Skip to main content
SpringerLink
Log in

Genetic Dissection of PARylation in the Filamentous Fungus Neurospora crassa

  • Protocol
  • First Online:
Poly(ADP-ribose) Polymerase

Part of the book series: Methods in Molecular Biology ((MIMB,volume 780))

  • 2596 Accesses

Abstract

PARylation is a posttranslational protein modification carried out by PAR polymerases (PARPs). These enzymes function as ADP-ribose transferases that add polymers of ADP-ribose (PAR) to target proteins. PARP proteins have critical functions impacting the aspects of normal human health, such as aging, as well as disease development, particularly cancer. Recently, the powerful antitumor PARP inhibitor Olaparib was shown to be effective in blocking the progression of BRCA1/2-associated tumors, prompting Bruce Alberts to call for an expansion of cancer research beyond utilization of cancer cell lines to include model organisms, such as bacteria, yeast, worms, flies, and mice. Although Dr. Alberts did not specifically mention the filamentous fungus Neurospora crassa, it is now known that Neurospora is the only genetically tractable model eukaryote with completely dispensable PARylation. PARylation in Neurospora can be entirely eliminated by disruption of a single predicted ORF, encoding a nuclear localized PARP protein termed Neurospora PARP ortholog (NPO). We, thus, present this initial genetic characterization of PARylation in N. crassa as evidence of the supreme advantage of using Neurospora as a tool for the genetic dissection of PARP and PARylation and emphasize the power of this system to advance unparalleled contributions to knowledge in this field.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. 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(Pt 2):249–268

    Article  PubMed  Google Scholar 

  2. Burkle A (2000) Poly(ADP-ribosyl)ation: a posttranslational protein modification linked with genome protection and mammalian longevity. Biogerontology 1:41–46

    Article  PubMed  CAS  Google Scholar 

  3. Kraus WL, Lis JT (2003) PARP goes transcription. Cell 113:677–683

    Article  PubMed  CAS  Google Scholar 

  4. Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG (2005) The macro domain is an ADP-ribose binding module. EMBO J 24:1911–1920

    Article  PubMed  CAS  Google Scholar 

  5. 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 451:81–85

    Article  PubMed  CAS  Google Scholar 

  6. Ame JC, Fouquerel E, Gauthier LR, Biard D, Boussin FD, Dantzer F, de Murcia G, Schreiber V (2009) Radiation-induced mitotic catastrophe in PARG-deficient cells. J Cell Sci 122:1990–2002

    Article  PubMed  CAS  Google Scholar 

  7. Ame JC, Hakme A, Quenet D, Fouquerel E, Dantzer F, Schreiber V (2009) Detection of the nuclear poly(ADP-ribose)-metabolizing enzymes and activities in response to DNA damage. Methods Mol Biol 464:267–283

    Article  PubMed  Google Scholar 

  8. Boulu RG, Mesenge C, Charriaut-Marlangue C, Verrecchia C, Plotkine M (2001) [Neuronal death: potential role of the nuclear enzyme, poly (ADP-ribose) polymerase]. Bull Acad Natl Med 185:555–563, discussion 564–555

    PubMed  CAS  Google Scholar 

  9. Bouchard VJ, Rouleau M, Poirier GG (2003) PARP-1, a determinant of cell survival in response to DNA damage. Exp Hematol 31:446–454

    Article  PubMed  CAS  Google Scholar 

  10. Pieper AA, Verma A, Zhang J, Snyder SH (1999) Poly (ADP-ribose) polymerase, nitric oxide and cell death. Trends Pharmacol Sci 20:171–181

    Article  PubMed  CAS  Google Scholar 

  11. Smulson ME, Simbulan-Rosenthal CM, Boulares AH, Yakovlev A, Stoica B, Iyer S, Luo R, Haddad B, Wang ZQ, Pang T, Jung M, Dritschilo A, Rosenthal DS (2000) Roles of poly(ADP-ribosyl)ation and PARP in apoptosis, DNA repair, genomic stability and functions of p53 and E2F-1. Adv Enzyme Regul 40:183–215

    Article  PubMed  CAS  Google Scholar 

  12. Herceg Z, Wang ZQ (2001) Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death. Mutat Res 477:97–110

    Article  PubMed  CAS  Google Scholar 

  13. Hong SJ, Dawson TM, Dawson VL (2004) Nuclear and mitochondrial conversations in cell death: PARP-1 and AIF signaling. Trends Pharmacol Sci 25:259–264

    Article  PubMed  CAS  Google Scholar 

  14. Jeggo PA (1998) DNA repair: PARP – another guardian angel? Curr Biol 8:R49–R51

    Article  PubMed  CAS  Google Scholar 

  15. Burkle A (2001) PARP-1: a regulator of genomic stability linked with mammalian longevity. Chembiochem 2:725–728

    Article  PubMed  CAS  Google Scholar 

  16. Burkle A, Diefenbach J, Brabeck C, Beneke S (2005) Ageing and PARP. Pharmacol Res 52:93–99

    Article  PubMed  Google Scholar 

  17. Beneke S, Burkle A (2004) Poly(ADP-ribosyl)ation, PARP, and aging. Sci Aging Knowl Environ 2004:re9

    Article  Google Scholar 

  18. Chiarugi A, Moskowitz MA (2002) Cell biology. PARP-1 – a perpetrator of apoptotic cell death? Science 297:200–201

    Article  PubMed  CAS  Google Scholar 

  19. Kim MY, Zhang T, Kraus WL (2005) Poly(ADP-ribosyl)ation by PARP-1: ‘PAR-laying’ NAD+ into a nuclear signal. Genes Dev 19:1951–1967

    Article  PubMed  CAS  Google Scholar 

  20. Beneke S, Burkle A (2007) Poly(ADP-ribosyl)ation in mammalian ageing. Nucleic Acids Res 35:7456–7465

    Article  PubMed  CAS  Google Scholar 

  21. Zhang J (2003) Are poly(ADP-ribosyl)ation by PARP-1 and deacetylation by Sir2 linked? Bioessays 25:808–814

    Article  PubMed  CAS  Google Scholar 

  22. Smith KM, Kothe GO, Matsen CB, Khlafallah TK, Adhvaryu KK, Hemphill M, Freitag M, Motamedi MR, Selker EU (2008) The fungus Neurospora crassa displays telomeric silencing mediated by multiple sirtuins and by methylation of histone H3 lysine 9. Epigenetics Chromatin 1(1):5

    Article  PubMed  Google Scholar 

  23. Kothe GO, Kitamura M, Masutani M, Selker EU, Inoue H (2010) PARP is involved in replicative aging in Neurospora crassa. Fungal Genet Biol 47:297–309

    Article  PubMed  CAS  Google Scholar 

  24. Metzenberg RL (1993) Do protoperithecia smell perithecia? Fungal Genet Newslett 40:83

    Google Scholar 

  25. He Q, Liu Y (2005) Molecular mechanism of light responses in Neurospora: from light-induced transcription to photoadaptation. Genes Dev 19:2888–2899

    Article  PubMed  CAS  Google Scholar 

  26. Read ND, Lichius A, Shoji JY, Goryachev AB (2009) Self-signalling and self-fusion in filamentous fungi. Curr Opin Microbiol 12:608–615

    Article  PubMed  Google Scholar 

  27. Dunlap JC, Loros JJ, Colot HV, Mehra A, Belden WJ, Shi M, Hong CI, Larrondo LF, Baker CL, Chen CH, Schwerdtfeger C, Collopy PD, Gamsby JJ, Lambreghts R (2007) A circadian clock in Neurospora: how genes and proteins cooperate to produce a sustained, entrainable, and compensated biological oscillator with a period of about a day. Cold Spring Harb Symp Quant Biol 72:57–68

    Article  PubMed  CAS  Google Scholar 

  28. Spingola M, Grate L, Haussler D, Ares M Jr (1999) Genome-wide bioinformatic and molecular analysis of introns in Saccharomyces cerevisiae. RNA 5:221–234

    Article  PubMed  CAS  Google Scholar 

  29. Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, Jaffe D, FitzHugh W, Ma LJ, Smirnov S, Purcell S, Rehman B, Elkins T, Engels R, Wang S, Nielsen CB, Butler J, Endrizzi M, Qui D, Ianakiev P, Bell-Pedersen D, Nelson MA, Werner-Washburne M, Selitrennikoff CP, Kinsey JA, Braun EL, Zelter A, Schulte U, Kothe GO, Jedd G, Mewes W, Staben C, Marcotte E, Greenberg D, Roy A, Foley K, Naylor J, Stange-Thomann N, Barrett R, Gnerre S, Kamal M, Kamvysselis M, Mauceli E, Bielke C, Rudd S, Frishman D, Krystofova S, Rasmussen C, Metzenberg RL, Perkins DD, Kroken S, Cogoni C, Macino G, Catcheside D, Li W, Pratt RJ, Osmani SA, DeSouza CP, Glass L, Orbach MJ, Berglund JA, Voelker R, Yarden O, Plamann M, Seiler S, Dunlap J, Radford A, Aramayo R, Natvig DO, Alex LA, Mannhaupt G, Ebbole DJ, Freitag M, Paulsen I, Sachs MS, Lander ES, Nusbaum C, Birren B (2003) The genome sequence of the filamentous fungus Neurospora crassa. Nature 422:859–868

    Article  PubMed  CAS  Google Scholar 

  30. Davis RD, DeSerres FJ (1970) Genetic and microbiological research techniques for Neurospora crassa. Methods Enzymol 17:79–143

    Article  Google Scholar 

  31. Semighini CP, Savoldi M, Goldman GH, Harris SD (2006) Functional characterization of the putative Aspergillus nidulans poly(ADP-ribose) polymerase homolog PrpA. Genetics 173:87–98

    Article  PubMed  CAS  Google Scholar 

  32. Davis RH, de Serres FJ (1970) Genetic and microbial research techniques for Neurospora crassa. Methods Enzymol 17A:79–143

    Article  Google Scholar 

  33. Ninomiya Y, Suzuki K, Ishii C, Inoue H (2004) Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc Natl Acad Sci USA 101:12248–12253

    Article  PubMed  CAS  Google Scholar 

  34. de Serres FJ, Inoue H, Schupbach ME (1980) Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. I. Development of isogenic strains and spontaneous mutability. Mutat Res 71:53–65

    Article  PubMed  Google Scholar 

  35. Loros JJ, Denome SA, Dunlap JC (1989) Molecular cloning of genes under control of the circadian clock in Neurospora. Science 243:385–388

    Article  PubMed  CAS  Google Scholar 

  36. McNally MT, Free SJ (1988) Isolation and characterization of a Neurospora glucose-repressible gene. Curr Genet 14:545–551

    Article  PubMed  CAS  Google Scholar 

  37. Freitag M, Hickey PC, Raju NB, Selker EU, Read ND (2004) GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa. Fungal Genet Biol 41:897–910

    Article  PubMed  CAS  Google Scholar 

  38. Freitag M, Hickey PC, Khlafallah TK, Read ND, Selker EU (2004) HP1 is essential for DNA methylation in neurospora. Mol Cell 13:427–434

    Article  PubMed  CAS  Google Scholar 

  39. Tulin A, Stewart D, Spradling AC (2002) The Drosophila heterochromatic gene encoding poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during development. Genes Dev 16:2108–2119

    Article  PubMed  CAS  Google Scholar 

  40. Tulin A, Spradling A (2003) Chromatin loosening by poly(ADP)-ribose polymerase (PARP) at Drosophila puff loci. Science 299:560–562

    Article  PubMed  CAS  Google Scholar 

  41. Menissier de Murcia J, Ricoul M, Tartier L, Niedergang C, Huber A, Dantzer F, Schreiber V, Ame JC, Dierich A, LeMeur M, Sabatier L, Chambon P, de Murcia G (2003) Functional interaction between PARP-1 and PARP-2 in chromosome stability and embryonic development in mouse. EMBO J 22:2255–2263

    Article  PubMed  CAS  Google Scholar 

  42. Smogorzewska A, de Lange T (2004) Regulation of telomerase by telomeric proteins. Annu Rev Biochem 73:177–208

    Article  PubMed  CAS  Google Scholar 

  43. Smith S, Giriat I, Schmitt A, de Lange T (1998) Tankyrase, a poly(ADP-ribose) polymerase at human telomeres. Science 282:1484–1487

    Article  PubMed  CAS  Google Scholar 

  44. Pillai JB, Isbatan A, Imai S, Gupta MP (2005) Poly(ADP-ribose) polymerase-1-dependent cardiac myocyte cell death during heart failure is mediated by NAD+ depletion and reduced Sir2alpha deacetylase activity. J Biol Chem 280:43121–43130

    Article  PubMed  CAS  Google Scholar 

  45. Kolthur-Seetharam U, Dantzer F, McBurney MW, de Murcia G, Sassone-Corsi P (2006) Control of AIF-mediated cell death by the functional interplay of SIRT1 and PARP-1 in response to DNA damage. Cell Cycle 5:873–877

    Article  PubMed  CAS  Google Scholar 

  46. Sassone-Corsi P (2009) SIRT1/PARP-1 functional interplay. Cell Cycle 8:1649

    Article  PubMed  CAS  Google Scholar 

  47. Honda S, Selker E (2009) Tools for fungal proteomics: multifunctional neurospora vectors for gene replacement, protein expression and protein purification. Genetics 182(1):11–23

    Article  PubMed  CAS  Google Scholar 

  48. Lee HC, Chang SS, Choudhary S, Aalto AP, Maiti M, Bamford DH, Liu Y (2009) qiRNA is a new type of small interfering RNA induced by DNA damage. Nature 459:274–277

    Article  PubMed  CAS  Google Scholar 

  49. Howard CA, Baker TI (1988) Relationship of histidine sensitivity to DNA damage and stress induced responses in mutagen sensitive mutants of Neurospora crassa. Curr Genet 13:391–399

    Article  PubMed  CAS  Google Scholar 

  50. Schroder M, Kaufman RJ (2005) The mammalian unfolded protein response. Annu Rev Biochem 74:739–789

    Article  PubMed  Google Scholar 

  51. Rose MD, Misra LM, Vogel JP (1989) KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene. Cell 57:1211–1221

    Article  PubMed  CAS  Google Scholar 

  52. Malling HV, de Serres FJ (1968) Identification of genetic alterations induced by ethyl methanesulfonate in Neurospora crassa. Mutat Res 6:181–193

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Molecular Biology, University of Oregon, Eugene, OR, USA

    Gregory O. Kothe

Authors
  1. Gregory O. Kothe
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Keystone Program in Epigenetics &, Progenitor Cells, Fox Chase Cancer Center, Cottman Avenue 333, Philadelphia, 19111, Pennsylvania, USA

    Alexei V. Tulin

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Kothe, G.O. (2011). Genetic Dissection of PARylation in the Filamentous Fungus Neurospora crassa . 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_26

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-270-0_26

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-269-4

  • Online ISBN: 978-1-61779-270-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation