Presentation Title

Inhibition of Oxidative DNA-protein Crosslinking via Vitamin C

Start Date

November 2016

End Date

November 2016

Location

HUB 379

Type of Presentation

Oral Talk

Abstract

Oxidative damage is involved in the formation of free radicals, which can cause various diseases. In DNA, this damage is observed primarily at guanine (G) because it is the most easily oxidized base and one form of oxidative damage is DNA-protein crosslinking. Previous projects exclusively tested the effects of bottled green tea, which contains vitamin C as a preservative, for its antioxidative properties. Here, we examined whether vitamin C alone can inhibit oxidative DNA damage. The flash-quench technique is a method that is used for guanine oxidation and it can induce DNA-protein crosslinking. In the flash quench technique, the intercalator, Ru(phen)2dppz 2+[phen = phenanthroline,dppz = dipyridophenazine], is excited with a laser and gives an electron to the quencher, Co(NH3)5Cl2+ . The intercalator takes an electron from guanine, creating the guanine radical, which then interacts with protein. After the performance of a chloroform extraction assay proved ineffective with vitamin C, another method was used. In our experiment, samples containing Ru(phen)2dppz 2+,Co(NH3)5Cl2+, histone protein, pUC19 DNA and either water or vitamin C (at 3 different concentrations) were irradiated for 4 minutes. Here, the level of crosslinking was observed via a gel shift assay: a 2% agarose gel was made, SDS was added, as was blue dye to facilitate loading the gel run for 150 minutes at 85V, and finally imaging the gel to view the results. Standardizing the vitamin C to pH of 7 converts the ascorbic acid into ascorbate ion, a form that is hypothesized not to interact likewise with DNA. Our results demonstrated a greater percentage of crosslinking material at lower concentrations of vitamin C than at the higher concentrations. Future work includes performance of a comet assay to observe the effects of oxidative DNA damage and the inhibition via vitamin C at the cellular level.

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Nov 12th, 10:30 AM Nov 12th, 10:45 AM

Inhibition of Oxidative DNA-protein Crosslinking via Vitamin C

HUB 379

Oxidative damage is involved in the formation of free radicals, which can cause various diseases. In DNA, this damage is observed primarily at guanine (G) because it is the most easily oxidized base and one form of oxidative damage is DNA-protein crosslinking. Previous projects exclusively tested the effects of bottled green tea, which contains vitamin C as a preservative, for its antioxidative properties. Here, we examined whether vitamin C alone can inhibit oxidative DNA damage. The flash-quench technique is a method that is used for guanine oxidation and it can induce DNA-protein crosslinking. In the flash quench technique, the intercalator, Ru(phen)2dppz 2+[phen = phenanthroline,dppz = dipyridophenazine], is excited with a laser and gives an electron to the quencher, Co(NH3)5Cl2+ . The intercalator takes an electron from guanine, creating the guanine radical, which then interacts with protein. After the performance of a chloroform extraction assay proved ineffective with vitamin C, another method was used. In our experiment, samples containing Ru(phen)2dppz 2+,Co(NH3)5Cl2+, histone protein, pUC19 DNA and either water or vitamin C (at 3 different concentrations) were irradiated for 4 minutes. Here, the level of crosslinking was observed via a gel shift assay: a 2% agarose gel was made, SDS was added, as was blue dye to facilitate loading the gel run for 150 minutes at 85V, and finally imaging the gel to view the results. Standardizing the vitamin C to pH of 7 converts the ascorbic acid into ascorbate ion, a form that is hypothesized not to interact likewise with DNA. Our results demonstrated a greater percentage of crosslinking material at lower concentrations of vitamin C than at the higher concentrations. Future work includes performance of a comet assay to observe the effects of oxidative DNA damage and the inhibition via vitamin C at the cellular level.