J R Milligan1, J A Aguilera, J F Ward. 1. Department of Radiology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0610, USA. jmilligan@ucsd.edu
Abstract
PURPOSE: Gamma irradiation of an aqueous solution containing thiocyanate ions produces the strongly oxidizing intermediate (SCN)2*-. Reaction of this species with plasmid DNA produces damage that is revealed as strand breaks after incubation with the Escherichia coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). It has been previously reported that the yield of damage is highly sensitive to the experimental conditions, leading to the suspicion that electron transfer between DNA and (SCN)2*- is reversible. In principle this makes it possible to determine the oxidation potential for plasmid DNA (more formally the reduction potential of one-electron oxidized plasmid DNA), a fundamental parameter describing the reactivity of DNA towards electron transfer reactions. MATERIALS AND METHODS: Aqueous solutions of plasmid DNA and thiocyanate ions were subjected to 137Cs gamma-irradiation. After irradiation, the plasmid was incubated with the E. coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). The yield of this damage was quantified by using agarose gel electrophoresis to identify the fraction of the plasmid population that contains strand breaks. RESULTS: The yield of FPG-sensitive sites decreases with increasing thiocyanate concentration, decreasing DNA concentration, and increasing dose rate. By making some simple assumptions about the chemical reactions that produce DNA damage, it is possible to derive a quantitative mathematical model for the yield of FPG-sensitive sites. A good agreement was found between this model and the experimental observations over a wide range of conditions (thiocyanate concentrations, DNA concentrations, and dose rates that vary by 20-, 40-, and 150-fold respectively). CONCLUSIONS: It was possible to assign a value to the equilibrium constant for the one electron transfer reaction between the two radical species (SCN)2*- and DNA-G*+. This leads to an estimate of the reduction potential at pH 7 for the couple DNA G*+/DNA of E7 = +1.39+/-0.01V.
PURPOSE: Gamma irradiation of an aqueous solution containing thiocyanate ions produces the strongly oxidizing intermediate (SCN)2*-. Reaction of this species with plasmid DNA produces damage that is revealed as strand breaks after incubation with the Escherichia coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). It has been previously reported that the yield of damage is highly sensitive to the experimental conditions, leading to the suspicion that electron transfer between DNA and (SCN)2*- is reversible. In principle this makes it possible to determine the oxidation potential for plasmid DNA (more formally the reduction potential of one-electron oxidized plasmid DNA), a fundamental parameter describing the reactivity of DNA towards electron transfer reactions. MATERIALS AND METHODS: Aqueous solutions of plasmid DNA and thiocyanate ions were subjected to 137Cs gamma-irradiation. After irradiation, the plasmid was incubated with the E. coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). The yield of this damage was quantified by using agarose gel electrophoresis to identify the fraction of the plasmid population that contains strand breaks. RESULTS: The yield of FPG-sensitive sites decreases with increasing thiocyanate concentration, decreasing DNA concentration, and increasing dose rate. By making some simple assumptions about the chemical reactions that produce DNA damage, it is possible to derive a quantitative mathematical model for the yield of FPG-sensitive sites. A good agreement was found between this model and the experimental observations over a wide range of conditions (thiocyanate concentrations, DNA concentrations, and dose rates that vary by 20-, 40-, and 150-fold respectively). CONCLUSIONS: It was possible to assign a value to the equilibrium constant for the one electron transfer reaction between the two radical species (SCN)2*- and DNA-G*+. This leads to an estimate of the reduction potential at pH 7 for the couple DNA G*+/DNA of E7 = +1.39+/-0.01V.
Authors: Lata I Shukla; Amitava Adhikary; Robert Pazdro; David Becker; Michael D Sevilla Journal: Nucleic Acids Res Date: 2004-12-15 Impact factor: 16.971
Authors: Amitava Adhikary; Aramice Y S Malkhasian; Sean Collins; Jessica Koppen; David Becker; Michael D Sevilla Journal: Nucleic Acids Res Date: 2005-10-04 Impact factor: 16.971