DNA damage excision repair in microplate wells with chemiluminescence detection: Development and perspectives

The development of in vitro repair assays with human cell-free extracts led to new insights on the mechanism of excision of DNA damage which consists of incision/excision and repair synthesis/ligation. We have adapted the rep air synthesis reaction with cells extracts incubated with damaged plasmid D NA performed in liquid phase to solid phase by DNA adsorption into micropla te wells. Since cells extracts are repair competent in base excision and nu cleotide excision repair, all types of substrate DNA lesions were detected with chemiluminescence measurement after incorporation of biotin-deoxynucle otide during the repair synthesis step. Derivatives of our initial 3D-assay (DNA damage detection) have been set up to: i) screen antioxidative compou nds and NER inhibitors; ii) capture genomic DNA (3D(Cell)-assay) that allow s detection of alkylated base and consequently determines the kinetics of t he cellular repair; and iii) immunodetect the repair proteins in an ELISA r eaction (3D(Rec)-assay). The 3D derived assays are presented and discussed. (C) Societe francaise de biochimie et biologie moleculaire / Elsevier, Par is.