Dictyostelium as a Model to Assess Site-Specific ADP-Ribosylation Events

Part of the Methods in Molecular Biology book series (MIMB, volume 1813)


The amoeba Dictyostelium discoideum is a single-cell organism that can undergo a simple developmental program, making it an excellent model to study the molecular mechanisms of cell motility, signal transduction, and cell-type differentiation. A variety of human genes that are absent or show limited conservation in other invertebrate models have been identified in this organism. This includes ADP-ribosyltransferases, also known as poly-ADP-ribose polymerases (PARPs), a family of proteins that catalyze the addition of single or poly-ADP-ribose moieties onto target proteins. The genetic tractability of Dictyostelium and its relatively simple genome structure makes it possible to disrupt PARP gene combinations, in addition to specific ADP-ribosylation sites at endogenous loci. Together, this makes Dictyostelium an attractive model to assess how ADP-ribosylation regulates a variety of cellular processes including DNA repair, transcription, and cell-type specification. Here we describe a range of techniques to study ADP-ribosylation in Dictyostelium, including analysis of ADP-ribosylation events in vitro and in vivo, in addition to approaches to assess the functional roles of this modification in vivo.

Key words

ADP-ribosylation PARP ADP-ribosyltransferase Dictyostelium DNA repair 



We thank members of the Lakin and Pears laboratories for constructive comments during the preparation of this manuscript. N.L.’s laboratory is supported by Cancer Research UK (; grant C1521/A12353), Medical Research Council (; MR/L000164/1), and NC3Rs (; NC/K00137X/1). AR was supported by a Clarendon Award (University of Oxford). C.P.’s laboratory is supported by NC3Rs (; NC/M000834/1).


  1. 1.
    Citarelli M, Teotia S, Lamb RS (2010) Evolutionary history of the poly(ADP-ribose) polymerase gene family in eukaryotes. BMC Evol Biol 10:308. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Otto H, Reche PA, Bazan F, Dittmar K, Haag F, Koch-Nolte F (2005) In silico characterization of the family of PARP-like poly(ADP-ribosyl)transferases (pARTs). BMC Genomics 6:139. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Vyas S, Matic I, Uchima L, Rood J, Zaja R, Hay RT, Ahel I, Chang P (2014) Family-wide analysis of poly(ADP-ribose) polymerase activity. Nat Commun 5:4426. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Block WD, Lees-Miller SP (2005) Putative homologues of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and other components of the non-homologous end joining machinery in Dictyostelium discoideum. DNA Repair (Amst) 4(10):1061–1065. CrossRefGoogle Scholar
  5. 5.
    Hsu DW, Gaudet P, Hudson JJ, Pears CJ, Lakin ND (2006) DNA damage signaling and repair in Dictyostelium discoideum. Cell Cycle 5(7):702–708CrossRefGoogle Scholar
  6. 6.
    Hudson JJ, Hsu DW, Guo K, Zhukovskaya N, Liu PH, Williams JG, Pears CJ, Lakin ND (2005) DNA-PKcs-dependent signaling of DNA damage in Dictyostelium discoideum. Curr Biol 15(20):1880–1885. CrossRefPubMedGoogle Scholar
  7. 7.
    Muramoto T, Chubb JR (2008) Live imaging of the Dictyostelium cell cycle reveals widespread S phase during development, a G2 bias in spore differentiation and a premitotic checkpoint. Development 135(9):1647–1657. CrossRefPubMedGoogle Scholar
  8. 8.
    Zhang XY, Langenick J, Traynor D, Babu MM, Kay RR, Patel KJ (2009) Xpf and not the Fanconi anaemia proteins or Rev3 accounts for the extreme resistance to cisplatin in Dictyostelium discoideum. PLoS Genet 5(9):e1000645. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Pontel LB, Langenick J, Rosado IV, Zhang XY, Traynor D, Kay RR, Patel KJ (2016) Xpf suppresses the mutagenic consequences of phagocytosis in Dictyostelium. J Cell Sci 129(24):4449–4454. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Pears CJ, Couto CA, Wang HY, Borer C, Kiely R, Lakin ND (2012) The role of ADP-ribosylation in regulating DNA double-strand break repair. Cell Cycle 11(1):48–56. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Couto CA, Hsu DW, Teo R, Rakhimova A, Lempidaki S, Pears CJ, Lakin ND (2013) Nonhomologous end-joining promotes resistance to DNA damage in the absence of an ADP-ribosyltransferase that signals DNA single strand breaks. J Cell Sci 126(Pt 15):3452–3461. CrossRefPubMedGoogle Scholar
  12. 12.
    Couto CA, Wang HY, Green JC, Kiely R, Siddaway R, Borer C, Pears CJ, Lakin ND (2011) PARP regulates nonhomologous end joining through retention of Ku at double-strand breaks. J Cell Biol 194(3):367–375. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Rakhimova A, Ura S, Hsu DW, Wang HY, Pears CJ, Lakin ND (2017) Site-specific ADP-ribosylation of histone H2B in response to DNA double strand breaks. Sci Rep 7:43750. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Ahel I, Ahel D, Matsusaka T, Clark AJ, Pines J, Boulton SJ, West SC (2008) Poly(ADP-ribose)-binding zinc finger motifs in DNA repair/checkpoint proteins. Nature 451(7174):81–85. CrossRefPubMedGoogle Scholar
  15. 15.
    Gunn AR, Banos-Pinero B, Paschke P, Sanchez-Pulido L, Ariza A, Day J, Emrich M, Leys D, Ponting CP, Ahel I, Lakin ND (2016) The role of ADP-ribosylation in regulating DNA interstrand crosslink repair. J Cell Sci 129(20):3845–3858. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Strmecki L, Greene DM, Pears CJ (2005) Developmental decisions in Dictyostelium discoideum. Dev Biol 284(1):25–36CrossRefGoogle Scholar
  17. 17.
    Robinson DN, Spudich JA (2000) Dynacortin, a genetic link between equatorial contractility and global shape control discovered by library complementation of a Dictyostelium discoideum cytokinesis mutant. J Cell Biol 150(4):823–838CrossRefGoogle Scholar
  18. 18.
    Chen L, Iijima M, Tang M, Landree MA, Huang YE, Xiong Y, Iglesias PA, Devreotes PN (2007) PLA2 and PI3K/PTEN pathways act in parallel to mediate chemotaxis. Dev Cell 12(4):603–614. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Li G, Alexander H, Schneider N, Alexander S (2000) Molecular basis for resistance to the anticancer drug cisplatin in Dictyostelium. Microbiology 146(Pt 9):2219–2227CrossRefGoogle Scholar
  20. 20.
    Van Driessche N, Alexander H, Min J, Kuspa A, Alexander S, Shaulsky G (2007) Global transcriptional responses to cisplatin in Dictyostelium discoideum identify potential drug targets. Proc Natl Acad Sci U S A 104(39):15406–15411. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Williams RS, Eames M, Ryves WJ, Viggars J, Harwood AJ (1999) Loss of a prolyl oligopeptidase confers resistance to lithium by elevation of inositol (1,4,5) trisphosphate. EMBO J 18(10):2734–2745. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Hsu DW, Chubb JR, Muramoto T, Pears CJ, Mahadevan LC (2012) Dynamic acetylation of lysine-4-trimethylated histone H3 and H3 variant biology in a simple multicellular eukaryote. Nucleic Acids Res 40(15):7247–7256. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Stevense M, Chubb JR, Muramoto T (2011) Nuclear organization and transcriptional dynamics in Dictyostelium. Develop Growth Differ 53(4):576–586. CrossRefGoogle Scholar
  24. 24.
    Leiting B, Noegel A (1988) Construction of an extrachromosomally replicating transformation vector for Dictyostelium discoideum. Plasmid 20(3):241–248CrossRefGoogle Scholar
  25. 25.
    Manstein DJ, Schuster HP, Morandini P, Hunt DM (1995) Cloning vectors for the production of proteins in Dictyostelium discoideum. Gene 162(1):129–134CrossRefGoogle Scholar
  26. 26.
    D'Amours D, Desnoyers S, D'Silva I, Poirier GG (1999) Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions. Biochem J 342(Pt2):249–268CrossRefGoogle Scholar
  27. 27.
    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(7):411–424. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Slade D, Dunstan MS, Barkauskaite E, Weston R, Lafite P, Dixon N, Ahel M, Leys D, Ahel I (2011) The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase. Nature 477(7366):616–620. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Barkauskaite E, Brassington A, Tan ES, Warwicker J, Dunstan MS, Banos B, Lafite P, Ahel M, Mitchison TJ, Ahel I, Leys D (2013) Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities. Nat Commun 4:2164. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Sharifi R, Morra R, Denise Appel C, Tallis M, Chioza B, Jankevicius G, Simpson MA, Matic I, Ozkan E, Golia B, Schellenberg MJ, Weston R, Williams JG, Rossi MN, Galehdari H, Krahn J, Wan A, Trembath RC, Crosby AH, Ahel D, Hay R, Ladurner AG, Timinszky G, Williams RS, Ahel I (2013) Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease. EMBO J 32:1225. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Rosenthal F, Feijs KL, Frugier E, Bonalli M, Forst AH, Imhof R, Winkler HC, Fischer D, Caflisch A, Hassa PO, Luscher B, Hottiger MO (2013) Macrodomain-containing proteins are new mono-ADP-ribosylhydrolases. Nat Struct Mol Biol 20(4):502–507. CrossRefPubMedGoogle Scholar
  32. 32.
    Jankevicius G, Hassler M, Golia B, Rybin V, Zacharias M, Timinszky G, Ladurner AG (2013) A family of macrodomain proteins reverses cellular mono-ADP-ribosylation. Nat Struct Mol Biol 20(4):508–514. CrossRefPubMedGoogle Scholar
  33. 33.
    Messner S, Hottiger MO (2011) Histone ADP-ribosylation in DNA repair, replication and transcription. Trends Cell Biol 21(9):534–542. CrossRefPubMedGoogle Scholar
  34. 34.
    Couto AM, Lakin ND, Pears CJ (2013) Investigation of DNA repair pathway activity. Methods Mol Biol 983:295–310. CrossRefPubMedGoogle Scholar
  35. 35.
    Faix J, Linkner J, Nordholz B, Platt JL, Liao XH, Kimmel AR (2013) The application of the Cre-loxP system for generating multiple knock-out and knock-in targeted loci. Methods Mol Biol 983:249–267. CrossRefPubMedGoogle Scholar
  36. 36.
    Daniels CM, Ong SE, Leung AK (2014) Phosphoproteomic approach to characterize protein mono- and poly(ADP-ribosyl)ation sites from cells. J Proteome Res 13(8):3510–3522. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Daniels CM, Ong SE, Leung AK (2015) The promise of proteomics for the study of ADP-ribosylation. Mol Cell 58(6):911–924. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Gagne JP, Isabelle M, Lo KS, Bourassa S, Hendzel MJ, Dawson VL, Dawson TM, Poirier GG (2008) Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes. Nucleic Acids Res 36(22):6959–6976. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Isabelle M, Gagne JP, Gallouzi IE, Poirier GG (2012) Quantitative proteomics and dynamic imaging reveal that G3BP-mediated stress granule assembly is poly(ADP-ribose)-dependent following exposure to MNNG-induced DNA alkylation. J Cell Sci 125(Pt 19):4555–4566. CrossRefPubMedGoogle Scholar
  40. 40.
    Jungmichel S, Rosenthal F, Altmeyer M, Lukas J, Hottiger MO, Nielsen ML (2013) Proteome-wide identification of poly(ADP-ribosyl)ation targets in different genotoxic stress responses. Mol Cell 52(2):272–285. CrossRefPubMedGoogle Scholar
  41. 41.
    Zhang Y, Wang J, Ding M, Yu Y (2013) Site-specific characterization of the Asp- and Glu-ADP-ribosylated proteome. Nat Methods 10(10):981–984. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Bonfiglio JJ, Fontana P, Zhang Q, Colby T, Gibbs-Seymour I, Atanassov I, Bartlett E, Zaja R, Ahel I, Matic I (2017) Serine ADP-ribosylation depends on HPF1. Mol Cell 65(5):932–940 e936. CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Leidecker O, Bonfiglio JJ, Colby T, Zhang Q, Atanassov I, Zaja R, Palazzo L, Stockum A, Ahel I, Matic I (2016) Serine is a new target residue for endogenous ADP-ribosylation on histones. Nat Chem Biol 12(12):998–1000. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Faix J, Kreppel L, Shaulsky G, Schleicher M, Kimmel AR (2004) A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system. Nucleic Acids Res 32(19):e143. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Garcia MX, Alexander H, Mahadeo D, Cotter DA, Alexander S (2003) The Dictyostelium discoideum prespore-specific catalase B functions to control late development and to protect spore viability. Biochim Biophys Acta 1641(1):55–64CrossRefGoogle Scholar
  46. 46.
    Gaudet P, Tsang A (1999) Regulation of the ribonucleotide reductase small subunit gene by DNA-damaging agents in Dictyostelium discoideum. Nucleic Acids Res 27(15):3042–3048CrossRefGoogle Scholar
  47. 47.
    Podgorski G, Deering RA (1980) Effect of methyl methanesulfonate on survival of radiation-sensitive strains of Dictyostelium discoideum. Mutat Res 73(2):415–418CrossRefGoogle Scholar
  48. 48.
    Bronner CE, Welker DL, Deering RA (1992) Mutations affecting sensitivity of the cellular slime mold Dictyostelium discoideum to DNA-damaging agents. Mutat Res 274(3):187–200CrossRefGoogle Scholar
  49. 49.
    Freeland TM, Guyer RB, Ling AZ, Deering RA (1996) Apurinic/apyrimidinic (AP) endonuclease from Dictyostelium discoideum: cloning, nucleotide sequence and induction by sublethal levels of DNA damaging agents. Nucleic Acids Res 24(10):1950–1953CrossRefGoogle Scholar
  50. 50.
    Ling AZ, Guyer RB, Deering RA (2001) Dictyostelium discoideum plasmid containing an AP-endonuclease upstream sequence: bleomycin induction of a luciferase reporter. Environ Mol Mutagen 38(2–3):244–247CrossRefGoogle Scholar
  51. 51.
    Deering RA, Guyer RB, Stevens L, Watson-Thais TE (1996) Some repair-deficient mutants of Dictyostelium discoideum display enhanced susceptibilities to bleomycin. Antimicrob Agents Chemother 40(2):464–467CrossRefGoogle Scholar
  52. 52.
    Hsu DW, Kiely R, Couto CA, Wang HY, Hudson JJ, Borer C, Pears CJ, Lakin ND (2011) DNA double-strand break repair pathway choice in Dictyostelium. J Cell Sci 124(Pt 10):1655–1663. CrossRefPubMedGoogle Scholar
  53. 53.
    Lee SK, Yu SL, Alexander H, Alexander S (1998) A mutation in repB, the dictyostelium homolog of the human xeroderma pigmentosum B gene, has increased sensitivity to UV-light but normal morphogenesis. Biochim Biophys Acta 1399(2–3):161–172CrossRefGoogle Scholar
  54. 54.
    Hurley DL, Skantar AM, Deering RA (1989) Nuclear DNA synthesis is blocked by UV irradiation in Dictyostelium discoideum. Mutat Res 217(1):25–32CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of OxfordOxfordUK

Personalised recommendations