunits H1 and H3 has also been investigated. Initial one-electron and 1O2 oxidation of guanine can lead to DNA-protein cross-links. DNA-protein crosslinks between histone subunits H1 and H3 and oligonucleotides were explored with chemically modified histones with methylated lysine residues, a post-translational modification important in regulation of cellular processes including transciption. DNA-protein cross-links are observed between unmodified and methylated histone subunits. The amount of DNA oxidation is the same in the presence of histone H1 and methylated H1 but is decreased in the presence of H3 and methylate H3. This may be due to the amount and type of lysine methylation of the histone subunits. These results further show the importance of environment on DNA oxidation.