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.
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Acknowledgments
The authors would like to acknowledge the past and current members of the Chow laboratory for their input, discussion, and trouble shooting of the protocol. We are also grateful for the assistance from the Laboratory of Macromolecular Analysis and Proteomics (Einstein) with mass spectrometry analysis. O.A.O. was supported by 1F31-GM66607.
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Olabisi, O.A., Chow, CW. (2011). Assay for Protein Modification by Poly-ADP-Ribose In Vitro. In: Tulin, A. (eds) Poly(ADP-ribose) Polymerase. Methods in Molecular Biology, vol 780. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-270-0_3
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DOI: https://doi.org/10.1007/978-1-61779-270-0_3
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Publisher Name: Humana Press, Totowa, NJ
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