Presentation Title

Inhibition of oxidative DNA-protein crosslinking via vitamin C

Faculty Mentor

Dr. Eric Stemp

Start Date

18-11-2017 2:15 PM

End Date

18-11-2017 3:15 PM

Location

BSC-Ursa Minor 28

Session

Poster 3

Type of Presentation

Poster

Subject Area

physical_mathematical_sciences

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. 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 this methodology, 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 chloroform extraction assay proved ineffective at detecting oxidative DNA-protein crosslinking in the presence of vitamin C, the gel shift assay was employed. Samples containing Ru(phen)2dppz2+,Co(NH3)5Cl2+, histone protein, pUC19 DNA and either water or vitamin C (at different concentrations) in a pH 7 sodium phosphate buffer were irradiated with the 442 nm output of a HeCd laser. Samples were then analyzed by agarose gel electrophoresis, and the extent of crosslinking was evident from both the disappearance of the free DNA band and the appearance of material with retarded mobility. A modified version of the Comet Assay was also used, in order to detect where the DNA is cleaved at the sites of damage. Our results demonstrated a lower percentage of crosslinking in the presence of vitamin C, and also that aged vitamin C was still effective at preventing oxidative damage. The inhibition of oxidative damage afforded by vitamin C was surprising, given that vitamin C at pH 7 is predominantly in the negatively charged ascorbate ion form, and as such would not be expected to bind to DNA because of electrostatic repulsion.

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Nov 18th, 2:15 PM Nov 18th, 3:15 PM

Inhibition of oxidative DNA-protein crosslinking via vitamin C

BSC-Ursa Minor 28

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. 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 this methodology, 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 chloroform extraction assay proved ineffective at detecting oxidative DNA-protein crosslinking in the presence of vitamin C, the gel shift assay was employed. Samples containing Ru(phen)2dppz2+,Co(NH3)5Cl2+, histone protein, pUC19 DNA and either water or vitamin C (at different concentrations) in a pH 7 sodium phosphate buffer were irradiated with the 442 nm output of a HeCd laser. Samples were then analyzed by agarose gel electrophoresis, and the extent of crosslinking was evident from both the disappearance of the free DNA band and the appearance of material with retarded mobility. A modified version of the Comet Assay was also used, in order to detect where the DNA is cleaved at the sites of damage. Our results demonstrated a lower percentage of crosslinking in the presence of vitamin C, and also that aged vitamin C was still effective at preventing oxidative damage. The inhibition of oxidative damage afforded by vitamin C was surprising, given that vitamin C at pH 7 is predominantly in the negatively charged ascorbate ion form, and as such would not be expected to bind to DNA because of electrostatic repulsion.