Biochemical and biophysical assays of PAR-WWE domain interactions and production of iso-ADPr for PAR-binding analysis

Abstract

The poly(ADP-ribose) polymerase (PARP) family of proteins utilize NAD + as the substrate to modify protein acceptors with either mono(ADP-ribose) (MAR) or poly(ADP-ribose) (PAR). MAR and PAR have been shown to regulate distinct cellular processes. Iso-ADP-ribose (iso-ADPr) is the smallest internal PAR structural unit containing the characteristic ribose-ribose glycosidic bond formed during poly(ADP-ribosyl)ation. The WWE domain of RNF146 specifically recognizes the iso-ADPr moiety in PAR but does not interact with MAR. This provides a way to distinguish PAR from MAR modification and to isolate PARylated proteins. Iso-ADPr can be used to detect the PAR-specific binding properties of interested proteins. Here we describe the detailed method to generate and purify iso-ADPr and its use in PAR-binding analysis through isothermal titration calorimetry (ITC) analysis.