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

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


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) 



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.


  1. 1.
    Bonfiglio JJ, Fontana P, Zhang Q et al (2017) Serine ADP-ribosylation depends on HPF1. Mol Cell 65:932–940 e936CrossRefGoogle Scholar
  2. 2.
    Gibson BA, Kraus WL (2012) New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs. Nat Rev Mol Cell Biol 13:411–424CrossRefGoogle Scholar
  3. 3.
    Laing S, Unger M, Koch-Nolte F et al (2011) ADP-ribosylation of arginine. Amino Acids 41:257–269CrossRefGoogle Scholar
  4. 4.
    Schreiber V, Dantzer F, Ame JC et al (2006) Poly(ADP-ribose): novel functions for an old molecule. Nat Rev Mol Cell Biol 7:517–528CrossRefGoogle Scholar
  5. 5.
    Deng Q, Barbieri JT (2008) Molecular mechanisms of the cytotoxicity of ADP-ribosylating toxins. Annu Rev Microbiol 62:271–288CrossRefGoogle Scholar
  6. 6.
    Simon NC, Aktories K, Barbieri JT (2014) Novel bacterial ADP-ribosylating toxins: structure and function. Nat Rev Microbiol 12:599–611CrossRefGoogle Scholar
  7. 7.
    Glowacki G, Braren R, Firner K et al (2002) The family of toxin-related ecto-ADP-ribosyltransferases in humans and the mouse. Protein Sci 11:1657–1670CrossRefGoogle Scholar
  8. 8.
    Hawse WF, Wolberger C (2009) Structure-based mechanism of ADP-ribosylation by sirtuins. J Biol Chem 284:33654–33661CrossRefGoogle Scholar
  9. 9.
    Rack JG, Morra R, Barkauskaite E et al (2015) Identification of a class of protein ADP-Ribosylating sirtuins in microbial pathogens. Mol Cell 59:309–320CrossRefGoogle Scholar
  10. 10.
    Van Meter M, Mao Z, Gorbunova V et al (2011) Repairing split ends: SIRT6, mono-ADP ribosylation and DNA repair. Aging (Albany NY) 3:829–835CrossRefGoogle Scholar
  11. 11.
    Ame JC, Spenlehauer C, De Murcia G (2004) The PARP superfamily. BioEssays 26:882–893CrossRefGoogle Scholar
  12. 12.
    Hottiger MO (2016) SnapShot: ADP-ribosylation signaling. Mol Cell 62:472CrossRefGoogle Scholar
  13. 13.
    Vyas S, Matic I, Uchima L et al (2014) Family-wide analysis of poly(ADP-ribose) polymerase activity. Nat Commun 5:4426CrossRefGoogle Scholar
  14. 14.
    Kiehlbauch CC, Aboul-Ela N, Jacobson EL et al (1993) High resolution fractionation and characterization of ADP-ribose polymers. Anal Biochem 208:26–34CrossRefGoogle Scholar
  15. 15.
    Gupte R, Liu Z, Kraus WL (2017) PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes. Genes Dev 31:101–126CrossRefGoogle Scholar
  16. 16.
    Luo X, Kraus WL (2012) On PAR with PARP: cellular stress signaling through poly(ADP-ribose) and PARP-1. Genes Dev 26:417–432CrossRefGoogle Scholar
  17. 17.
    Ryu KW, Kim DS, Kraus WL (2015) New facets in the regulation of gene expression by ADP-ribosylation and poly(ADP-ribose) polymerases. Chem Rev 115:2453–2481CrossRefGoogle Scholar
  18. 18.
    Barkauskaite E, Jankevicius G, Ladurner AG et al (2013) The recognition and removal of cellular poly(ADP-ribose) signals. FEBS J 280:3491–3507CrossRefGoogle Scholar
  19. 19.
    Fontana P, Bonfiglio JJ, Palazzo L et al (2017) Serine ADP-ribosylation reversal by the hydrolase ARH3. elife 6Google Scholar
  20. 20.
    Teloni F, Altmeyer M (2016) Readers of poly(ADP-ribose): designed to be fit for purpose. Nucleic Acids Res 44:993–1006CrossRefGoogle Scholar
  21. 21.
    Karras GI, Kustatscher G, Buhecha HR et al (2005) The macro domain is an ADP-ribose binding module. EMBO J 24:1911–1920CrossRefGoogle Scholar
  22. 22.
    Kustatscher G, Hothorn M, Pugieux C et al (2005) Splicing regulates NAD metabolite binding to histone macroH2A. Nat Struct Mol Biol 12:624–625CrossRefGoogle Scholar
  23. 23.
    Timinszky G, Till S, Hassa PO et al (2009) A macrodomain-containing histone rearranges chromatin upon sensing PARP1 activation. Nat Struct Mol Biol 16:923–929CrossRefGoogle Scholar
  24. 24.
    Kang HC, Lee YI, Shin JH et al (2011) Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates DNA damage. Proc Natl Acad Sci U S A 108:14103–14108CrossRefGoogle Scholar
  25. 25.
    Wang Z, Michaud GA, Cheng Z et al (2012) Recognition of the iso-ADP-ribose moiety in poly(ADP-ribose) by WWE domains suggests a general mechanism for poly(ADP-ribosyl)ation-dependent ubiquitination. Genes Dev 26:235–240CrossRefGoogle Scholar
  26. 26.
    Zhang Y, Liu S, Mickanin C et al (2011) RNF146 is a poly(ADP-ribose)-directed E3 ligase that regulates axin degradation and Wnt signalling. Nat Cell Biol 13:623–629CrossRefGoogle Scholar
  27. 27.
    Aguilera Gomez A, Van Oorschot MM, Veenendaal T et al (2016) In vivo vizualisation of mono-ADP-ribosylation by dPARP16 upon amino-acid starvation. elife 5:e21475CrossRefGoogle Scholar
  28. 28.
    Bartolomei G, Leutert M, Manzo M et al (2016) Analysis of chromatin ADP-ribosylation at the genome-wide level and at specific loci by ADPr-ChAP. Mol Cell 61:474–485CrossRefGoogle Scholar
  29. 29.
    Gibson BA, Zhang Y, Jiang H et al (2016) Chemical genetic discovery of PARP targets reveals a role for PARP-1 in transcription elongation. Science 353:45–50CrossRefGoogle Scholar
  30. 30.
    Luo X, Ryu KW, Kim DS et al (2017) PARP-1 controls the adipogenic transcriptional program by PARylating C/EBPβ and modulating its transcriptional activity. Mol Cell 65:260–271CrossRefGoogle Scholar
  31. 31.
    Martello R, Leutert M, Jungmichel S et al (2016) Proteome-wide identification of the endogenous ADP-ribosylome of mammalian cells and tissue. Nat Commun 7:12917CrossRefGoogle Scholar
  32. 32.
    Murawska M, Hassler M, Renkawitz-Pohl R et al (2011) Stress-induced PARP activation mediates recruitment of Drosophila Mi-2 to promote heat shock gene expression. PLoS Genet 7:e1002206CrossRefGoogle Scholar
  33. 33.
    Griesenbeck J, Oei SL, Mayer-Kuckuk P et al (1997) Protein-protein interaction of the human poly(ADP-ribosyl)transferase depends on the functional state of the enzyme. Biochemistry 36:7297–7304CrossRefGoogle Scholar
  34. 34.
    Haince JF, Poirier GG, Kirkland JB (2004) Nonisotopic methods for determination of poly(ADP-ribose) levels and detection of poly(ADP-ribose) polymerase. Curr Protoc Cell Biol Chapter 18:Unit 18.17Google Scholar
  35. 35.
    Malanga M, Bachmann S, Panzeter PL et al (1995) Poly(ADP-ribose) quantification at the femtomole level in mammalian cells. Anal Biochem 228:245–251CrossRefGoogle Scholar

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

Personalised recommendations