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Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2–7 during G1 phase

Abstract

Cdt1 is essential for loading Mcm2–7 proteins into prereplicative complexes (pre-RCs) during replication licensing and has been found in organisms as diverse as fission yeast and humans. We have identified a homologue of Cdt1 in Saccharomyces cerevisiae, which is required for pre-RC assembly. We show that, like Mcm2–7p, Cdt1p accumulates in the nucleus during G1 phase and is excluded from the nucleus later in the cell cycle by cyclin dependent kinases (cdks). Cdt1p interacts with the Mcm2–7p complex, and the nuclear accumulation of these proteins during G1 is interdependent. This coregulation of Cdt1p and Mcm2–7p represents a novel level of pre-RC control.

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Figure 1: Cdt1p is the budding yeast Cdt1 orthologue.
Figure 2: Cdt1p is not required after hydroxyurea-induced arrest.
Figure 3: Cdt1p levels and nuclear localization during the cell cycle.
Figure 4: Cdt1p relocalizes to the cytoplasm before initiation.
Figure 5: The function of Cdc28p and Cdc6p in Cdt1p-GFP nuclear localization.
Figure 6: Functional interactions between Cdt1p and the Mcm2–7p complex.
Figure 7: Steps in the regulation of budding yeast pre-RCs.

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Acknowledgements

We thank K. Labib, L. Drury, L. Noton, and T. Seki for helpful discussions and advice, and other members of our laboratory for discussion and encouragement. We also thank K. Labib for critically reading this manuscript, S. Hiraga and A. Hayashi-Hagihara for helpful information about GFP and J. Li for plasmids. This work is supported by the Imperial Cancer Research Fund. S.T. is the recipient of a Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad.

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Correspondence to John F.X. Diffley.

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Supplementary tables and figures

Supplemental Table 1 Yeast strains (PDF 875 kb)

Supplemental Table 2 Oligonucleotides

Figure S1 cdt1-td mutant.

Figure S2 Flow cytometry of the CDT1+ strain corresponding to Fig. 1D.

Figure S3 FACS analysis of cells in Fig. 6D and E.

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Tanaka, S., Diffley, J. Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2–7 during G1 phase. Nat Cell Biol 4, 198–207 (2002). https://doi.org/10.1038/ncb757

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