MULTIWELL COLORIMETRIC ASSAY FOR DNA REPAIR PROTEIN.

DESCRIPTION (provided by applicant): DNA damages are key events during carcinogenesis. Deficiencies in repairing damaged DNA represent a poor prognosis during tumorigenesis and/or cancer treatment. Besides monitoring DNA damage formation, it would be helpful to monitor DNA repair protein activity. We propose to develop a new technology that allows for high-throughput and fast analysis of the most common DNA repair proteins. We will use a damaged DNA probe immobilized onto a 96-well microplate to capture DNA repair proteins in an extract from cells or tissues. Six biotinylated oligonucleotides containing different DNA damage types will be synthesized. The DNA-protein binding conditions will be developed by using purified recombinant DNA repair proteins. Antibodies raised specifically against DNA repair proteins will identify the bound protein and yield a quantitative result in two hours. Finally, the procedure will be applied to relevant samples such as extracts from damaging agent-treated cell lines and disease models. Such assay will help elucidate the molecular mechanisms of the DNA damage and repair pathways. It would also provide scientists and clinicians with a unique tool to answer seminal questions in risk assessment in a fraction of the time normally required.