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Non-proteolytic inactivation of geminin requires CDK-dependent ubiquitination

Nature Cell Biology volume 6, pages 260–267 (2004)Cite this article

Abstract

In late mitosis and G1, a complex of the essential initiation proteins Mcm2–7 are assembled onto replication origins to 'license' them for initiation. At other times licensing is inhibited by cyclin-dependent kinases (CDKs) and geminin, thus ensuring that origins fire only once per cell cycle. Here we show that, paradoxically, CDKs are also required to inactivate geminin and activate the licensing system. On exit from metaphase in Xenopus laevis egg extracts, CDK-dependent activation of the anaphase-promoting complex (APC/C) results in the transient polyubiquitination of geminin. This ubiquitination triggers geminin inactivation without requiring ubiquitin-dependent proteolysis, and is essential for replication origins to become licensed. This reveals an unexpected role for CDKs and ubiquitination in activating chromosomal DNAreplication.

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Figure 1: Roscovitine inhibits licensing at concentrations where MPF is inhibited.
Figure 2: 6-DMAP and roscovitine prevent release of geminin from Cdt1.
Figure 3: Geminin is the only inhibitory activity in treated extracts.
Figure 4: Activation of licensing depends on the APC/C, but not on proteolysis.
Figure 5: Geminin is only transiently ubiquitinated on exit from metaphase.

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References

  1. Blow, J.J. & Hodgson, B. Replication licensing – defining the proliferative state? Trends Cell Biol. 12, 72–78 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Nishitani, H. & Lygerou, Z. Control of DNA replication licensing in a cell cycle. Genes Cells 7, 523–534 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Tada, S., Li, A., Maiorano, D., Mechali, M. & Blow, J.J. Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. Nature Cell Biol. 3, 107–113 (2001).

    Article  CAS  PubMed  Google Scholar 

  4. Hodgson, B., Li, A., Tada, S. & Blow, J.J. Geminin becomes activated as an inhibitor of Cdt1/RLF-B following nuclear import. Current Biol. 12, 678–683 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  7. Chong, J.P., Mahbubani, H.M., Khoo, C.Y. & Blow, J.J. Purification of an MCM-containing complex as a component of the DNA replication licensing system. Nature 375, 418–421 (1995).

    Article  CAS  PubMed  Google Scholar 

  8. Kubota, Y., Mimura, S., Nishimoto, S., Takisawa, H. & Nojima, H. Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. Cell 81, 601–609 (1995).

    Article  CAS  PubMed  Google Scholar 

  9. Madine, M.A., Khoo, C.-Y., Mills, A.D. & Laskey, R.A. MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells. Nature 375, 421–424 (1995).

    Article  CAS  PubMed  Google Scholar 

  10. Blow, J.J. & Laskey, R.A. A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature 332, 546–548 (1988).

    Article  CAS  PubMed  Google Scholar 

  11. Blow, J.J. Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor. J. Cell Biol. 122, 993–1002 (1993).

    Article  CAS  PubMed  Google Scholar 

  12. Kubota, Y. & Takisawa, H. Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts. J. Cell Biol. 123, 1321–1331 (1993).

    Article  CAS  PubMed  Google Scholar 

  13. Gillespie, P.J. & Blow, J.J. Nucleoplasmin-mediated chromatin remodelling is required for Xenopus sperm nuclei to become licensed for DNA replication. Nucleic Acids Res. 28, 472–480 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gillespie, P.J., Li, A. & Blow, J.J. Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins. BioMed Central Biochem. 2, 15 (2001).

    CAS  Google Scholar 

  15. Mahbubani, H.M., Chong, J.P., Chevalier, S., Thömmes, P. & Blow, J.J. Cell cycle regulation of the replication licensing system: involvement of a Cdk-dependent inhibitor. J. Cell Biol. 136, 125–135 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Meijer, L. et al. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur. J. Biochem. 243, 527–536 (1997).

    Article  CAS  PubMed  Google Scholar 

  17. Blow, J.J. & Nurse, P. A cdc2-like protein is involved in the initiation of DNA replication in Xenopus egg extracts. Cell 62, 855–862 (1990).

    Article  CAS  PubMed  Google Scholar 

  18. Zachariae, W. & Nasmyth, K. Whose end is destruction: cell division and the anaphase-promoting complex. Genes Dev. 13, 2039–2058 (1999).

    Article  CAS  PubMed  Google Scholar 

  19. Peters, J.M. The anaphase-promoting complex: proteolysis in mitosis and beyond. Mol. Cell 9, 931–943 (2002).

    Article  CAS  PubMed  Google Scholar 

  20. Luca, F.C. & Ruderman, J.V. Control of programmed cyclin destruction in a cell-free system. J. Cell Biol. 109, 1895–1909 (1989).

    Article  CAS  PubMed  Google Scholar 

  21. Peter, M. et al. The APC is dispensable for first meiotic anaphase in Xenopus oocytes. Nature Cell Biol. 3, 83–87 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Nishiyama, A. et al. A non-proteolytic function of the proteasome is required for the dissociation of Cdc2 and cyclin B at the end of M phase. Genes Dev. 14, 2344–2357 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Mahaffey, D., Yoo, Y. & Rechsteiner, M. Ubiquitin metabolism in cycling Xenopus egg extracts. J. Biol. Chem. 268, 21205–21211 (1993).

    CAS  PubMed  Google Scholar 

  24. Bach, I. & Ostendorff, H.P. Orchestrating nuclear functions: ubiquitin sets the rhythm. Trends Biochem. Sci. 28, 189–195 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Schnell, J.D. & Hicke, L. Non-traditional functions of ubiquitin and ubiquitin-binding proteins. J. Biol. Chem. 278, 35857–35860 (2003).

    Article  CAS  PubMed  Google Scholar 

  26. Su, T.T. & O'Farrell, P.H. Chromosome association of minichromosome maintenance proteins in Drosophila endoreplication cycles. J. Cell Biol. 140, 451–460 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Coverley, D., Laman, H. & Laskey, R.A. Distinct roles for cyclins E and A during DNA replication complex assembly and activation. Nature Cell Biol. 4, 523–528 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Chong, J.P., Thömmes, P., Rowles, A., Mahbubani, H.M. & Blow, J.J. Characterization of the Xenopus replication licensing system. Methods Enzymol. 283, 549–564 (1997).

    Article  CAS  PubMed  Google Scholar 

  29. Strausfeld, U.P. et al. Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts. J. Cell Sci. 109, 1555–1563 (1996).

    CAS  PubMed  Google Scholar 

  30. 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  PubMed  Google Scholar 

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Acknowledgements

Thanks to C. Nieduszynski, S. Shreeram and A. Woodward for comments on the manuscript; to J. Hutchins and P. Clarke for the D-box peptide; to J. Kirk and T. Hunt for the anti-cyclin B antibody; and to J. Walter and T. Prokhorova for the anti-Cdk1 antibody. This work was supported by Cancer Research UK grants SP2385/0101 and C303/A3135.

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  1. Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee, DD1 5EH, UK

    Anatoliy Li & J. Julian Blow

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Correspondence to J. Julian Blow.

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Li, A., Blow, J. Non-proteolytic inactivation of geminin requires CDK-dependent ubiquitination. Nat Cell Biol 6, 260–267 (2004). https://doi.org/10.1038/ncb1100

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