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 γ-irradiated DNA

Add time:08/11/2019    Source:sciencedirect.com

The endonuclease III from Escherichia coli is a repair enzyme which exhibits both a glycosylase and an endonuclease functions. The activity of the enzyme can be assayed by measuring the released targeted bases in solution from a sample of modified DNA. In the present study, gas chromatography–mass spectrometry was used together with an HPLC prepurification step in order to single out the released bases. The prepurification was found to enhance the specificity and the sensitivity of the assay. Thus, the overall method allowed us to analyze separately 5-hydroxy-5,6-dihydrothymine from the cis and trans isomers of 6-hydroxy-5,6-dihydrothymine. Examples of application of the assay are provided with the measurement of the E. coli endonuclease III-mediated excision of 5-hydroxy-5,6-dihydrothymine and 5,6-dihydrothymine from samples of γ-irradiated DNA in the presence of cysteine.

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