GAS-CHROMATOGRAPHY MASS-SPECTROMETRY WITH HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY PREPURIFICATION FOR MONITORING THE ENDONUCLEASE-III MEDIATED EXCISION OF 5-HYDROXY-5,6-DIHYDROTHYMINE AND 5,6-DIHYDROTHYMINE FROM GAMMA-IRRADIATED DNA

The endonuclease III from Escherichia coli is a repair enzyme which ex hibits both a glycosylase and an endonuclease function. The activity o f the enzyme can be assayed by measuring the released targeted bases i n solution from a sample of modified DNA. In the present study, gas ch romatography-mass spectrometry was used together with an HPLC prepurif ication step in order to single out the released bases, The prepurific ation was found to enhance the specificity and the sensitivity of the assay. Thus, the overall method allowed us to analyze separately 5-hyd roxy-5,6-dihydrothymine from the cis and trans isomers of 6-hydroxy-5, 6-dihydrothymine. Examples of application of the assay are provided wi th the measurement of the E. coli endonuclease III-mediated excision o f 5-hydroxy-5,6-dihydrothymine and 5,6-dihydrothymine from samples of gamma-irradiated DNA in the presence of cysteine. (C) 1998 Elsevier Sc ience B.V. All rights reserved.