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:

HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability

Nature Cell Biology volume 9, pages 391–401 (2007)Cite this article

Abstract

Here, we show that the human homologue of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation. ATR phosphorylation is known to both activate Chk1 and target it for proteolytic degradation, and depleting ATR or mutation of Chk1 at Ser 345 restored Chk1 protein levels in HCLK2-depleted cells. We conclude that HCLK2 promotes activation of the S-phase checkpoint and downstream repair responses by preventing unscheduled Chk1 degradation by the proteasome.

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: HCLK2 interacts with components of the S-phase checkpoint.
Figure 2: HCLK2-deficient cells exhibit S-phase checkpoint defects.
Figure 3: HCLK2 is required for damage-induced FANCD2 monoubiquitination and recruitment to sites of replication stress.
Figure 4: C. elegans clk-2 mutants and HCLK2-deficient human cells exhibit similar defects in FANCD2 function and ICL repair.
Figure 5: HCLK2 is required for homologous-recombination repair following replication stress.
Figure 6: HCLK2 depletion compromises claspin phosphorylation and Cdc25A degradation in the absence of HCLK2, but not Chk2 pThr 68 phosphorylation.
Figure 7: Chk1 instability in HCLK2-depleted cells is dependent on ATR phosphorylation of Chk1 at Ser 345.
Figure 8: Schematic representation of the role of HCLK2 in regulating the S-phase checkpoint.

Similar content being viewed by others

References

  1. Bekker-Jensen, S. et al. Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J. Cell Biol. 173, 195–206 (2006).

    Article  CAS  Google Scholar 

  2. Stucki, M. & Jackson, S. P. γH2AX and MDC1: anchoring the DNA-damage-response machinery to broken chromosomes. DNA Repair 5, 534–543 (2006).

    Article  CAS  Google Scholar 

  3. Kastan, M. B. & Bartek, J. Cell-cycle checkpoints and cancer. Nature 432, 316–323 (2004).

    Article  CAS  Google Scholar 

  4. Zhou, B. B. & Elledge, S. J. The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Shiloh, Y. ATM and related protein kinases: safeguarding genome integrity. Nature Rev. Cancer 3, 155–168 (2003).

    Article  CAS  Google Scholar 

  7. Kennedy, R. D. & D'Andrea, A. D. The Fanconi Anemia/BRCA pathway: new faces in the crowd. Genes Dev. 19, 2925–2940 (2005).

    Article  CAS  Google Scholar 

  8. Antoch, M. P., Kondratov, R. V. & Takahashi, J. S. Circadian clock genes as modulators of sensitivity to genotoxic stress. Cell Cycle 4, 901–907 (2005).

    Article  CAS  Google Scholar 

  9. Fu, L., Pelicano, H., Liu, J., Huang, P. & Lee, C. The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo. Cell 111, 41–50 (2002).

    Article  CAS  Google Scholar 

  10. Gery, S. et al. The circadian gene per1 plays an important role in cell growth and DNA damage control in human cancer cells. Mol. Cell 22, 375–382 (2006).

    Article  CAS  Google Scholar 

  11. Pregueiro, A. M., Liu, Q., Baker, C. L., Dunlap, J. C. & Loros, J. J. The Neurospora checkpoint kinase 2: a regulatory link between the circadian and cell cycles. Science 313, 644–649 (2006).

    Article  CAS  Google Scholar 

  12. Ahmed, S., Alpi, A., Hengartner, M. O. & Gartner, A. C. elegans RAD-5/CLK-2 defines a new DNA damage checkpoint protein. Curr. Biol. 11, 1934–1944 (2001).

    Article  CAS  Google Scholar 

  13. Garcia-Muse, T. & Boulton, S. J. Distinct modes of ATR activation after replication stress and DNA double-strand breaks in Caenorhabditis elegans. EMBO J. 24, 4345–4355 (2005).

    Article  CAS  Google Scholar 

  14. Early, A., Drury, L. S. & Diffley, J. F. Mechanisms involved in regulating DNA replication origins during the cell cycle and in response to DNA damage. Philos. Trans. R. Soc. Lond. B Biol. Sci. 359, 31–38 (2004).

    Article  CAS  Google Scholar 

  15. Polanowska, J., Martin, J. S., Garcia-Muse, T., Petalcorin, M. I. & Boulton, S. J. A conserved pathway to activate BRCA1-dependent ubiquitylation at DNA damage sites. EMBO J. 25, 2178–2188 (2006).

    Article  CAS  Google Scholar 

  16. Brown, E. J. & Baltimore, D. ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev. 14, 397–402 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Kim, J. E., McAvoy, S. A., Smith, D. I. & Chen, J. Human TopBP1 ensures genome integrity during normal S phase. Mol. Cell Biol. 25, 10907–10915 (2005).

    Article  CAS  Google Scholar 

  18. Syljuasen, R. G. et al. Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. Mol. Cell Biol. 25, 3553–3562 (2005).

    Article  CAS  Google Scholar 

  19. Jaspers, N. G. & Zdzienicka, M. Z. Inhibition of DNA synthesis by ionizing radiation: a marker for an S-phase checkpoint. Methods Mol. Biol. 314, 51–59 (2006).

    Article  CAS  Google Scholar 

  20. Brown, E. J. & Baltimore, D. Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance. Genes Dev. 17, 615–628 (2003).

    Article  CAS  Google Scholar 

  21. Chini, C. C. & Chen, J. Human claspin is required for replication checkpoint control. J. Biol. Chem. 278, 30057–30062 (2003).

    Article  CAS  Google Scholar 

  22. Goldberg, M. et al. MDC1 is required for the intra-S-phase DNA damage checkpoint. Nature 421, 952–956 (2003).

    Article  CAS  Google Scholar 

  23. Lou, Z., Minter-Dykhouse, K., Wu, X. & Chen, J. MDC1 is coupled to activated CHK2 in mammalian DNA damage response pathways. Nature 421, 957–961 (2003).

    Article  CAS  Google Scholar 

  24. Stewart, G. S., Wang, B., Bignell, C. R., Taylor, A. M. & Elledge, S. J. MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature 421, 961–966 (2003).

    Article  CAS  Google Scholar 

  25. Xu, B., Kim, S. & Kastan, M. B. Involvement of Brca1 in S-phase and G(2)-phase checkpoints after ionizing irradiation. Mol. Cell Biol. 21, 3445–3450 (2001).

    Article  CAS  Google Scholar 

  26. Liu, Q. et al. Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev. 14, 1448–1459 (2000).

    Article  CAS  Google Scholar 

  27. Lam, M. H., Liu, Q., Elledge, S. J. & Rosen, J. M. Chk1 is haploinsufficient for multiple functions critical to tumor suppression. Cancer Cell 6, 45–59 (2004).

    Article  CAS  Google Scholar 

  28. Takai, H. et al. Aberrant cell cycle checkpoint function and early embryonic death in Chk1−/− mice. Genes Dev. 14, 1439–1447 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Andreassen, P. R., D'Andrea, A. D. & Taniguchi, T. ATR couples FANCD2 monoubiquitination to the DNA-damage response. Genes Dev. 18, 1958–1963 (2004).

    Article  CAS  Google Scholar 

  30. Stiff, T. et al. Nbs1 is required for ATR-dependent phosphorylation events. EMBO J. 24, 199–208 (2005).

    Article  CAS  Google Scholar 

  31. Wang, X. et al. Chk1 mediated phosphorylation of FANCE is required for the Fanconi Anemia/BRCA pathway. Mol. Cell Biol. doi: 10.1128/MCB.02357-06 (2007).

  32. Garcia-Higuera, I. et al. Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol. Cell 7, 249–262 (2001).

    Article  CAS  Google Scholar 

  33. Collis, S. J., Barber, L. J., Ward, J. D., Martin, J. S. & Boulton, S. J. C. elegans FANCD2 responds to replication stress and functions in interstrand cross-link repair. DNA Repair 5, 1398–1406 (2006).

    Article  CAS  Google Scholar 

  34. Sorensen, C. S. et al. The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair. Nature Cell Biol. 7, 195–201 (2005).

    Article  CAS  Google Scholar 

  35. Johnson, R. D. & Jasin, M. Double-strand-break-induced homologous recombination in mammalian cells. Biochem. Soc. Trans. 29, 196–201 (2001).

    Article  CAS  Google Scholar 

  36. Saleh-Gohari, N. et al. Spontaneous homologous recombination is induced by collapsed replication forks that are caused by endogenous DNA single-strand breaks. Mol. Cell Biol. 25, 7158–7169 (2005).

    Article  CAS  Google Scholar 

  37. Zhao, H. & Piwnica-Worms, H. ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol. Cell Biol. 21, 4129–4139 (2001).

    Article  CAS  Google Scholar 

  38. Bartek, J., Lukas, C. & Lukas, J. Checking on DNA damage in S phase. Nature Rev. Mol. Cell Biol. 5, 792–804 (2004).

    Article  CAS  Google Scholar 

  39. Chini, C. C., Wood, J. & Chen, J. Chk1 is required to maintain Claspin stability. Oncogene 25, 4165–4171 (2006).

    Article  CAS  Google Scholar 

  40. Kumagai, A. & Dunphy, W. G. Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts. Mol. Cell 6, 839–849 (2000).

    Article  CAS  Google Scholar 

  41. Rao, V. A. et al. Phosphorylation of BLM, dissociation from topoisomerase IIIα, and colocalization with γ-H2AX after topoisomerase I-induced replication damage. Mol. Cell Biol. 25, 8925–8937 (2005).

    Article  CAS  Google Scholar 

  42. Zhang, Y. W. et al. Genotoxic stress targets human Chk1 for degradation by the ubiquitin-proteasome pathway. Mol. Cell 19, 607–618 (2005).

    Article  CAS  Google Scholar 

  43. Tercero, J. A. & Diffley, J. F. Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint. Nature 412, 553–557 (2001).

    Article  CAS  Google Scholar 

  44. Howlett, N. G., Taniguchi, T., Durkin, S. G., D'Andrea, A. D. & Glover, T. W. The Fanconi anemia pathway is required for the DNA replication stress response and for the regulation of common fragile site stability. Hum. Mol. Genet. 14, 693–701 (2005).

    Article  CAS  Google Scholar 

  45. O'Driscoll, M., Ruiz-Perez, V. L., Woods, C. G., Jeggo, P. A. & Goodship, J. A. A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome. Nature Genet. 33, 497–501 (2003).

    Article  CAS  Google Scholar 

  46. Dimitrova, D. S. & Gilbert, D. M. Temporally coordinated assembly and disassembly of replication factories in the absence of DNA synthesis. Nature Cell Biol. 2, 686–694 (2000).

    Article  CAS  Google Scholar 

  47. Feijoo, C. et al. Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing. J. Cell Biol. 154, 913–923 (2001).

    Article  CAS  Google Scholar 

  48. Jiang, N., Benard, C. Y., Kebir, H., Shoubridge, E. A. & Hekimi, S. Human CLK2 links cell cycle progression, apoptosis, and telomere length regulation. J. Biol. Chem. 278, 21678–21684 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We wish to thank: A. Jones and H. Cooper for mass-spectrometry; Y. Murakawa and S. Takeda for laser micro-irradiation experiments; S. Hekimi, S. West, N. Lakin, G. Smith, S. Jackson and A. Jazayeri for HCLK2, Rad51, ATR, ATRIP and Cdc25A antibodies, respectively; N. Mailand, J. Bartek, H. Piwnica-Worms, Y.-W. Zhang and B. Abraham for Chk1 constructs; H. Bryant and T. Helleday for SW480sn3 cells, ISce-1 and GFP-reporter plasmids; N. O'Reilly for peptide synthesis; and R. Peat and R. Horton-Harpin for cell culture. Thanks to J. Svejstrup, P.Zegerman, A. Jazayeri, G. Alderton, J. Falck, P. Robins, M. Segurado and members of the Boulton lab for technical advice and comments on the manuscript. This work was funded by Breast Cancer Campaign (GA3221) and Cancer Research UK.

Author information

Authors and Affiliations

  1. DNA Damage Response Laboratory, Cancer Research UK, The London Research Institute, Clare Hall Laboratories, South Mimms, EN6 3LD, UK

    Spencer J. Collis, Louise J. Barber, Allison J. Clark, Julie S. Martin, Jordan D. Ward & Simon J. Boulton

Authors
  1. Spencer J. Collis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Louise J. Barber
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Allison J. Clark
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Julie S. Martin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jordan D. Ward
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Simon J. Boulton
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.J.C. performed the majority of experiments. L.J.B., A.J.C., J.S.M. and J.D.W. all contributed to the experiments. S.J.B., S.J.C. and L.J.B. contributed intellectually to this work.

Corresponding author

Correspondence to Simon J. Boulton.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures S1, S2, S3, S4 and S5 (PDF 612 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Collis, S., Barber, L., Clark, A. et al. HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability. Nat Cell Biol 9, 391–401 (2007). https://doi.org/10.1038/ncb1555

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb1555

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
pFad - Phonifier reborn

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.

Note: This service is not intended for secure transactions such as banking, social media, email, or purchasing. Use at your own risk. We assume no liability whatsoever for broken pages.


Alternative Proxies:

Alternative Proxy

pFad Proxy

pFad v3 Proxy

pFad v4 Proxy