Skip to main content

Thank you for visiting 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.

DNA-repair scaffolds dampen checkpoint signalling by counteracting the adaptor Rad9


In response to genotoxic stress, a transient arrest in cell-cycle progression enforced by the DNA-damage checkpoint (DDC) signalling pathway positively contributes to genome maintenance1. Because hyperactivated DDC signalling can lead to a persistent and detrimental cell-cycle arrest2,3, cells must tightly regulate the activity of the kinases involved in this pathway. Despite their importance, the mechanisms for monitoring and modulating DDC signalling are not fully understood. Here we show that the DNA-repair scaffolding proteins Slx4 and Rtt107 prevent the aberrant hyperactivation of DDC signalling by lesions that are generated during DNA replication in Saccharomyces cerevisiae. On replication stress, cells lacking Slx4 or Rtt107 show hyperactivation of the downstream DDC kinase Rad53, whereas activation of the upstream DDC kinase Mec1 remains normal. An Slx4–Rtt107 complex counteracts the checkpoint adaptor Rad9 by physically interacting with Dpb11 and phosphorylated histone H2A, two positive regulators of Rad9-dependent Rad53 activation. A decrease in DDC signalling results from hypomorphic mutations in RAD53 and H2A and rescues the hypersensitivity to replication stress of cells lacking Slx4 or Rtt107. We propose that the Slx4–Rtt107 complex modulates Rad53 activation by a competition-based mechanism that balances the engagement of Rad9 at replication-induced lesions. Our findings show that DDC signalling is monitored and modulated through the direct action of DNA-repair factors.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Slx4 counteracts Rad9-dependent Rad53 activation.
Figure 2: Slx4 binding to Dpb11 counteracts the Dpb11–Rad9 interaction and Rad53 activation.
Figure 3: Rtt107 counteracts Rad9-dependent Rad53 activation by binding to phosphorylated histone H2A.
Figure 4: Slx4 sensitizes mrc1 Δ cells to hydroxyurea-induced replication stress.


  1. Weinert, T. A. & Hartwell, L. H. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae . Science 241, 317–322 (1988)

    ADS  CAS  Article  Google Scholar 

  2. Clerici, M. et al. Hyperactivation of the yeast DNA damage checkpoint by TEL1 and DDC2 overexpression. EMBO J. 20, 6485–6498 (2001)

    CAS  Article  Google Scholar 

  3. 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)

    CAS  Article  Google Scholar 

  4. Fekairi, S. et al. Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases. Cell 138, 78–89 (2009)

    CAS  Article  Google Scholar 

  5. Svendsen, J. M. et al. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair. Cell 138, 63–77 (2009)

    CAS  Article  Google Scholar 

  6. Muñoz, I. M. et al. Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair. Mol. Cell 35, 116–127 (2009)

    Article  Google Scholar 

  7. Fricke, W. M. & Brill, S. J. Slx1–Slx4 is a second structure-specific endonuclease functionally redundant with Sgs1–Top3. Genes Dev. 17, 1768–1778 (2003)

    CAS  Article  Google Scholar 

  8. Stoepker, C. et al. SLX4, a coordinator of structure-specific endonucleases, is mutated in a new Fanconi anemia subtype. Nature Genet. 43, 138–141 (2011)

    CAS  Article  Google Scholar 

  9. Kim, Y. et al. Mutations of the SLX4 gene in Fanconi anemia. Nature Genet. 43, 142–146 (2011)

    CAS  Article  Google Scholar 

  10. Roberts, T. M. et al. Slx4 regulates DNA damage checkpoint-dependent phosphorylation of the BRCT domain protein Rtt107/Esc4. Mol. Biol. Cell 17, 539–548 (2006)

    CAS  Article  Google Scholar 

  11. Schwartz, M. F. et al. Rad9 phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint. Mol. Cell 9, 1055–1065 (2002)

    CAS  Article  Google Scholar 

  12. Schwartz, M. F., Lee, S. J., Duong, J. K., Eminaga, S. & Stern, D. F. FHA domain-mediated DNA checkpoint regulation of Rad53. Cell Cycle 2, 381–394 (2003)

    Article  Google Scholar 

  13. Tercero, J. A., Longhese, M. P. & Diffley, J. F. A central role for DNA replication forks in checkpoint activation and response. Mol. Cell 11, 1323–1336 (2003)

    CAS  Article  Google Scholar 

  14. Ohouo, P. Y., Bastos de Oliveira, F. M., Almeida, B. S. & Smolka, M. B. DNA damage signaling recruits the Rtt107–Slx4 scaffolds via Dpb11 to mediate replication stress response. Mol. Cell 39, 300–306 (2010)

    CAS  Article  Google Scholar 

  15. Navadgi-Patil, V. M. & Burgers, P. M. Yeast DNA replication protein Dpb11 activates the Mec1/ATR checkpoint kinase. J. Biol. Chem. 283, 35853–35859 (2008)

    CAS  Article  Google Scholar 

  16. Mordes, D. A., Nam, E. A. & Cortez, D. Dpb11 activates the Mec1–Ddc2 complex. Proc. Natl Acad. Sci. USA 105, 18730–18734 (2008)

    ADS  CAS  Article  Google Scholar 

  17. Granata, M. et al. Dynamics of Rad9 chromatin binding and checkpoint function are mediated by its dimerization and are cell cycle-regulated by CDK1 activity. PLoS Genet. 6, e1001047 (2010)

    Article  Google Scholar 

  18. Pfander, B. & Diffley, J. F. Dpb11 coordinates Mec1 kinase activation with cell cycle-regulated Rad9 recruitment. EMBO J. 30, 4897–4907 (2011)

    CAS  Article  Google Scholar 

  19. Tanaka, S. et al. CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast. Nature 445, 328–332 (2007)

    ADS  CAS  Article  Google Scholar 

  20. Zegerman, P. & Diffley, J. F. Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast. Nature 445, 281–285 (2007)

    ADS  CAS  Article  Google Scholar 

  21. Puddu, F. et al. Phosphorylation of the budding yeast 9-1-1 complex is required for Dpb11 function in the full activation of the UV-induced DNA damage checkpoint. Mol. Cell. Biol. 28, 4782–4793 (2008)

    CAS  Article  Google Scholar 

  22. Li, X. et al. Structure of C-terminal tandem BRCT repeats of Rtt107 protein reveals critical role in interaction with phosphorylated histone H2A during DNA damage repair. J. Biol. Chem. 287, 9137–9146 (2012)

    CAS  Article  Google Scholar 

  23. Javaheri, A. et al. Yeast G1 DNA damage checkpoint regulation by H2A phosphorylation is independent of chromatin remodeling. Proc. Natl Acad. Sci. USA 103, 13771–13776 (2006)

    ADS  CAS  Article  Google Scholar 

  24. Alcasabas, A. A. et al. Mrc1 transduces signals of DNA replication stress to activate Rad53. Nature Cell Biol. 3, 958–965 (2001)

    CAS  Article  Google Scholar 

  25. Smolka, M. B. et al. An FHA domain-mediated protein interaction network of Rad53 reveals its role in polarized cell growth. J. Cell Biol. 175, 743–753 (2006)

    CAS  Article  Google Scholar 

  26. Albuquerque, C. P. et al. A multidimensional chromatography technology for in-depth phosphoproteome analysis. Mol. Cell. Proteomics 7, 1389–1396 (2008)

    CAS  Article  Google Scholar 

  27. Smolka, M. B., Albuquerque, C. P., Chen, S. H. & Zhou, H. Proteome-wide identification of in vivo targets of DNA damage checkpoint kinases. Proc. Natl Acad. Sci. USA 104, 10364–10369 (2007)

    ADS  CAS  Article  Google Scholar 

  28. Smolka, M. B. et al. Dynamic changes in protein–protein interaction and protein phosphorylation probed with amine-reactive isotope tag. Mol. Cell. Proteomics 4, 1358–1369 (2005)

    CAS  Article  Google Scholar 

  29. Chen, S. H., Albuquerque, C. P., Liang, J., Suhandynata, R. T. & Zhou, H. A proteome-wide analysis of kinase-substrate network in the DNA damage response. J. Biol. Chem. 285, 12803–12812 (2010)

    CAS  Article  Google Scholar 

  30. Petesch, S. J. & Lis, J. T. Rapid, transcription-independent loss of nucleosomes over a large chromatin domain at Hsp70 loci. Cell 134, 74–84 (2008)

    CAS  Article  Google Scholar 

Download references


This work was supported by grants from the National Institutes of Health (RO1-GM097272 to M.B.S. and F31-GM093588 to P.Y.O.). F.M.B.O. was supported by a Cornell Fleming Research Fellowship. C.J.M. was supported by an HHMI Institutional Undergraduate Education Grant to Cornell. The authors thank B. Almeida for technical assistance and R. Weiss, S. Emr, A. Bretscher, G. Balmus and P. Russell for comments on the manuscript.

Author information

Authors and Affiliations



P.Y.O., F.M.B.O. and M.B.S. designed and performed experiments and analysed the data. P.Y.O. and M.B.S. performed the mass spectrometry experiments. F.M.B.O. performed the chromatin immunoprecipitation analysis and generated the slx4 mutants. Y.L. and P.Y.O. performed co-immunoprecipitations between Dpb11 and Rad9. Y.L. performed pull-down experiments with the BRCT domains of Dpb11. C.J.M. performed the Rtt107–H2A binding assay and the experiments with the Rtt107 BRCT domains. P.Y.O. and M.B.S. performed experiments involving the overexpression of Slx4. P.Y.O. and M.B.S. wrote the paper.

Corresponding author

Correspondence to Marcus B. Smolka.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-12, Supplementary Tables 1-4 and additional references. (PDF 8516 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ohouo, P., Bastos de Oliveira, F., Liu, Y. et al. DNA-repair scaffolds dampen checkpoint signalling by counteracting the adaptor Rad9. Nature 493, 120–124 (2013).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


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.


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