Quantitative Measurement of the Diastereoisomers of CIS Thymidine Glycol in Gamma-Irradiated DNA

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Abstract
A technique for determing the relative content of each of the diastereoisomers of cis thymidine glycol (dTG) in DNA exposed to ionizing radiation has been developed. [3H]thymidine DNA was gamma-irradiated, digested to 2′-deoxyribonucleosides, authentic [14C] (+, -) cis dTG added to the digestate and the mixture resolved by HPLC. 3H fractions coeluting with [14C] (+, -) dTG were collected and acetylated. The acetoxy derivatives.of (+) and (-) cis dTG were easily resolved by a second HPLC analysis and their absolute configuration determined by NMR amd mass spectroscopies. We have constructed a dose-response curve for formation of each isomer in gamma-irradiated DNA and shown that they are formed in equal amounts. This technique may be used to determine the relative formation of cis dTG isomers in DNA resulting from other oxidative stresses and whether repair of these is influenced by their configuration.