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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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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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