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The yeast Fun30 and human SMARCAD1 chromatin remodellers promote DNA end resection

Nature volume 489, pages 581–584 (2012)Cite this article

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Abstract

Several homology-dependent pathways can repair potentially lethal DNA double-strand breaks (DSBs). The first step common to all homologous recombination reactions is the 5′–3′ degradation of DSB ends that yields the 3′ single-stranded DNA required for the loading of checkpoint and recombination proteins. In yeast, the Mre11–Rad50–Xrs2 complex (Xrs2 is known as NBN or NBS1 in humans) and Sae2 (known as RBBP8 or CTIP in humans) initiate end resection, whereas long-range resection depends on the exonuclease Exo1, or the helicase–topoisomerase complex Sgs1–Top3–Rmi1 together with the endonuclease Dna2 (refs 1–6). DSBs occur in the context of chromatin, but how the resection machinery navigates through nucleosomal DNA is a process that is not well understood7. Here we show that the yeast Saccharomyces cerevisiae Fun30 protein and its human counterpart SMARCAD1 (ref. 8), two poorly characterized ATP-dependent chromatin remodellers of the Snf2 ATPase family, are directly involved in the DSB response. Fun30 physically associates with DSB ends and directly promotes both Exo1- and Sgs1-dependent end resection through a mechanism involving its ATPase activity. The function of Fun30 in resection facilitates the repair of camptothecin-induced DNA lesions, although it becomes dispensable when Exo1 is ectopically overexpressed. Interestingly, SMARCAD1 is also recruited to DSBs, and the kinetics of recruitment is similar to that of EXO1. The loss of SMARCAD1 impairs end resection and recombinational DNA repair, and renders cells hypersensitive to DNA damage resulting from camptothecin or poly(ADP-ribose) polymerase inhibitor treatments. These findings unveil an evolutionarily conserved role for the Fun30 and SMARCAD1 chromatin remodellers in controlling end resection, homologous recombination and genome stability in the context of chromatin.

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Figure 1: fun30 Δ and DNA end-resection mutants show high BIR efficiencies.
Figure 2: Fun30 promotes long-range 5′−3′ DNA end resection and is recruited to DSBs.
Figure 3: SMARCAD1 promotes end resection, homologous recombination and cell survival after genotoxic insults in U2OS cells.
Figure 4: Model for Fun30 and SMARCAD1 control of end resection through DSB-associated nucleosomes.

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Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data have been deposited in the NCBI Gene Expression Omnibus and are accessible through accession numbers GSE38715 (BIR screen) and GSE38735 (fun30∆ transcriptome).

Change history

  • 26 September 2012

    Reference numbering in the online-only Methods section was corrected.

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Acknowledgements

We thank G. Ira for sharing unpublished data and S. Janicki, R. Greenberg, L. Symington and all the laboratories from the Centre National de la Recherche Scientifique (CNRS) UPR3081 for providing reagents. We thank S. Coulon for help in the analysis of the fun30 repressible allele, I. Lafontaine for support in statistical analyses, C. V. Camacho for generating the V5-EXO1 constructs and A. Guénolé, R. Srivas, T. Ideker, K. Vreeken and M. Vermeulen for help in searching for Fun30 interactors. B.L. is grateful to B. Dujon for hosting him and providing the opportunity to perform the BIR screen. S.B. is supported by grants from the National Institutes of Health (RO1 CA149461), National Aeronautics and Space Administration (NNX10AE08G) and the Cancer Prevention and Research Institute of Texas (RP100644). H.v.A. receives funding from the Netherlands Organization for Scientific Research (NWO-VIDI grant) and Human Frontiers Science Program (HFSP-CDA grant). B.L. is supported by grants from the CNRS (ATIP) and the Agence Nationale de la Recherche (ANR-10-BLAN-1606-03).

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Author notes

  1. Thomas Costelloe, Raphaël Louge and Nozomi Tomimatsu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands,

    Thomas Costelloe, Wouter W. Wiegant & Haico van Attikum

  2. Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7258, Centre de Recherche en Cancérologie de Marseille, F-13009, Marseille, France

    Raphaël Louge, Basheer Khadaroo, Kenny Dubois & Bertrand Llorente

  3. Aix-Marseille University, Unité Mixte de Recherche 7258, Marseille, F-13284, France

    Raphaël Louge, Basheer Khadaroo, Kenny Dubois & Bertrand Llorente

  4. Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.214E, Dallas, Texas 75390, USA,

    Nozomi Tomimatsu, Bipasha Mukherjee & Sandeep Burma

  5. CEA-DSV-Institut de Radiobiologie Cellulaire et Moléculaire, Fontenay aux Roses, 92265, France

    Emmanuelle Martini

  6. Institut Pasteur, Unité de Génétique Moléculaire des Levures, Centre National de la Recherche Scientifique and University Pierre and Marie Curie-Paris, 25 rue du Docteur Roux, F75724 Paris Cedex 15, France ,

    Agnès Thierry

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Contributions

B.L. and A.T. performed the genetic screen and B.L. identified the resection defect of fun30Δ. T.C. constructed yeast strains and plasmids and performed the yeast ChIP experiments. R.L. constructed yeast strains and performed ssDNA analysis by alkaline gels, BIR and gap-repair assays. R.L. and T.C. analysed SSA defects. N.T. and B.M. performed all of the SMARCAD1 knockdown experiments in human cells and the DR-GFP assays. E.M. designed and built the strain containing the inducible I-SceI cut site at HIS3, performed the micrococcal nuclease assay and contributed to data analysis. B.K. performed the analysis of survivors in the absence of telomerase. K.D. assisted R.L. and K.D., R.L. and T.C. performed fun30 DNA-damage-sensitivity assays. W.W.W. examined the localization of SMARCAD1 at FokI-induced DSBs. T.C., S.B., H.v.A. and B.L. designed the experiments and analysed the data. H.v.A. and B.L. wrote the manuscript.

Corresponding authors

Correspondence to Haico van Attikum or Bertrand Llorente.

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The authors declare no competing financial interests.

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Supplementary Information

This file contains Supplementary Figures 1-13, the full legend for Supplementary Table 1, Supplementary Tables 2 and 3 and Supplementary References. This file was replaced on 26 September 2012 to correct the numbering of the references. (PDF 5558 kb)

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Costelloe, T., Louge, R., Tomimatsu, N. et al. The yeast Fun30 and human SMARCAD1 chromatin remodellers promote DNA end resection. Nature 489, 581–584 (2012). https://doi.org/10.1038/nature11353

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