Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Increased mobility of double-strand breaks requires Mec1, Rad9 and the homologous recombination machinery

Nature Cell Biology volume 14, pages 502–509 (2012)Cite this article

Subjects

Abstract

Chromatin mobility is thought to facilitate homology search during homologous recombination and to shift damage either towards or away from specialized repair compartments. However, unconstrained mobility of double-strand breaks could also promote deleterious chromosomal translocations. Here we use live time-lapse fluorescence microscopy to track the mobility of damaged DNA in budding yeast. We found that a Rad52–YFP focus formed at an irreparable double-strand break moves in a larger subnuclear volume than the undamaged locus. In contrast, Rad52–YFP bound at damage arising from a protein–DNA adduct shows no increase in movement. Mutant analysis shows that enhanced double-strand-break mobility requires Rad51, the ATPase activity of Rad54, the ATR homologue Mec1 and the DNA-damage-response mediator Rad9. Consistent with a role for movement in the homology-search step of homologous recombination, we show that recombination intermediates take longer to form in cells lacking Rad9.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The mobility of spontaneous and damage-induced Rad52–YFP in live cells.
Figure 2: An I-SceI-induced Rad52–YFP focus is less constrained than the same undamaged locus.
Figure 3: The increase in the radius of constraint after I-SceI-induced DSB depends on Rad51 and Rad54.
Figure 4: Rad52–YFP foci induced by a Flp-nick move differently and have different genetic requirements than I-SceI-induced foci.
Figure 5: RAD9 and the DNA-damage response promote the movement of I-SceI-induced Rad52–YFP foci and the kinetics of homologous recombination.

Similar content being viewed by others

References

  1. Gehlen, L., Gasser, S. M. & Dion, V. How broken DNA finds a template for repair: a computational approach. Prog. Theor. Phys. Suppl. 191, 20–29 (2011).

    Article  CAS  Google Scholar 

  2. Savage, J. R. Insight into sites. Mutat. Res. 366, 81–95 (1996).

    Article  CAS  Google Scholar 

  3. Dimitrova, N., Chen, Y. C., Spector, D. L. & de Lange, T. 53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility. Nature 456, 524–528 (2008).

    Article  CAS  Google Scholar 

  4. Noon, A. T. & Goodarzi, A. A. 53BP1-mediated DNA double strand break repair: insert bad pun here. DNA Repair (Amst) 10, 1071–1076 (2011).

    Article  CAS  Google Scholar 

  5. Aten, J. A. et al. Dynamics of DNA double-strand breaks revealed by clustering of damaged chromosome domains. Science 303, 92–95 (2004).

    Article  CAS  Google Scholar 

  6. Lisby, M., Mortensen, U. H. & Rothstein, R. Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre. Nat. Cell Biol. 5, 572–577 (2003).

    Article  CAS  Google Scholar 

  7. Jakob, B., Splinter, J., Durante, M. & Taucher-Scholz, G. Live cell microscopy analysis of radiation-induced DNA double-strand break motion. Proc. Natl Acad. Sci. USA 106, 3172–3177 (2009).

    Article  CAS  Google Scholar 

  8. Jakob, B., Splinter, J. & Taucher-Scholz, G. Positional stability of damaged chromatin domains along radiation tracks in mammalian cells. Radiat. Res. 171, 405–418 (2009).

    Article  CAS  Google Scholar 

  9. Kruhlak, M. J. et al. Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks. J. Cell Biol. 172, 823–834 (2006).

    Article  CAS  Google Scholar 

  10. Soutoglou, E. et al. Positional stability of single double-strand breaks in mammalian cells. Nat. Cell Biol. 9, 675–682 (2007).

    Article  CAS  Google Scholar 

  11. Nelms, B. E., Maser, R. S., MacKay, J. F., Lagally, M. G. & Petrini, J. H. In situ visualization of DNA double-strand break repair in human fibroblasts. Science 280, 590–592 (1998).

    Article  CAS  Google Scholar 

  12. Gartenberg, M. R., Neumann, F. R., Laroche, T., Blaszczyk, M. & Gasser, S. M. Sir-mediated repression can occur independently of chromosomal and subnuclear contexts. Cell 119, 955–967 (2004).

    Article  CAS  Google Scholar 

  13. Meister, P., Gehlen, L. R., Varela, E., Kalck, V. & Gasser, S. M. Visualizing yeast chromosomes and nuclear architecture. Methods Enzymol. 470, 535–567 (2010).

    Article  CAS  Google Scholar 

  14. Neumann, F. R. et al. INO80 promotes chromatin movement and functionally impacts homologous recombination. Genes Dev. 26, 369–383 (2012).

    Article  CAS  Google Scholar 

  15. Straight, A. F., Belmont, A. S., Robinett, C. C. & Murray, A. W. GFP tagging of budding yeast chromosomes reveals that protein–protein interactions can mediate sister chromatid cohesion. Curr. Biol. 6, 1599–1608 (1996).

    Article  CAS  Google Scholar 

  16. Lisby, M., Barlow, J. H., Burgess, R. C. & Rothstein, R. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell 118, 699–713 (2004).

    Article  CAS  Google Scholar 

  17. Heun, P., Laroche, T., Shimada, K., Furrer, P. & Gasser, S. M. Chromosome dynamics in the yeast interphase nucleus. Science 294, 2181–2186 (2001).

    Article  CAS  Google Scholar 

  18. Povirk, L. F. DNA damage and mutagenesis by radiomimetic DNA-cleaving agents: bleomycin, neocarzinostatin and other enediynes. Mutat. Res. 355, 71–89 (1996).

    Article  Google Scholar 

  19. Mazin, A. V., Mazina, O. M., Bugreev, D. V. & Rossi, M. J. Rad54, the motor of homologous recombination. DNA Repair (Amst) 9, 286–302 (2010).

    Article  CAS  Google Scholar 

  20. Clever, B., Schmuckli-Maurer, J., Sigrist, M., Glassner, B. J. & Heyer, W. D. Specific negative effects resulting from elevated levels of the recombinational repair protein Rad54p in Saccharomyces cerevisiae. Yeast 15, 721–740 (1999).

    Article  CAS  Google Scholar 

  21. Nielsen, I. et al. A Flp-nick system to study repair of a single protein-bound nick in vivo. Nat. Methods 6, 753–757 (2009).

    Article  CAS  Google Scholar 

  22. Mochan, T. A., Venere, M., DiTullio, R. A. Jr & Halazonetis, T. D. 53BP1, an activator of ATM in response to DNA damage. DNA Repair (Amst) 3, 945–952 (2004).

    Article  CAS  Google Scholar 

  23. Nyberg, K. A., Michelson, R. J., Putnam, C. W. & Weinert, T. A. Toward maintaining the genome: DNA damage and replication checkpoints. Annu. Rev. Genet. 36, 617–656 (2002).

    Article  CAS  Google Scholar 

  24. Stucki, M. & Jackson, S.P. MDC1/NFBD1: a key regulator of the DNA damage response in higher eukaryotes. DNA Repair (Amst) 3, 953–957 (2004).

    Article  CAS  Google Scholar 

  25. Vialard, J. E., Gilbert, C. S., Green, C. M. & Lowndes, N. F. The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage. EMBO J. 17, 5679–5688 (1998).

    Article  CAS  Google Scholar 

  26. Sweeney, F. D. et al. Saccharomyces cerevisiae Rad9 acts as a Mec1 adaptor to allow Rad53 activation. Curr. Biol. 15, 1364–1375 (2005).

    Article  CAS  Google Scholar 

  27. Usui, T., Foster, S. S. & Petrini, J. H. Maintenance of the DNA-damage checkpoint requires DNA-damage-induced mediator protein oligomerization. Mol. Cell 33, 147–159 (2009).

    Article  CAS  Google Scholar 

  28. Conde, F. et al. The Dot1 histone methyltransferase and the Rad9 checkpoint adaptor contribute to cohesin-dependent double-strand break repair by sister chromatid recombination in Saccharomyces cerevisiae. Genetics 182, 437–446 (2009).

    Article  Google Scholar 

  29. Zhao, X., Muller, E. G. & Rothstein, R. A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools. Mol Cell 2, 329–340 (1998).

    Article  CAS  Google Scholar 

  30. Barlow, J. H. & Rothstein, R. Rad52 recruitment is DNA replication independent and regulated by Cdc28 and the Mec1 kinase. EMBO J. 28, 1121–1130 (2009).

    Article  CAS  Google Scholar 

  31. White, C. I. & Haber, J. E. Intermediates of recombination during mating type switching in Saccharomyces cerevisiae. EMBO J. 9, 663–673 (1990).

    Article  CAS  Google Scholar 

  32. Frank-Vaillant, M. & Marcand, S. Transient stability of DNA ends allows nonhomologous end joining to precede homologous recombination. Mol. Cell 10, 1189–1199 (2002).

    Article  CAS  Google Scholar 

  33. Nagai, S. et al. Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase. Science 322, 597–602 (2008).

    Article  CAS  Google Scholar 

  34. Pellicioli, A., Lee, S. E., Lucca, C., Foiani, M. & Haber, J. E. Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest. Mol. Cell 7, 293–300 (2001).

    Article  CAS  Google Scholar 

  35. Miné-Hattab, J. & Rothstein, R. Increased chromosome mobility: a model for homology search during homologous recombination. Nat. Cell Biol.http://dx.doi.org/10.1038/ncb2472 (2012).

  36. Dubrana, K., van Attikum, H., Hediger, F. & Gasser, S. M. The processing of double-strand breaks and binding of single-strand-binding proteins RPA and Rad51 modulate the formation of ATR-kinase foci in yeast. J. Cell Sci. 120, 4209–4220 (2007).

    Article  CAS  Google Scholar 

  37. Zou, L. & Elledge, S. J. Sensing DNA damage through ATRIP recognition of RPA–ssDNA complexes. Science 300, 1542–1548 (2003).

    Article  CAS  Google Scholar 

  38. Bennett, C. B., Lewis, A. L., Baldwin, K. K. & Resnick, M. A. Lethality induced by a single site-specific double-strand break in a dispensable yeast plasmid. Proc. Natl Acad. Sci. USA 90, 5613–5617 (1993).

    Article  CAS  Google Scholar 

  39. Chiolo, I. et al. Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair. Cell 144, 732–744 (2011).

    Article  CAS  Google Scholar 

  40. Torres-Rosell, J. et al. The Smc5–Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus. Nat. Cell Biol. 9, 923–931 (2007).

    Article  CAS  Google Scholar 

  41. Ponti, A., Gulati, A., Bäcker, V. & Schwarb, P. Huygens Remote Manager: a web interface for high-volume batch deconvolution. Imaging Microscopy 9, 57–58 (2007).

    Article  Google Scholar 

  42. Sage, D., Neumann, F. R., Hediger, F., Gasser, S. M. & Unser, M. Automatic tracking of individual fluorescence particles: application to the study of chromosome dynamics. IEEE Trans. Image Process 14, 1372–1383 (2005).

    Article  Google Scholar 

  43. Kim, Y. H. et al. Chromosome XII context is important for rDNA function in yeast. Nucleic Acids Res. 34, 2914–2924 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Schmid for imaging assistance, H. van Attikum for cloning assistance and L. Bjergbaek for the Flp-nick strain and sharing unpublished data. We thank W. Heyer for the Rad54 mutant construct, J. E. Haber for JKM154 and R. Rothstein and J. Mine-Hattab for sharing unpublished reagents and results. We thank B. Pike, K. Shimada, A. Gonzalez, N. Hustedt, M. Oppikofer, A. Seeber and F. Hamaratoglu for reading the manuscript and the Friedrich Miescher Institute Facility for Advanced Imaging and Microscopy for technical help. V.D. is supported in part by a postdoctoral award from the Terry Fox Foundation (award no. 19759) and work in S.M.G.’s laboratory is supported by the Novartis Research Foundation and the Swiss National Science Foundation National Centre of Competence in Research ‘Frontiers in genetics’ programme.

Author information

Authors and Affiliations

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

    Vincent Dion, Véronique Kalck, Chihiro Horigome, Benjamin D. Towbin & Susan M. Gasser

  2. University of Basel, Faculty of Sciences, CH-4056 Basel, Switzerland

    Benjamin D. Towbin & Susan M. Gasser

Authors
  1. Vincent Dion
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Véronique Kalck
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chihiro Horigome
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Benjamin D. Towbin
    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

Contributions

V.D. and S.M.G. designed the experiments, analysed the results and wrote the paper. V.D. and V.K. carried out the experiments. C.H. provided the cutting-efficiency data. B.D.T. tested and optimized the I-SceI cleavage.

Corresponding author

Correspondence to Susan M. Gasser.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 431 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dion, V., Kalck, V., Horigome, C. et al. Increased mobility of double-strand breaks requires Mec1, Rad9 and the homologous recombination machinery. Nat Cell Biol 14, 502–509 (2012). https://doi.org/10.1038/ncb2465

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2465

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing