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Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin

Nature Cell Biology volume 3, pages 107–113 (2001)Cite this article

Abstract

Eukaryotic replication origins are 'licensed' for replication early in the cell cycle by loading Mcm(2–7) proteins. As chromatin replicates, Mcm(2–7) are removed, thus preventing the origin from firing again. Here we report the purification of the RLF-B component of the licensing system and show that it corresponds to Cdt1. RLF-B/Cdt1 was inhibited by geminin, a protein that is degraded during late mitosis. Immunodepletion of geminin from metaphase extracts allowed them to assemble licensed replication origins. Inhibition of CDKs in metaphase stimulated origin assembly only after the depletion of geminin. These experiments suggest that geminin-mediated inhibition of RLF-B/Cdt1 is essential for repressing origin assembly late in the cell cycle of higher eukaryotes.

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Figure 1: Inhibition of DNA replication by geminin is mediated by inhibition of RLF-B.
Figure 2: Purification of RLF-B.
Figure 3: Recombinant Cdt1 can provide RLF-B activity.
Figure 4: RLF-B/Cdt1 can associate with geminin in interphase extracts.
Figure 5: Proteins associated with chromatin in different extracts.
Figure 6: Characterization of the licensing inhibitor in metaphase extracts.
Figure 7: Licensing of sperm nuclei in geminin-depleted metaphase extract.
Figure 8: Model for licensing control during mitosis.

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Acknowledgements

We thank Jason Swedlow, Tom Owen-Hughes, Neil Perkins, Margret Michalski and members of the laboratory for comments on the manuscript. This work was supported by the Cancer Research Campaign (grant SP2385/1001) and by a Uehara Fellowship to S.T.

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  1. Shusuke Tada

    Present address: Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai, Miyagi, 980–8578, Japan

Authors and Affiliations

  1. CRC Chromosome Replication Research Group, Wellcome Trust Biocentre, Dow Street University of Dundee, Dundee, DD1 5EH, UK

    Shusuke Tada, Anatoliy Li & J. Julian Blow

  2. Institut of Human Genetics, CNRS, Genome Dynamics and Development, 141 rue de la Cardonille, Montpellier, 34396, Cedex 5, France

    Domenico Maiorano & Marcel Méchali

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Correspondence to J. Julian Blow.

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Tada, S., Li, A., Maiorano, D. et al. Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. Nat Cell Biol 3, 107–113 (2001). https://doi.org/10.1038/35055000

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