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

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ADP-ribosylation and NAD+ Utilizing Enzymes

Part of the book series: Methods in Molecular Biology ((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.

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Acknowledgment

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

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Authors and Affiliations

  1. Department of Biological Structure, University of Washington, Seattle, WA, USA

    Zhizhi Wang & Wenqing Xu

Authors
  1. Zhizhi Wang
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  2. Wenqing Xu
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Correspondence to Wenqing Xu .

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Editors and Affiliations

  1. Department of Biology, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Paul Chang

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Wang, Z., Xu, W. (2018). Biochemical and Biophysical Assays of PAR-WWE Domain Interactions and Production of iso-ADPr for PAR-Binding Analysis. In: Chang, P. (eds) ADP-ribosylation and NAD+ Utilizing Enzymes. Methods in Molecular Biology, vol 1813. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8588-3_5

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  • DOI: https://doi.org/10.1007/978-1-4939-8588-3_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-8587-6

  • Online ISBN: 978-1-4939-8588-3

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