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Biochemical and Biophysical Assays of PAR-WWE Domain Interactions and Production of iso-ADPr for PAR-Binding Analysis

  • Zhizhi Wang
  • Wenqing Xu
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1813)

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.

Key words

PARP PAR PARylation ADP-ribose MAR iso-ADPr WWE domain 

Notes

Acknowledgment

This work was supported by NIH grant R01 GM099766 to W.X.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biological StructureUniversity of WashingtonSeattleUSA

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