The removal of DNA damages by dedicated repair pathways plays a key role in the maintenance of the integrity of genomes and is involved in several pathologies (cancer, neurodegenerative diseases�). In order to detect enzymatic activities of the base excision repair pathway (BER), we devised a new tool based on a set of fluorescent and hairpin-shaped DNA probes immobilized on magnetic microbeads. Each probe is made substrate for a specific DNA-N-glycosylase or AP-endonuclease by incorporating a defined lesion in the double-stranded structure. The excision/incision activity of targeted enzymes, leading to the probe's cleavage, is detected and quantified by spectrofluorometry. This device allows the activities' detection of purified enzymes and within cellular extracts. Likewise, application of such molecular tools to search for inhibitors of repair enzymes was investigated. Altogether, these results validate our bioanalytical device and the associated functional assay to analyze DNA repair activities in a parallelized/miniaturized on support format.

On beads fluorescent assay based on functionalized damaged oligonucleotides to monitor specific DNA repair activities

244th National Meeting (2012)

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