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.

  • Letter
  • Published:

The Fun30 nucleosome remodeller promotes resection of DNA double-strand break ends

Abstract

Chromosomal double-strand breaks (DSBs) are resected by 5′ nucleases to form 3′ single-stranded DNA substrates for binding by homologous recombination and DNA damage checkpoint proteins. Two redundant pathways of extensive resection have been described both in cells1,2,3 and in vitro4,5,6, one relying on Exo1 exonuclease and the other on Sgs1 helicase and Dna2 nuclease. However, it remains unknown how resection proceeds within the context of chromatin, where histones and histone-bound proteins represent barriers for resection enzymes. Here we identify the yeast nucleosome-remodelling enzyme Fun30 as a factor promoting DSB end resection. Fun30 is the major nucleosome remodeller promoting extensive Exo1- and Sgs1-dependent resection of DSBs. The RSC and INO80 chromatin-remodelling complexes and Fun30 have redundant roles in resection adjacent to DSB ends. ATPase and helicase domains of Fun30, which are needed for nucleosome remodelling7, are also required for resection. Fun30 is robustly recruited to DNA breaks and spreads along the DSB coincident with resection. Fun30 becomes less important for resection in the absence of the histone-bound Rad9 checkpoint adaptor protein known to block 5′ strand processing8 and in the absence of either histone H3 K79 methylation or γ-H2A, which mediate recruitment of Rad9 (refs 9, 10). Together these data suggest that Fun30 helps to overcome the inhibitory effect of Rad9 on DNA resection.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: A genome-wide screen identifies novel genes that regulate gene integration in yeast.
Figure 2: fun30 Δ mutants are deficient in resection.
Figure 3: Fun30 is directly involved in DSB end resection.
Figure 4: Fun30 chromatin-remodelling factor promotes resection within Rad9-bound nucleosomes.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data for gene expression and gene targeting have been deposited in the Gene Expression Omnibus under accession number GSE38601.

References

  1. Gravel, S., Chapman, J. R., Magill, C. & Jackson, S. P. DNA helicases Sgs1 and BLM promote DNA double-strand break resection. Genes Dev. 22, 2767–2772 (2008)

    Article  CAS  PubMed  Google Scholar 

  2. Mimitou, E. P. & Symington, L. S. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455, 770–774 (2008)

    Article  ADS  CAS  PubMed  Google Scholar 

  3. Zhu, Z., Chung, W. H., Shim, E. Y., Lee, S. E. & Ira, G. Sgs1 helicase and two nucleases dna2 and exo1 resect DNA double-strand break ends. Cell 134, 981–994 (2008)

    Article  CAS  PubMed  Google Scholar 

  4. Cejka, P. et al. DNA end resection by Dna2–Sgs1–RPA and its stimulation by Top3–Rmi1 and Mre11–Rad50–Xrs2. Nature 467, 112–116 (2010)

    Article  ADS  CAS  PubMed  Google Scholar 

  5. Nicolette, M. L. et al. Mre11–Rad50–Xrs2 and Sae2 promote 5′ strand resection of DNA double-strand breaks. Nature Struct. Mol. Biol. 17, 1478–1485 (2010)

    Article  CAS  Google Scholar 

  6. Niu, H. et al. Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae . Nature 467, 108–111 (2010)

    Article  ADS  CAS  PubMed  Google Scholar 

  7. Awad, S., Ryan, D., Prochasson, P., Owen-Hughes, T. & Hassan, A. H. The Snf2 homolog Fun30 acts as a homodimeric ATP-dependent chromatin-remodeling enzyme. J. Biol. Chem. 285, 9477–9484 (2010)

    Article  CAS  PubMed  Google Scholar 

  8. Lazzaro, F. et al. Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres. EMBO J. 27, 1502–1512 (2008)

    CAS  PubMed Central  PubMed  Google Scholar 

  9. Hammet, A., Magill, C., Heierhorst, J. & Jackson, S. P. Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast. EMBO Rep. 8, 851–857 (2007)

    Article  CAS  PubMed  Google Scholar 

  10. Toh, G. W. et al. Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation. DNA Repair (Amst.) 5, 693–703 (2006)

    Article  CAS  Google Scholar 

  11. Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391 (2002)

    Article  ADS  CAS  PubMed  Google Scholar 

  12. Chung, W. H., Zhu, Z., Papusha, A., Malkova, A. & Ira, G. Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting. PLoS Genet. 6, e1000948 (2010)

    Article  PubMed  Google Scholar 

  13. Prakash, R. et al. Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination. Genes Dev. 23, 67–79 (2009)

    Article  CAS  PubMed  Google Scholar 

  14. Flaus, A., Martin, D. M., Barton, G. J. & Owen-Hughes, T. Identification of multiple distinct Snf2 subfamilies with conserved structural motifs. Nucleic Acids Res. 34, 2887–2905 (2006)

    Article  CAS  PubMed  Google Scholar 

  15. Neves-Costa, A., Will, W. R., Vetter, A. T., Miller, J. R. & Varga-Weisz, P. The SNF2-family member Fun30 promotes gene silencing in heterochromatic loci. PLoS ONE 4, e8111 (2009)

    Article  ADS  PubMed  Google Scholar 

  16. Yu, Q., Zhang, X. & Bi, X. Roles of chromatin remodeling factors in the formation and maintenance of heterochromatin structure. J. Biol. Chem. 286, 14659–14669 (2011)

    Article  CAS  PubMed  Google Scholar 

  17. Vaze, M. et al. Recovery from checkpoint-mediated arrest after repair of a double-strand break requires srs2 helicase. Mol. Cell 10, 373–385 (2002)

    Article  CAS  Google Scholar 

  18. Kalocsay, M., Hiller, N. J. & Jentsch, S. 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 (2009)

    Article  CAS  PubMed  Google Scholar 

  19. Shim, E. Y. et al. RSC mobilizes nucleosomes to improve accessibility of repair machinery to the damaged chromatin. Mol. Cell. Biol. 27, 1602–1613 (2007)

    Article  CAS  PubMed  Google Scholar 

  20. van Attikum, H., Fritsch, O., Hohn, B. & Gasser, S. M. Recruitment of the INO80 complex by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair. Cell 119, 777–788 (2004)

    Article  CAS  PubMed  Google Scholar 

  21. Ira, G., Malkova, A., Liberi, G., Foiani, M. & Haber, J. E. Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell 115, 401–411 (2003)

    Article  CAS  PubMed  Google Scholar 

  22. Ira, G. et al. DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1. Nature 431, 1011–1017 (2004)

    Article  ADS  CAS  PubMed  Google Scholar 

  23. Ivanov, E. L., Sugawara, N., White, C. I., Fabre, F. & Haber, J. E. Mutations in XRS2 and RAD50 delay but do not prevent mating-type switching in Saccharomyces cerevisiae . Mol. Cell. Biol. 14, 3414–3425 (1994)

    Article  CAS  PubMed  Google Scholar 

  24. Chen, C. C. et al. Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair. Cell 134, 231–243 (2008)

    Article  CAS  PubMed  Google Scholar 

  25. Alexeev, A., Mazin, A. & Kowalczykowski, S. C. Rad54 protein possesses chromatin-remodeling activity stimulated by the Rad51-ssDNA nucleoprotein filament. Nature Struct. Biol. 10, 182–186 (2003)

    Article  CAS  PubMed  Google Scholar 

  26. Lydall, D. & Weinert, T. Yeast checkpoint genes in DNA damage processing: implications for repair and arrest. Science 270, 1488–1491 (1995)

    Article  ADS  CAS  PubMed  Google Scholar 

  27. Bothmer, A. et al. Regulation of DNA end joining, resection, and immunoglobulin class switch recombination by 53BP1. Mol. Cell 42, 319–329 (2011)

    Article  CAS  PubMed  Google Scholar 

  28. Bunting, S. F. et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141, 243–254 (2010)

    Article  CAS  PubMed  Google Scholar 

  29. Okazaki, N. et al. The novel protein complex with SMARCAD1/KIAA1122 binds to the vicinity of TSS. J. Mol. Biol. 382, 257–265 (2008)

    Article  CAS  PubMed  Google Scholar 

  30. Wach, A., Brachat, A., Pohlmann, R. & Philippsen, P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae . Yeast 10, 1793–1808 (1994)

    Article  CAS  Google Scholar 

  31. Chen, X. et al. Cell cycle regulation of DNA double-strand break end resection by Cdk1-dependent Dna2 phosphorylation. Nature Struct. Mol. Biol. 18, 1015–1019 (2011)

    Article  Google Scholar 

  32. Sugawara, N., Wang, X. & Haber, J. E. In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination. Mol. Cell 12, 209–219 (2003)

    Article  CAS  Google Scholar 

  33. Lambert, J. P. et al. Defining the budding yeast chromatin-associated interactome. Mol. Syst. Biol. 6, 448 (2010)

    Article  CAS  PubMed  Google Scholar 

  34. Church, G. M. & Gilbert, W. Genomic sequencing. Proc. Natl Acad. Sci. USA 81, 1991–1995 (1984)

    Article  ADS  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank P. Sung, W.-D. Heyer and A. Pellicioli for antibodies; S.-E. Lee, S. Kron and M. Osley for strains; B. Llorente for sharing unpublished data; and J. Tyler, P. Sung and S. E. Lee for comments on the manuscript. This work was supported by the National Institutes of Health grants GM080600 (to G.I.) and HG004840 (to X.P.).

Author information

Authors and Affiliations

Authors

Contributions

X.C. and D.C. contributed equally to this work. X.C. constructed most of the strains and analysed chromatin structure, protein interactions, protein recruitment to DSBs, histone loss at DSBs, activation of the DNA damage checkpoint and DNA damage sensitivity. X.Z. and K.C. performed microarray analysis of gene expression. D.C., J.T. and X.P. performed the genetic screen and constructed FUN30 point mutants. A.P. and C.-D.C. analysed resection and crossover frequency. X.C., X.P. and G.I. designed the experiments, discussed the data and wrote the manuscript.

Corresponding authors

Correspondence to Xuewen Pan or Grzegorz Ira.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 2-3, Supplementary References and Supplementary Figures 1-10. (PDF 7524 kb)

Supplementary Data

This file contains Supplementary Table 1 containing all data obtained from the genetic screen. Selected data from this supplemental table are presented in Figure 1 of the main text. (XLS 1268 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, X., Cui, D., Papusha, A. et al. The Fun30 nucleosome remodeller promotes resection of DNA double-strand break ends. Nature 489, 576–580 (2012). https://doi.org/10.1038/nature11355

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature11355

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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