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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

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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.

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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.

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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for imaging assistance, H. van Attikum for cloning assistance and L. Bjergbaek for the Flp-nick strain and sharing unpublished data.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thank W. Heyer for the Rad54 mutant construct, J. E. Haber for JKM154 and R. Rothstein and J. Mine-Hattab for sharing unpublished reagents and results.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.

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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

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  1. Vincent Dion
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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.

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Correspondence to Susan M. Gasser.

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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

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