Detection of Oxidised Purines and UV-induced Photoproducts in DNA of Single Cells,by Inclusion of Lesion-specific Enzymes in the Comet Assay

Abstract

The comet assay (single cell gel electrophoresis) is a rapid, very sensitive method for the detection of DNA strand breaks at the level of single cells, which is now being applied in genotojricity testing. We modified this method for the detection of a variety of kinds of DNA lesion, by treating nucleoid DNA in the gel with either formamidopyrimidine-DNA glycosylase (which recognises ring opened purines, 8-hydroxyguanine and apurinic/apyrimidinic sites), or uvrABC excinuclease (uvrABC; which has a rather broad specificity, including bulky lesions and UV photoproducts). By using this modified assay, we demonstrate the removal of DNA strand breaks and oxidised purines upon incubating cells after treatment with hydrogen peroxide. This modification clearly increases the usefulness of the assay for the analysis of DNA damage and repair, for screening human populations for DNA damage, and for testing novel chemicals for genotoxicity.

Keywords

DNA damage comet assay formamidopyrimidine-DNA glycosylase uvrABC excinuclease

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