Assay for protein modification by poly-ADP-ribose in vitro

Opeyemi A. Olabisi, Chi Wing Chow

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

The enzymatic function of poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) is central to many of its function as a component of DNA repair machinery, modulator of gene transcription, and cell differentiation. While the auto-modification domain of PARP has been shown to be a primary acceptor site of poly-ADP ribose (pADPr), other DNA binding nuclear proteins are also modified by pADPr. It is -generally accepted that pADPr polymer is built upon the carboxyl side chain of specific Glu, Asp, and/or Lys residues within the target protein. Identification of the unique amino acid acceptors of pADPr in these nuclear proteins is an active area of study. Because of the heterogeneity of pADPr chain on modified -protein targets, the resulting modified proteins have unpredictable final masses, making it difficult to -identify acceptor amino acids. Using recombinant proteins, in vitro pADP ribosylation assay and mass spectrometry, we have been able to identify conserved Glu residue in transcription factor NFAT that is enzymatically modified in vitro with pADPr by PARP-1. We discuss this protocol here as a model approach for identifying pADPr acceptors in other nuclear proteins.

Original languageEnglish (US)
Title of host publicationPoly(ADP-ribose) Polymerase
Subtitle of host publicationMethods and Protocols
EditorsAlexei V. Tulin
Pages47-55
Number of pages9
DOIs
Publication statusPublished - Oct 24 2011

Publication series

NameMethods in Molecular Biology
Volume780
ISSN (Print)1064-3745

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Keywords

  • Poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP)
  • Poly-ADP ribose (pADPr)
  • Transcription factor NFAT

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Olabisi, O. A., & Chow, C. W. (2011). Assay for protein modification by poly-ADP-ribose in vitro. In A. V. Tulin (Ed.), Poly(ADP-ribose) Polymerase: Methods and Protocols (pp. 47-55). (Methods in Molecular Biology; Vol. 780). https://doi.org/10.1007/978-1-61779-270-0_3