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Generating Protein-Linked and Protein-Free Mono-, Oligo-, and Poly(ADP-Ribose) In Vitro

  • Ken Y. Lin
  • Dan Huang
  • W. Lee Kraus
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1813)

Abstract

ADP-ribosylation is a covalent posttranslational modification of proteins that is catalyzed by various types of ADP-ribosyltransferase (ART) enzymes, including members of the poly(ADP-ribose) polymerase (PARP) family. ADP-ribose (ADPR) modifications can occur as mono(ADP-ribosyl)ation, oligo(ADP-ribosyl)ation, or poly(ADP-ribosyl)ation, depending on the particular ART enzyme catalyzing the reaction, as well as the specific reaction conditions. Understanding the biology of ADP-ribosylation requires facile and robust means of generating and detecting the modification in all of its forms. Here we describe how to generate protein-linked mono(ADP-ribose), oligo(ADP-ribose), and poly(ADP-ribose) (MAR, OAR, and PAR, respectively) in vitro as an automodification of PARPs 1 or 3. First, epitope-tagged PARP-1 (a PARP polyenzyme) and PARP-3 (a PARP monoenzyme) are expressed individually in insect cells using baculovirus expression vectors, and purified using immunoaffinity chromatography. Second, the purified recombinant PARPs are incubated individually in the presence of different concentrations of NAD+ (as a donor of ADPR groups) and sheared DNA (to activate their catalytic activities) resulting in various forms of auto-ADP-ribosylation. Third, the products are confirmed using ADPR detection reagents that can distinguish among MAR, OAR, and PAR. Finally, if desired, the OAR and PAR can be deproteinized. The protein-linked and free MAR, OAR, and PAR generated in these reactions can be used as standards, substrates, or binding partners in a variety of ADPR-related assays.

Key words

ADP-ribose (ADPR) ADPR binding domain (ARBD) ADP-ribosylation ADP-ribosyltransferase (ART) Automodification Mono(ADP-ribosyl)ation (MARylation) Nicotinamide adenosine dinucleotide (NAD+Oligo(ADP-ribosyl)ation (OARylation) Poly(ADP-ribose) polymerase (PARP) Poly(ADP-ribosyl)ation (PARylation) Posttranslational modification (PTM) 

Notes

Acknowledgments

The PARP-related research in the Kraus lab is supported by grants from the National Institutes of Health, NIDDK (DK069710), the Cancer Prevention and Research Institute of Texas (CPRIT) (RP160319), and the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowments.

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Copyright information

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

Authors and Affiliations

  1. 1.Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology SciencesUniversity of Texas Southwestern Medical CenterDallasUSA
  2. 2.Division of Basic Research, Department of Obstetrics and GynecologyUniversity of Texas Southwestern Medical CenterDallasUSA
  3. 3.Department of Cardiovascular Diseases, Clinical Center of Human Gene Research, Union Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanP.R. China

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