Oxidative damage to DNA plays a role in many diseases, and this damage is observed primarily at guanine (G) because it is the most easily oxidized base. The flash-quench technique is a method that is used for selective guanine oxidation in double-helical DNA and it can induce DNA-protein crosslinking. We investigated whether glutathione, a small cysteine-containing peptide responsible for redox buffering in the cell, to play a role in diminishing DNA damage (e.g. DNA-protein crosslinks) by engaging in redox repair of the 1-electron oxidized guanine base. Samples containing Ru(phen)2dppz 2+ [phen = phenanthroline, dppz = dipyridophenazine], Co(NH3)5Cl2+, histone, sonicated calf thymus DNA and glutathione (reduced form) were irradiated for 0-240 seconds with 442 nm light to effect guanine damage. The % crosslinked was determined by the chloroform extraction assay: NaCl and SDS were added to the samples to disrupt noncovalent interactions between DNA and protein, then the proteinaceous material was extracted into 24:1 chloroform:isoamyl alcohol. After centrifuging, the aqueous phase containing unreacted DNA was assayed at 260 nm by uv spectroscopy. Results showed as the irradiation time increased, the absorption of free DNA decreased, consistent with crosslinking. Less DNA-protein crosslinking was observed when glutathione was present in the samples. Using the gel shift assay, we saw pBR322 plasmid migrated to the wells (consistent with oxidative DNA-protein crosslinking) upon flash quench treatment in the presence of histone, but this behavior is strongly inhibited by glutathione. Interestingly, preliminary control experiments indicate that oxidized glutathione also shows a small inhibitory effect, possibly as a result of reduction by the photoexcited ruthenium complex.

Effect of glutathione on DNA-protein crosslinking caused by guanine oxidation

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.