Skip to main content
SpringerLink
Log in
Book cover

ATM Kinase pp 263–275Cite as

Studies of the DNA Damage Response by Using the Lac Operator/Repressor (LacO/LacR) Tethering System

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1599))

Abstract

Maintaining the integrity of genetic information is essential for the survival of cells. Recent advances in cell biological and microscopy methodologies have complemented traditional genetic and biochemical approaches, and they now permit the observation of spatiotemporal aspects of damaged chromosomal loci. In one of these approaches, integrated LacO/TetO operator sequences can be used as binding sites to physically tether onto chromatin any protein of interest when genetically fused to the respective repressors (LacR/TetR). This methodology has been the basis of several models to probe the spatial dynamics of DNA repair in the eukaryotic nucleus and to visualize genomic loci in yeast, fly, nematodes, and in mammalian cells. Further applications are the induction of localized DNA damage by immobilizing endonucleases at different genome sites in vivo, the assessment of the hierarchy of protein interactions within repair complexes, and the activation of the DNA damage response (DDR) by the physical tethering of DSB-repair factors on chromatin in the absence of damage. We outline here a protocol for the quantification of DDR activation upon the prolonged immobilization of single repair factors on chromatin or upon tethering of the endonuclease FokI. The outlined protocol requires basic cell culture and microscopy skills and allows the tethering of any protein of interest within 2–3 days.

This is a preview of subscription content, 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   129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   169.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Jackson SP, Bartek J (2009) The DNA-damage response in human biology and disease. Nature 461(7267):1071–1078. doi:10.1038/nature08467

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Alt FW, Zhang Y, Meng FL, Guo C, Schwer B (2013) Mechanisms of programmed DNA lesions and genomic instability in the immune system. Cell 152(3):417–429. doi:10.1016/j.cell.2013.01.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Polo SE, Jackson SP (2011) Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Genes Dev 25(5):409–433. doi:10.1101/gad.2021311

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Roukos V, Burman B, Misteli T (2013) The cellular etiology of chromosome translocations. Curr Opin Cell Biol 25(3):357–364. doi:10.1016/j.ceb.2013.02.015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Roukos V, Misteli T (2014) The biogenesis of chromosome translocations. Nat Cell Biol 16(4):293–300. doi:10.1038/ncb2941

    Article  CAS  PubMed  Google Scholar 

  6. Stracker TH, Petrini JH (2011) The MRE11 complex: starting from the ends. Nat Rev Mol Cell Biol 12(2):90–103. doi:10.1038/nrm3047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kastan MB, Lim DS (2000) The many substrates and functions of ATM. Nat Rev Mol Cell Biol 1(3):179–186. doi:10.1038/35043058

    Article  CAS  PubMed  Google Scholar 

  8. Coster G, Goldberg M (2010) The cellular response to DNA damage: a focus on MDC1 and its interacting proteins. Nucleus 1(2):166–178. doi:10.4161/nucl.1.2.11176

    Article  PubMed  Google Scholar 

  9. Reinhardt HC, Yaffe MB (2013) Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response. Nat Rev Mol Cell Biol 14(9):563–580. doi:10.1038/nrm3640

    Article  CAS  PubMed  Google Scholar 

  10. Lukas C, Falck J, Bartkova J, Bartek J, Lukas J (2003) Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat Cell Biol 5(3):255–260. doi:10.1038/ncb945

    Article  CAS  PubMed  Google Scholar 

  11. Ciccia A, Elledge SJ (2010) The DNA damage response: making it safe to play with knives. Mol Cell 40(2):179–204. doi:10.1016/j.molcel.2010.09.019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Carney JP, Maser RS, Olivares H, Davis EM, Le Beau M, Yates JR 3rd, Hays L, Morgan WF, Petrini JH (1998) The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93(3):477–486

    Article  CAS  PubMed  Google Scholar 

  13. Nelms BE, Maser RS, MacKay JF, Lagally MG, Petrini JH (1998) In situ visualization of DNA double-strand break repair in human fibroblasts. Science 280(5363):590–592

    Article  CAS  PubMed  Google Scholar 

  14. Jakob B, Scholz M, Taucher-Scholz G (2003) Biological imaging of heavy charged-particle tracks. Radiat Res 159(5):676–684

    Article  CAS  PubMed  Google Scholar 

  15. Stap J, Krawczyk PM, Van Oven CH, Barendsen GW, Essers J, Kanaar R, Aten JA (2008) Induction of linear tracks of DNA double-strand breaks by alpha-particle irradiation of cells. Nat Methods 5(3):261–266. doi:10.1038/nmeth.f.206

    Article  CAS  PubMed  Google Scholar 

  16. Povirk LF (1996) DNA damage and mutagenesis by radiomimetic DNA-cleaving agents: bleomycin, neocarzinostatin and other enediynes. Mutat Res 355(1–2):71–89

    Article  PubMed  Google Scholar 

  17. Bekker-Jensen S, Lukas C, Kitagawa R, Melander F, Kastan MB, Bartek J, Lukas J (2006) Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J Cell Biol 173(2):195–206. doi:10.1083/jcb.200510130

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kong X, Mohanty SK, Stephens J, Heale JT, Gomez-Godinez V, Shi LZ, Kim JS, Yokomori K, Berns MW (2009) Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells. Nucleic Acids Res 37(9):e68. doi:10.1093/nar/gkp221

    Article  PubMed  PubMed Central  Google Scholar 

  19. Essers J, Houtsmuller AB, van Veelen L, Paulusma C, Nigg AL, Pastink A, Vermeulen W, Hoeijmakers JH, Kanaar R (2002) Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage. EMBO J 21(8):2030–2037. doi:10.1093/emboj/21.8.2030

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Essers J, Vermeulen W, Houtsmuller AB (2006) DNA damage repair: anytime, anywhere? Curr Opin Cell Biol 18(3):240–246. doi:10.1016/j.ceb.2006.03.004

    Article  CAS  PubMed  Google Scholar 

  21. Berkovich E, Monnat RJ Jr, Kastan MB (2008) Assessment of protein dynamics and DNA repair following generation of DNA double-strand breaks at defined genomic sites. Nat Protoc 3(5):915–922. doi:10.1038/nprot.2008.54

    Article  CAS  PubMed  Google Scholar 

  22. Iacovoni JS, Caron P, Lassadi I, Nicolas E, Massip L, Trouche D, Legube G (2010) High-resolution profiling of gammaH2AX around DNA double strand breaks in the mammalian genome. EMBO J 29(8):1446–1457. doi:10.1038/emboj.2010.38

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Rouet P, Smih F, Jasin M (1994) Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol Cell Biol 14(12):8096–8106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Aymard F, Bugler B, Schmidt CK, Guillou E, Caron P, Briois S, Iacovoni JS, Daburon V, Miller KM, Jackson SP, Legube G (2014) Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks. Nat Struct Mol Biol 21(4):366–374. doi:10.1038/nsmb.2796

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Lemaitre C, Grabarz A, Tsouroula K, Andronov L, Furst A, Pankotai T, Heyer V, Rogier M, Attwood KM, Kessler P, Dellaire G, Klaholz B, Reina-San-Martin B, Soutoglou E (2014) Nuclear position dictates DNA repair pathway choice. Genes Dev 28(22):2450–2463. doi:10.1101/gad.248369.114

    Article  PubMed  PubMed Central  Google Scholar 

  26. Clouaire T, Legube G (2015) DNA double strand break repair pathway choice: a chromatin based decision? Nucleus 6(2):107–113. doi:10.1080/19491034.2015.1010946

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. van Sluis M, McStay B (2015) A localized nucleolar DNA damage response facilitates recruitment of the homology-directed repair machinery independent of cell cycle stage. Genes Dev 29(11):1151–1163. doi:10.1101/gad.260703.115

    Article  PubMed  PubMed Central  Google Scholar 

  28. Roukos V, Voss TC, Schmidt CK, Lee S, Wangsa D, Misteli T (2013) Spatial dynamics of chromosome translocations in living cells. Science 341(6146):660–664. doi:10.1126/science.1237150

    Article  CAS  PubMed  Google Scholar 

  29. Soutoglou E, Dorn JF, Sengupta K, Jasin M, Nussenzweig A, Ried T, Danuser G, Misteli T (2007) Positional stability of single double-strand breaks in mammalian cells. Nat Cell Biol 9(6):675–682. doi:10.1038/ncb1591

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Belmont AS, Li G, Sudlow G, Robinett C (1999) Visualization of large-scale chromatin structure and dynamics using the lac operator/lac repressor reporter system. Methods Cell Biol 58:203–222

    Article  CAS  PubMed  Google Scholar 

  31. Gonzalez-Serricchio AS, Sternberg PW (2006) Visualization of C. elegans transgenic arrays by GFP. BMC Genet 7:36. doi:10.1186/1471-2156-7-36

    Article  PubMed  PubMed Central  Google Scholar 

  32. Lassadi I, Bystricky K (2011) Tracking of single and multiple genomic loci in living yeast cells. Methods Mol Biol 745:499–522. doi:10.1007/978-1-61779-129-1_29

    Article  CAS  PubMed  Google Scholar 

  33. Lisby M, Mortensen UH, Rothstein R (2003) Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre. Nat Cell Biol 5(6):572–577. doi:10.1038/ncb997

    Article  CAS  PubMed  Google Scholar 

  34. Meister P, Gehlen LR, Varela E, Kalck V, Gasser SM (2010) Visualizing yeast chromosomes and nuclear architecture. Methods Enzymol 470:535–567. doi:10.1016/S0076-6879(10)70021-5

    Article  CAS  PubMed  Google Scholar 

  35. Masui O, Bonnet I, Le Baccon P, Brito I, Pollex T, Murphy N, Hupe P, Barillot E, Belmont AS, Heard E (2011) Live-cell chromosome dynamics and outcome of X chromosome pairing events during ES cell differentiation. Cell 145(3):447–458. doi:10.1016/j.cell.2011.03.032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Dion V, Gasser SM (2013) Chromatin movement in the maintenance of genome stability. Cell 152(6):1355–1364. doi:10.1016/j.cell.2013.02.010

    Article  CAS  PubMed  Google Scholar 

  37. Dion V, Kalck V, Horigome C, Towbin BD, Gasser SM (2012) Increased mobility of double-strand breaks requires Mec1, Rad9 and the homologous recombination machinery. Nat Cell Biol 14(5):502–509. doi:10.1038/ncb2465

    Article  CAS  PubMed  Google Scholar 

  38. Mine-Hattab J, Rothstein R (2012) Increased chromosome mobility facilitates homology search during recombination. Nat Cell Biol 14(5):510–517. doi:10.1038/ncb2472

    Article  CAS  PubMed  Google Scholar 

  39. Chubb JR, Boyle S, Perry P, Bickmore WA (2002) Chromatin motion is constrained by association with nuclear compartments in human cells. Curr Biol 12(6):439–445

    Article  CAS  PubMed  Google Scholar 

  40. Thomson I, Gilchrist S, Bickmore WA, Chubb JR (2004) The radial positioning of chromatin is not inherited through mitosis but is established de novo in early G1. Curr Biol 14(2):166–172

    Article  CAS  PubMed  Google Scholar 

  41. Vazquez J, Belmont AS, Sedat JW (2001) Multiple regimes of constrained chromosome motion are regulated in the interphase Drosophila nucleus. Curr Biol 11(16):1227–1239

    Article  CAS  PubMed  Google Scholar 

  42. Reddy KL, Zullo JM, Bertolino E, Singh H (2008) Transcriptional repression mediated by repositioning of genes to the nuclear lamina. Nature 452(7184):243–247. doi:10.1038/nature06727

    Article  CAS  PubMed  Google Scholar 

  43. Chuang CH, Carpenter AE, Fuchsova B, Johnson T, de Lanerolle P, Belmont AS (2006) Long-range directional movement of an interphase chromosome site. Curr Biol 16(8):825–831. doi:10.1016/j.cub.2006.03.059

    Article  CAS  PubMed  Google Scholar 

  44. Janicki SM, Tsukamoto T, Salghetti SE, Tansey WP, Sachidanandam R, Prasanth KV, Ried T, Shav-Tal Y, Bertrand E, Singer RH, Spector DL (2004) From silencing to gene expression: real-time analysis in single cells. Cell 116(5):683–698

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Shanbhag NM, Rafalska-Metcalf IU, Balane-Bolivar C, Janicki SM, Greenberg RA (2010) ATM-dependent chromatin changes silence transcription in cis to DNA double-strand breaks. Cell 141(6):970–981. doi:10.1016/j.cell.2010.04.038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Sadler JR, Sasmor H, Betz JL (1983) A perfectly symmetric lac operator binds the lac repressor very tightly. Proc Natl Acad Sci U S A 80(22):6785–6789

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bergmann JH, Rodriguez MG, Martins NM, Kimura H, Kelly DA, Masumoto H, Larionov V, Jansen LE, Earnshaw WC (2011) Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore. EMBO J 30(2):328–340. doi:10.1038/emboj.2010.329

    Article  CAS  PubMed  Google Scholar 

  48. Bonilla CY, Melo JA, Toczyski DP (2008) Colocalization of sensors is sufficient to activate the DNA damage checkpoint in the absence of damage. Mol Cell 30(3):267–276. doi:10.1016/j.molcel.2008.03.023

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Soutoglou E, Misteli T (2008) Activation of the cellular DNA damage response in the absence of DNA lesions. Science 320(5882):1507–1510. doi:10.1126/science.1159051

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Luijsterburg MS, Acs K, Ackermann L, Wiegant WW, Bekker-Jensen S, Larsen DH, Khanna KK, van Attikum H, Mailand N, Dantuma NP (2012) A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure. EMBO J 31(11):2511–2527. doi:10.1038/emboj.2012.104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Verschure PJ, van der Kraan I, de Leeuw W, van der Vlag J, Carpenter AE, Belmont AS, van Driel R (2005) In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation. Mol Cell Biol 25(11):4552–4564. doi:10.1128/MCB.25.11.4552-4564.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Burgess RC, Burman B, Kruhlak MJ, Misteli T (2014) Activation of DNA damage response signaling by condensed chromatin. Cell Rep 9(5):1703–1717. doi:10.1016/j.celrep.2014.10.060

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Burman B, Zhang ZZ, Pegoraro G, Lieb JD, Misteli T (2015) Histone modifications predispose genome regions to breakage and translocation. Genes Dev 29(13):1393–1402. doi:10.1101/gad.262170.115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Kaiser TE, Intine RV, Dundr M (2008) De novo formation of a subnuclear body. Science 322(5908):1713–1717. doi:10.1126/science.1165216

    Article  CAS  PubMed  Google Scholar 

  55. Shevtsov SP, Dundr M (2011) Nucleation of nuclear bodies by RNA. Nat Cell Biol 13(2):167–173. doi:10.1038/ncb2157

    Article  CAS  PubMed  Google Scholar 

  56. Zolghadr K, Mortusewicz O, Rothbauer U, Kleinhans R, Goehler H, Wanker EE, Cardoso MC, Leonhardt H (2008) A fluorescent two-hybrid assay for direct visualization of protein interactions in living cells. Mol Cell Proteomics 7(11):2279–2287. doi:10.1074/mcp.M700548-MCP200

    Article  CAS  PubMed  Google Scholar 

  57. Zolghadr K, Rothbauer U, Leonhardt H (2012) The fluorescent two-hybrid (F2H) assay for direct analysis of protein-protein interactions in living cells. Methods Mol Biol 812:275–282. doi:10.1007/978-1-61779-455-1_16

    Article  CAS  PubMed  Google Scholar 

  58. Roukos V, Burgess RC, Misteli T (2014) Generation of cell-based systems to visualize chromosome damage and translocations in living cells. Nat Protoc 9(10):2476–2492. doi:10.1038/nprot.2014.167

    Article  CAS  PubMed  Google Scholar 

  59. Jacome A, Fernandez-Capetillo O (2011) Lac operator repeats generate a traceable fragile site in mammalian cells. EMBO Rep 12(10):1032–1038. doi:10.1038/embor.2011.158

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank members of the Roukos group for helpful comments and Gianluca Pegoraro for critical reading of the manuscript. This work is supported by the “DFG Major Research Instrumentation Programme” (INST 247/845-1 FUGG).

Author information

Authors and Affiliations

  1. Institute of Molecular Biology, Ackermannweg 4, 55128, Mainz, Germany

    Rossana Piccinno, Marta Cipinska & Vassilis Roukos

Authors
  1. Rossana Piccinno
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marta Cipinska
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vassilis Roukos
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vassilis Roukos .

Editor information

Editors and Affiliations

  1. University of Queensland Centre for Clinical Research (UQCCR), University of Queensland, Herston, Queensland, Australia

    Sergei V. Kozlov

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Piccinno, R., Cipinska, M., Roukos, V. (2017). Studies of the DNA Damage Response by Using the Lac Operator/Repressor (LacO/LacR) Tethering System. In: Kozlov, S. (eds) ATM Kinase. Methods in Molecular Biology, vol 1599. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6955-5_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6955-5_19

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6953-1

  • Online ISBN: 978-1-4939-6955-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation