A scintillation proximity assay for poly(ADP-ribose) polymerase

Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear protein in most o f the eukaryotic tissues. When activated by DNA damage, PARP synthesizes po ly(ADP-ribose) from NAD, Conventional radioactive PARP enzyme assay require s the separation of the polymer product from the NAD substrate, a rate-limi ting step that hampers large-scale chemical library screening to identify n ovel small-molecule PARP inhibitors. By using biotinylated NAD, we have dev eloped a scintillation proximity assay (SPA) for PARP. We demonstrated that PARP can incorporate the biotinylated ADP-ribose units into the radioactiv e poly-(ADP-ribose) polymer, which can directly bind and excite the strepta vidin-conjugated scintillation beads. PARP-SPA can be readily adapted to a 96-well format for automatic high-throughput screening for PARP inhibitors. (C) 2000 Academic Press.