Abstract
Poly-ADP-ribose polymerases (PARPs) comprise a family of 17 distinct enzymes that catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to acceptor sites on protein targets. PARPs have been implicated in a number of essential signaling pathways regulating both normal cell function and pathophysiology. To understand the physiological role of each PARP family member in the cell we need to identify the direct targets for each unique PARP in a cellular context. PARP-family member-specific target identification is challenging because of their shared catalytic mechanism and functional redundancy. To address this challenge, we have engineered a PARP variant that efficiently uses an orthogonal NAD+ analog, an analog that endogenous PARPs cannot use, as a substrate for ADP-ribosylation. The protocols in this unit describe a general procedure for using engineered PARP variants-orthogonal NAD+ analog pairs for labeling and identifying the direct targets of the poly-subfamily of PARPs (PARPs 1-3, 5, and 6).