Advertisement

SMC Complexes pp 209-219 | Cite as

In Vitro Detection of Long Noncoding RNA Generated from DNA Double-Strand Breaks

  • Sheetal SharmaEmail author
  • Fabrizio d’Adda di FagagnaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2004)

Abstract

DNA damage response (DDR) is essential for the maintenance of genomic integrity. We have recently discovered the generation of noncoding RNA from a DNA double-strand break (DSB) in an MRE11-RAD50-NBS1 complex-dependent manner, which are necessary for full DDR activation. The low abundance of these noncoding RNA makes them difficult to identify and study. In this chapter, we describe an in vitro biochemical assay to study the generation of damage-induced long noncoding RNA (dilncRNA) from a DNA DSB. In this assay, transcriptionally competent cell-free extracts upon incubation with a linear DNA support RNA synthesis from DNA ends, as monitored by incorporation of 32P[UTP] in discrete products resolved on a denaturing polyacrylamide gel. This approach can be used to identify the role of different DDR proteins in generating dilncRNA.

Key words

Cell-free extracts In vitro transcription Double-strand break (DSB) DNA damage response (DDR) Damage-induced long noncoding RNA (dilncRNA) Plasmid DNA 

Notes

Acknowledgements

S.S., a Structured International Postdoctoral Fellow, received funding from the People Programme, (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7 under grant agreement n.600399. F.d’A.d.F. was supported by the Associazione Italiana per la Ricerca sul Cancro, AIRC (application 12971), Human Frontier Science Program (contract RGP 0014/2012), Cariplo Foundation (grant 2010.0818 and 2014-0812), Marie Curie Initial Training Networks [FP7 PEOPLE 2012 ITN (CodAge)], Fondazione Telethon (GGP12059), Association for International Cancer Research (AICR-Worldwide Cancer Research Rif. N. 14-1331), Progetti di Ricerca di Interesse Nazionale (PRIN) 2010–2011, the Italian Ministry of Education Universities and Research EPIGEN Project, European Research Council advanced grant (322726), and AriSLA (project “DDRNA and ALS”).

References

  1. 1.
    Jackson SP, Bartek J (2009) The DNA-damage response in human biology and disease. Nature 461:1071–1078CrossRefGoogle Scholar
  2. 2.
    Polo SE, Jackson SP (2011) Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Genes Dev 25:409–433CrossRefGoogle Scholar
  3. 3.
    Blackford AN, Jackson SP (2017) ATM, ATR, and DNA-PK: the trinity at the heart of the DNA damage response. Mol Cell 66:801–817CrossRefGoogle Scholar
  4. 4.
    Sharma S, Javadekar SM, Pandey M, Srivastava M, Kumari R, Raghavan SC (2015) Homology and enzymatic requirements of microhomology-dependent alternative end joining. Cell Death Dis 6:e1697CrossRefGoogle Scholar
  5. 5.
    Sharma S, Raghavan SC (2010) Nonhomologous DNA end joining in cell-free extracts. J Nucleic Acids 2010:389129CrossRefGoogle Scholar
  6. 6.
    Ciccia A, Elledge SJ (2010) The DNA damage response: making it safe to play with knives. Mol Cell 40:179–204CrossRefGoogle Scholar
  7. 7.
    d’Adda di Fagagna F (2014) A direct role for small non-coding RNAs in DNA damage response. Trends Cell Biol 24:171–178CrossRefGoogle Scholar
  8. 8.
    D’Alessandro G, d’Adda di Fagagna F (2016) Transcription and DNA damage: holding hands or crossing swords? J Mol Biol 429:3215–3229CrossRefGoogle Scholar
  9. 9.
    Paull TT (2010) Making the best of the loose ends: Mre11/Rad50 complexes and Sae2 promote DNA double-strand break resection. DNA Repair 9:1283–1291CrossRefGoogle Scholar
  10. 10.
    Stracker TH, Petrini JH (2011) The MRE11 complex: starting from the ends. Nat Rev Mol Cell Biol 12:90–103CrossRefGoogle Scholar
  11. 11.
    Assenmacher N, Hopfner KP (2004) MRE11/RAD50/NBS1: complex activities. Chromosoma 113:157–166CrossRefGoogle Scholar
  12. 12.
    Paull TT, Deshpande RA (2014) The Mre11/Rad50/Nbs1 complex: recent insights into catalytic activities and ATP-driven conformational changes. Exp Cell Res 329:139–147CrossRefGoogle Scholar
  13. 13.
    Kanaar R, Wyman C (2008) DNA repair by the MRN complex: break it to make it. Cell 135:14–16CrossRefGoogle Scholar
  14. 14.
    D’Amours D, Jackson SP (2002) The Mre11 complex: at the crossroads of DNA repair and checkpoint signalling. Nat Rev Mol Cell Biol 3:317–327CrossRefGoogle Scholar
  15. 15.
    Michelini F, Pitchiaya S, Vitelli V, Sharma S, Gioia U, Pessina F, Cabrini M, Wang Y, Capozzo I, Iannelli F et al (2017) Damage-induced lncRNAs control the DNA damage response through interaction with DDRNAs at individual double-strand breaks. Nat Cell Biol 19:1400–1411CrossRefGoogle Scholar
  16. 16.
    Vitelli V, Galbiati A, Iannelli F, Pessina F, Sharma S, d’Adda di Fagagna F (2017) Recent advancements in DNA damage-transcription crosstalk and high-resolution mapping of DNA breaks. Annu Rev Genomics Hum Genet 18:87–113CrossRefGoogle Scholar
  17. 17.
    Jackson SP (2002) Sensing and repairing DNA double-strand breaks. Carcinogenesis 23:687–696CrossRefGoogle Scholar
  18. 18.
    Francia S, Michelini F, Saxena A, Tang D, de Hoon M, Anelli V, Mione M, Carninci P, d’Adda di Fagagna F (2012) Site-specific DICER and DROSHA RNA products control the DNA-damage response. Nature 488:231–235CrossRefGoogle Scholar
  19. 19.
    Wei W, Ba Z, Gao M, Wu Y, Ma Y, Amiard S, White CI, Rendtlew Danielsen JM, Yang YG, Qi Y (2012) A role for small RNAs in DNA double-strand break repair. Cell 149:101–112CrossRefGoogle Scholar
  20. 20.
    Qi Y, Zhang Y, Baller JA, Voytas DF (2016) Histone H2AX and the small RNA pathway modulate both non-homologous end-joining and homologous recombination in plants. Mutat Res 783:9–14CrossRefGoogle Scholar
  21. 21.
    Wang Q, Goldstein M (2016) Small RNAs recruit chromatin-modifying enzymes MMSET and Tip60 to reconfigure damaged DNA upon double-strand break and facilitate repair. Cancer Res 76:1904–1915CrossRefGoogle Scholar
  22. 22.
    Francia S, Cabrini M, Matti V, Oldani A, d’Adda di Fagagna F (2016) DICER, DROSHA and DNA damage response RNAs are necessary for the secondary recruitment of DNA damage response factors. J Cell Sci 129:1468–1476CrossRefGoogle Scholar
  23. 23.
    Manley JL, Fire A, Cano A, Sharp PA, Gefter ML (1980) DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A 77:3855–3859CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.IFOM—The FIRC Institute of Molecular OncologyMilanItaly
  2. 2.Department of Experimental Medicine and BiotechnologyPostgraduate Institute of Medical Education and ResearchChandigarhIndia
  3. 3.Istituto di Genetica MolecolareCNR—Consiglio Nazionale delle RicerchePaviaItaly

Personalised recommendations