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. One common permanent damage product is 8-oxoguanine, which often leads to mutation and has a lower oxidation potential (~0.7 V) relative to guanine (~1.3 V). The flash-quench technique, in which a strong oxidant is prepared in situ by oxidative quenching of a photosensitizer, can be employed to oxidize guanine in double-helical DNA and it can induce DNA-protein crosslinking. In contrast to Ru(phen)2dppz2+, [phen = phenanthroline, dppz = dipyridophenazine], which oxidizes G by flash quench, the metallointercalator Os(phen)2dppz 2+ should selectively oxidize 8-oxo-G, but not G, via flash quench, because of its lower (3+/2+) potential of 1.15 V. To induce the production of 8-oxo-G in the DNA, Ru(NH3)63+ and Ru(bpy)32+ – the quencher and photosensitizer, respectively – were added to pBR322 plasmid DNA and irradiated for 30 minutes at 442 nm using a HeCd laser; analogous experiments done with Ru(phen)2dppz2+ as the photosensitizer have been shown to generate 8-oxo-G in duplex DNA . The photosensitizer and quencher were then removed from the sample via ultrafiltration at high ionic strength and the sample of DNA exchanged into a lower ionic strength phosphate buffer (pH 7). Subsequently, the ability of Os(phen)2dppz 2+ to induce oxidative DNA-protein crosslinking was then compared for pBR322 and pBR322 that contained 8-oxo-G sites. Samples contained Os(phen)2dppz2+, Co(NH3)5Cl2+, and histone or poly(lysine) as the protein, in addition to the DNA. Samples were irradiated with 442 nm light to induce crosslinking and it was observed that oxidative DNA-protein crosslinking occurred more readily in the 8-oxo-G containing pBR322 DNA and was also more efficient using poly(lysine) rather than histone protein.

Oxidative DNA-protein crosslinking induced by oxidation of 8-oxoguanine by Os(phen)2dppz3+ via the flash-quench technique

253rd National Meeting (2017)

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