Oxidative damage to DNA plays a role in the progression of many diseases, including cancer. 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. Glutathione is a small cysteine-containing peptide responsible for redox buffering in the cell. Here, we investigated whether it might be possible for glutathione, a strong reductant, 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 as formed by the flash-quench method. Samples containing Ru(phen)2dppz 2+ [phen = phenanthroline, dppz = dipyridophenazine], Co(NH3)5Cl2+, histone protein, salmon sperm 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. Our 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. We also used the gel shift assay: pBR322 plasmid was seen to migrate to the wells upon flash quench treatment in the presence of histone, but this behavior is strongly inhibited by glutathione. Lastly, we investigated whether or not glutathione interfered with the quenching process by doing emission spectroscopy experiments. We found that glutathione only slightly interfered with the quenching of Ru(phen)2dppz 2+ , indicating that it most likely reacts with the guanine radical directly.

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

249th National Meeting (2015)

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