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Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore–microtubule attachment


Cdt1, a protein critical for replication origin licensing in G1 phase, is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase owing to abnormally unstable kinetochore–microtubule (kMT) attachments and Mad1-dependent spindle-assembly-checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kMT attachment.

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Figure 1: Cells depleted of Cdt1 after S phase do not complete cell division.
Figure 2: G2-specific Cdt1 inhibition induces mitotic arrest.
Figure 3: Cdt1 transiently localizes to kinetochores during prometaphase and metaphase.
Figure 4: Hec1 is required for Cdt1 kinetochore localization.
Figure 5: Cdt1 targeting to kinetochores depends on the flexible loop region of Hec1.
Figure 6: Cdt1 and the Hec1 loop domain are required to satisfy the spindle-assembly checkpoint.
Figure 7: Cdt1 and the Hec1 loop domain are required for stable kMT attachments.
Figure 8: Cdt1 and the Hec1 loop domain are required for proper Ndc80 conformation in vivo.

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  1. DeLuca, J. G. et al. Kinetochore microtubule dynamics and attachment stability are regulated by Hec1. Cell 127, 969–982 (2006).

    Article  CAS  Google Scholar 

  2. Cheeseman, I. M., Chappie, J. S., Wilson-Kubalek, E. M. & Desai, A. The conserved KMN network constitutes the core microtubule-binding site of the kinetochore. Cell 127, 983–997 (2006).

    Article  CAS  Google Scholar 

  3. Alushin, G. M. et al. The Ndc80 kinetochore complex forms oligomeric arrays along microtubules. Nature 467, 805–810 (2010).

    Article  CAS  Google Scholar 

  4. Hsu, K. S. & Toda, T. Ndc80 internal loop interacts with Dis1/TOG to ensure proper kinetochore-spindle attachment in fission yeast. Curr. Biol. 21, 214–220 (2011).

    Article  CAS  Google Scholar 

  5. Maure, J. F. et al. The Ndc80 loop region facilitates formation of kinetochore attachment to the dynamic microtubule plus end. Curr. Biol. 21, 207–213 (2011).

    Article  CAS  Google Scholar 

  6. Machida, Y. J., Hamlin, J. L. & Dutta, A. Right place, right time, and only once: replication initiation in metazoans. Cell 123, 13–24 (2005).

    Article  CAS  Google Scholar 

  7. Sclafani, R. A. & Holzen, T. M. Cell cycle regulation of DNA replication. Annu. Rev. Genet. 41, 237–280 (2007).

    Article  CAS  Google Scholar 

  8. Snyder, M., He, W. & Zhang, J. J. The DNA replication factor MCM5 is essential for Stat1-mediated transcriptional activation. Proc. Natl Acad. Sci. USA 102, 14539–14544 (2005).

    Article  CAS  Google Scholar 

  9. Fitch, M. J., Donato, J. J. & Tye, B. K. Mcm7, a subunit of the presumptive MCM helicase, modulates its own expression in conjunction with Mcm1. J. Biol. Chem. 278, 25408–25416 (2003).

    Article  CAS  Google Scholar 

  10. Clay-Farrace, L., Pelizon, C., Santamaria, D., Pines, J. & Laskey, R. A. Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1. EMBO J. 22, 704–712 (2003).

    Article  CAS  Google Scholar 

  11. Yoshida, K. et al. CDC6 interaction with ATR regulates activation of a replication checkpoint in higher eukaryotic cells. J. Cell Sci. 123, 225–235 (2010).

    Article  CAS  Google Scholar 

  12. Tachibana, K. E., Gonzalez, M. A., Guarguaglini, G., Nigg, E. A. & Laskey, R. A. Depletion of licensing inhibitor geminin causes centrosome overduplication and mitotic defects. EMBO Rep. 6, 1052–1057 (2005).

    Article  CAS  Google Scholar 

  13. Prasanth, S. G., Prasanth, K. V., Siddiqui, K., Spector, D. L. & Stillman, B. Human Orc2 localizes to centrosomes, centromeres and heterochromatin during chromosome inheritance. EMBO J. 23, 2651–2663 (2004).

    Article  CAS  Google Scholar 

  14. Prasanth, S. G., Prasanth, K. V. & Stillman, B. Orc6 involved in DNA replication, chromosome segregation, and cytokinesis. Science 297, 1026–1031 (2002).

    Article  CAS  Google Scholar 

  15. Feng, D., Tu, Z., Wu, W. & Liang, C. Inhibiting the expression of DNA replication-initiation proteins induces apoptosis in human cancer cells. Cancer Res 63, 7356–7364 (2003).

    CAS  PubMed  Google Scholar 

  16. Nevis, K. R., Cordeiro-Stone, M. & Cook, J. G. Origin licensing and p53 status regulate Cdk2 activity during G1. Cell Cycle 8, 1952–1963 (2009).

    Article  CAS  Google Scholar 

  17. Nishitani, H., Taraviras, S., Lygerou, Z. & Nishimoto, T. The human licensing factor for DNA replication Cdt1 accumulates in G1 and is destabilized after initiation of S-phase. J. Biol. Chem. 276, 44905–44911 (2001).

    Article  CAS  Google Scholar 

  18. Chandrasekaran, S., Tan, T. X., Hall, J. R. & Cook, J. G. Stress-stimulated mitogen-activated protein kinases control the stability and activity of the Cdt1 DNA replication licensing factor. Mol. Cell. Biol. 31, 4405–4416 (2011).

    Article  CAS  Google Scholar 

  19. Wohlschlegel, J. A. et al. Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. Science 290, 2309–2312 (2000).

    Article  CAS  Google Scholar 

  20. McGarry, T. J. & Kirschner, M. W. Geminin, an inhibitor of DNA replication, is degraded during mitosis. Cell 93, 1043–1053 (1998).

    Article  CAS  Google Scholar 

  21. Maiorano, D., Moreau, J. & Mechali, M. XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis. Nature 404, 622–625 (2000).

    Article  CAS  Google Scholar 

  22. Chen, S., de Vries, M. A. & Bell, S. P. Orc6 is required for dynamic recruitment of Cdt1 during repeated Mcm2-7 loading. Genes Dev. 21, 2897–2907 (2007).

    Article  CAS  Google Scholar 

  23. Bernal, J. A. & Venkitaraman, A. R. A vertebrate N-end rule degron reveals that Orc6 is required in mitosis for daughter cell abscission. J. Cell Biol. 192, 969–978 (2011).

    Article  CAS  Google Scholar 

  24. Leonhardt, H. et al. Dynamics of DNA replication factories in living cells. J. Cell Biol. 149, 271–280 (2000).

    Article  CAS  Google Scholar 

  25. Meraldi, P., Draviam, V. M. & Sorger, P. K. Timing and checkpoints in the regulation of mitotic progression. Dev. Cell 7, 45–60 (2004).

    Article  CAS  Google Scholar 

  26. Wan, X. et al. Protein architecture of the human kinetochore microtubule attachment site. Cell 137, 672–684 (2009).

    Article  CAS  Google Scholar 

  27. Wei, R. R., Al-Bassam, J. & Harrison, S. C. The Ndc80/HEC1 complex is a contact point for kinetochore-microtubule attachment. Nat. Struct. Mol. Biol. 14, 54–59 (2007).

    Article  CAS  Google Scholar 

  28. Guimaraes, G. J., Dong, Y., McEwen, B. F. & Deluca, J. G. Kinetochore-microtubule attachment relies on the disordered N-terminal tail domain of Hec1. Curr. Biol. 18, 1778–1784 (2008).

    Article  CAS  Google Scholar 

  29. Miller, S. A., Johnson, M. L. & Stukenberg, P. T. Kinetochore attachments require an interaction between unstructured tails on microtubules and Ndc80(Hec1). Curr. Biol. 18, 1785–1791 (2008).

    Article  CAS  Google Scholar 

  30. Cimini, D., Wan, X., Hirel, C. B. & Salmon, E. D. Aurora kinase promotes turnover of kinetochore microtubules to reduce chromosome segregation errors. Curr. Biol. 16, 1711–1718 (2006).

    Article  CAS  Google Scholar 

  31. Yang, Z., Tulu, U. S., Wadsworth, P. & Rieder, C. L. Kinetochore dynein is required for chromosome motion and congression independent of the spindle checkpoint. Curr. Biol. 17, 973–980 (2007).

    Article  CAS  Google Scholar 

  32. Arias, E. E. & Walter, J. C. Replication-dependent destruction of Cdt1 limits DNA replication to a single round per cell cycle in Xenopus egg extracts. Genes Dev. 19, 114–126 (2005).

    Article  CAS  Google Scholar 

  33. Nishitani, H. et al. Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis. EMBO J. 25, 1126–1136 (2006).

    Article  CAS  Google Scholar 

  34. Devault, A. et al. Identification of Tah11/Sid2 as the ortholog of the replication licensing factor Cdt1 in Saccharomyces cerevisiae. Curr. Biol. 12, 689–694 (2002).

    Article  CAS  Google Scholar 

  35. Hofmann, J. F. & Beach, D. cdt1 is an essential target of the Cdc10/Sct1 transcription factor: requirement for DNA replication and inhibition of mitosis. EMBO J. 13, 425–434 (1994).

    Article  CAS  Google Scholar 

  36. Arentson, E. et al. Oncogenic potential of the DNA replication licensing protein CDT1. Oncogene 21, 1150–1158 (2002).

    Article  CAS  Google Scholar 

  37. Liontos, M. et al. Deregulated overexpression of hCdt1 and hCdc6 promotes malignant behavior. Cancer Res. 67, 10899–10909 (2007).

    Article  CAS  Google Scholar 

  38. Ciferri, C. et al. Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex. Cell 133, 427–439 (2008).

    Article  CAS  Google Scholar 

  39. Starr, D. A., Williams, B. C., Hays, T. S. & Goldberg, M. L. ZW10 helps recruit dynactin and dynein to the kinetochore. J. Cell Biol. 142, 763–774 (1998).

    Article  CAS  Google Scholar 

  40. Varma, D., Monzo, P., Stehman, S. A. & Vallee, R. B. Direct role of dynein motor in stable kinetochore-microtubule attachment, orientation, and alignment. J. Cell Biol. 182, 1045–1054 (2008).

    Article  CAS  Google Scholar 

  41. Cook, J. G., Chasse, D. A. & Nevins, J. R. The regulated association of Cdt1 with minichromosome maintenance proteins and Cdc6 in mammalian cells. J. Biol. Chem. 279, 9625–9633 (2004).

    Article  CAS  Google Scholar 

  42. Maddox, P. S., Moree, B., Canman, J. C. & Salmon, E. D. Spinning disk confocal microscope system for rapid high-resolution, multimode, fluorescence speckle microscopy and green fluorescent protein imaging in living cells. Methods Enzymol. 360, 597–617 (2003).

    Article  Google Scholar 

  43. James, P., Halladay, J. & Craig, E. A. Genomic libraries and a host strain designed for highly efficient two- hybrid selection in yeast. Genetics 144, 1425–1436 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

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We thank A. Desai for providing Knl1, Nsl1, Dsn1 and Spindly antibodies, A. Musacchio (Max Planck Institute of Molecular Physiology, Dortmund, Germany) for anti-Mad1, Zwint1 and ZW10 antibodies, B. Stillman (Cold Spring Harbor Laboratory, New York, USA) for anti-Orc6 antibody, S. Taylor (University of Manchester, UK) for anti-Bub1 and BubR1 antibodies, T. Stukenberg (University of Virginia at Charlottesville, Virginia, USA) for anti-Spc24 antibody, T. Yen (Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA) for anti-CENP-E antibody and I. Cheeseman (Whitehead Institute of Biomedical Research and MIT, Cambridge, Massachusetts, USA) for anti-Ska3 antibody. We are grateful to A. Desai (University of California San Diego, La Jolla, CA, USA), D. Cheerambathur, T. Stukenberg, K. Slep and T. Maresca for helpful discussions and to J. Mick for generating Hec1 constructs. We would also like to thank other members of the Salmon, Cook, A. Desai and J. Nevins laboratories for their support during this project. J.G.C. was supported by NIH K01 CA094907 and NIH GM083024, E.D.S. was supported by NIH GM24364 and J.G.D. was supported by NIH GM088371 and a grant from the Pew Scholars Program in the Biomedical Sciences.

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D.V. and J.G.C. designed and carried out experiments, analysed data and wrote the manuscript. S.C., L.J.R.S. and K.T.R. carried out experiments and analysed data. D.A.D.C. and J.G.C. conducted the two-hybrid screen. K.R.N. and S.C. characterized the arrest of Cdc6- and Cdt1-depleted normal fibroblasts. X.W. and D.V. conducted the Delta analyses. J.G.D., E.D.S. and J.G.C. designed experiments, analysed data and wrote the manuscript. All authors proofread the manuscript.

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Correspondence to E. D. Salmon or Jeanette Gowen Cook.

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Varma, D., Chandrasekaran, S., Sundin, L. et al. Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore–microtubule attachment. Nat Cell Biol 14, 593–603 (2012).

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