DETECTION OF OXIDIZED PURINES AND UV-INDUCED PHOTOPRODUCTS IN DNA OF SINGLE CELLS, BY INCLUSION OF LESION-SPECIFIC ENZYMES IN THE COMET ASSAY

The comet assay (single cell gel electrophoresis) is a rapid, very sen sitive method for the detection of DNA strand breaks at the level of s ingle cells, which is now being applied in genotoxicity testing. We mo dified this method for the detection of a variety of kinds of DNA lesi on, by treating nucleoid DNA in the gel with either formamidopyrimidin e-DNA glycosylase (which recognises ring opened purines, 8-hydroxyguan ine and apurinic/apyrimidinic sites), or uvrABC excinuclease (uvrABC; which has a rather broad specificity, including bulky lesions and UV p hotoproducts). By using this modified assay, we demonstrate the remova l of DNA strand breaks and oxidised purines upon incubating cells afte r treatment with hydrogen peroxide. This modification clearly increase s the usefulness of the assay for the analysis of DNA damage and repai r, for screening human populations for DNA damage, and for testing nov el chemicals for genotoxicity.