Skip to main content
SpringerLink
Log in

Visualizing the Spatiotemporal Dynamics of DNA Damage in Budding Yeast

  • Protocol
Stress Responses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1292))

Abstract

Fluorescence microscopy has enabled the analysis of both the spatial distribution of DNA damage and its dynamics during the DNA damage response (DDR). Three microscopic techniques can be used to study the spatiotemporal dynamics of DNA damage. In the first part we describe how we determine the position of DNA double-strand breaks (DSBs) relative to the nuclear envelope. The second part describes how to quantify the co-localization of DNA DSBs with nuclear pore clusters, or other nuclear subcompartments. The final protocols describe methods for the quantification of locus mobility over time.

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 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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. Nagai S, Dubrana K, Tsai-Pflugfelder M et al (2008) Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase. Science 322:597–602

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Oza P, Jaspersen SL, Miele A et al (2009) Mechanisms that regulate localization of a DNA double-strand break to the nuclear periphery. Genes Dev 23:912–927

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Oza P, Peterson CL (2010) Opening the DNA repair toolbox: localization of DNA double strand breaks to the nuclear periphery. Cell Cycle 9:43–49

    Article  CAS  PubMed  Google Scholar 

  4. Kalocsay M, Hiller NJ, Jentsch S (2009) Chromosome-wide Rad51 spreading and SUMO-H2A.Z-dependent chromosome fixation in response to a persistent DNA double-strand break. Mol Cell 33:335–343

    Article  CAS  PubMed  Google Scholar 

  5. Horigome C, Oma Y, Konishi T et al (2014) SWR1 and INO80 chromatin remodelers contribute to DNA double-strand break perinuclear anchorage site choice. Mol Cell 55:626–639

    Article  CAS  PubMed  Google Scholar 

  6. Meister P, Gehlen LR, Varela E et al (2010) Visualizing yeast chromosomes and nuclear architecture. Methods Enzymol 470:535–567

    Article  CAS  PubMed  Google Scholar 

  7. Belmont AS (2001) Visualizing chromosome dynamics with GFP. Trends Cell Biol 11:250–257

    Article  CAS  PubMed  Google Scholar 

  8. Straight AF, Belmont AS, Robinett CC, Murray AW (1996) GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion. Curr Biol 6:1599–1608

    Article  CAS  PubMed  Google Scholar 

  9. Bystricky K, Van Attikum H, Montiel MD et al (2009) Regulation of nuclear positioning and dynamics of the silent mating type loci by the yeast Ku70/Ku80 complex. Mol Cell Biol 29:835–848

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Dion V, Kalck V, Seeber A et al (2013) Cohesin and the nucleolus constrain the mobility of spontaneous repair foci. EMBO Rep 14:984–991

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Sugawara N, Haber JE (2012) Monitoring DNA recombination initiated by HO endonuclease. Methods Mol Biol 920:349–370

    Article  CAS  PubMed  Google Scholar 

  12. Horigome C, Okada T, Shimazu K et al (2011) Ribosome biogenesis factors bind a nuclear envelope SUN domain protein to cluster yeast telomeres. EMBO J 30:3799–3811

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Doye V, Wepf R, Hurt EC (1994) A novel nuclear pore protein Nup133p with distinct roles in poly(A) + RNA transport and nuclear pore distribution. EMBO J 13:6062–6075

    PubMed Central  CAS  PubMed  Google Scholar 

  14. Loeillet S, Palancade B, Cartron M et al (2005) Genetic network interactions among replication, repair and nuclear pore deficiencies in yeast. DNA Repair (Amst) 4:459–468

    Article  CAS  Google Scholar 

  15. Schober H, Ferreira H, Kalck V et al (2009) Yeast telomerase and the SUN domain protein Mps3 anchor telomeres and repress subtelomeric recombination. Genes Dev 23:928–938

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Heun P, Laroche T, Shimada K et al (2001) Chromosome dynamics in the yeast interphase nucleus. Science 294:2181–2186

    Article  CAS  PubMed  Google Scholar 

  17. Neumann FR, Dion V, Gehlen LR et al (2012) Targeted INO80 enhances subnuclear chromatin movement and ectopic homologous recombination. Genes Dev 26:369–383

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Dion V, Gasser SM (2013) Chromatin movement in the maintenance of genome stability. Cell 152:1355–1364

    Article  CAS  PubMed  Google Scholar 

  19. Hediger F, Dubrana K, Gasser SM (2002) Myosin-like proteins 1 and 2 are not required for silencing or telomere anchoring, but act in the Tel1 pathway of telomere length control. J Struct Biol 140:79–91

    Article  CAS  PubMed  Google Scholar 

  20. Rosa A, Maddocks JH, Neumann FR et al (2006) Measuring limits of telomere movement on nuclear envelope. Biophys J 90:L24–L26

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Taddei A, Van Houwe G, Hediger F et al (2006) Nuclear pore association confers optimal expression levels for an inducible yeast gene. Nature 441:774–778

    Article  CAS  PubMed  Google Scholar 

  22. Dion V, Kalck V, Horigome C et al (2012) Increased mobility of double-strand breaks requires Mec1, Rad9 and the homologous recombination machinery. Nat Cell Biol 14:502–509

    Article  CAS  PubMed  Google Scholar 

  23. Seeber A, Dion V, Gasser SM (2013) Checkpoint kinases and the INO80 nucleosome remodeling complex enhance global chromatin mobility in response to DNA damage. Genes Dev 27:1999–2008

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Gartenberg MR, Neumann FR, Laroche T et al (2004) Sir-mediated repression can occur independently of chromosomal and subnuclear contexts. Cell 119:955–967

    Article  CAS  PubMed  Google Scholar 

  25. Marshall WF, Straight A, Marko JF et al (1997) Interphase chromosomes undergo constrained diffusional motion in living cells. Curr Biol 7:930–939

    Article  CAS  PubMed  Google Scholar 

  26. Mine-Hattab J, Rothstein R (2012) Increased chromosome mobility facilitates homology search during recombination. Nat Cell Biol 14:510–517

    Article  CAS  PubMed  Google Scholar 

  27. Sage D, Neumann FR, Hediger F et al (2005) Automatic tracking of individual fluorescence particles: application to the study of chromosome dynamics. IEEE Trans Image Process 14:1372–1383

    Article  PubMed  Google Scholar 

  28. Jensen RE, Herskowitz I (1984) Directionality and regulation of cassette substitution in yeast. Cold Spring Harb Symp Quant Biol 49:97–104

    Article  CAS  PubMed  Google Scholar 

  29. Sandell LL, Zakian VA (1993) Loss of a yeast telomere: arrest, recovery, and chromosome loss. Cell 75:729–739

    Article  CAS  PubMed  Google Scholar 

  30. Plessis A, Perrin A, Haber JE, Dujon B (1992) Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. Genetics 130:451–460

    PubMed Central  CAS  PubMed  Google Scholar 

  31. Jacquier A, Dujon B (1985) An intron-encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene. Cell 41:383–394

    Article  CAS  PubMed  Google Scholar 

  32. Meister P, Towbin BD, Pike BL et al (2010) The spatial dynamics of tissue-specific promoters during C. elegans development. Genes Dev 24:766–782

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Taddei A, Hediger F, Neumann FR et al (2004) Separation of silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins. EMBO J 23:1301–1312

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgement

We thank J. E. Haber for yeast strains and the Friedrich Miescher Institute Microscopy Facility for technical help. C.H. thanks the Marie Curie International program and JSPS Research Abroad program for fellowships. The Gasser laboratory thanks the Novartis Research Foundation, the Swiss National Science Foundation “Sinergia grant,” NCCR “Frontiers in Genetics,” and the Human Frontier Science Program (RGP0017).

Author information

Authors and Affiliations

  1. Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058, Basel, Switzerland

    Chihiro Horigome, Vincent Dion, Andrew Seeber, Lutz R. Gehlen & Susan M. Gasser

  2. Center for Integrative Genomics, University of Lausanne, 1015, Lausanne, Switzerland

    Vincent Dion

  3. Faculty of Natural Sciences, University of Basel, 4056, Basel, Switzerland

    Andrew Seeber & Susan M. Gasser

Authors
  1. Chihiro Horigome
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vincent Dion
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andrew Seeber
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lutz R. Gehlen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Susan M. Gasser
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Susan M. Gasser .

Editor information

Editors and Affiliations

  1. Wentworth Institute of Technology, Boston, Massachusetts, USA

    Christine M. Oslowski

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Horigome, C., Dion, V., Seeber, A., Gehlen, L.R., Gasser, S.M. (2015). Visualizing the Spatiotemporal Dynamics of DNA Damage in Budding Yeast. In: Oslowski, C. (eds) Stress Responses. Methods in Molecular Biology, vol 1292. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2522-3_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2522-3_6

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2521-6

  • Online ISBN: 978-1-4939-2522-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation