The Cdt1 protein is required to license DNA for replication in fission yeast

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Abstract

To maintain genome stability in eukaryotic cells, DNA is licensed for replication only after the cell has completed mitosis, ensuring that DNA synthesis (S phase) occurs once every cell cycle1. This licensing control is thought to require the protein Cdc6 (Cdc18 in fission yeast) as a mediator for association of minichromosome maintenance (MCM) proteins with chromatin2,3,4,5,6,7,8,9,10. The control is overridden in fission yeast by overexpressing Cdc18 (ref. 11) which leads to continued DNA synthesis in the absence of mitosis12. Other factors acting in this control have been postulated13 and we have used a re-replication assay to identify Cdt1 (ref. 14) as one such factor. Cdt1 cooperates with Cdc18 to promote DNA replication, interacts with Cdc18, is located in the nucleus, and its concentration peaks as cells finish mitosis and proceed to S phase. Both Cdc18 and Cdt1 are required to load the MCM protein Cdc21 onto chromatin at the end of mitosis and this is necessary to initiate DNA replication. Genes related to Cdt1 have been found in Metazoa and plants (A. Whitaker, I. Roysman and T. Orr-Weaver, personal communication), suggesting that the cooperation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto chromatin may be a generally conserved feature of DNA licensing in eukaryotes.

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Figure 1: Cdt1 enhances the ability of Cdc18 to induce continuing DNA synthesis.
Figure 2: Cdt1 can form a complex with Cdc18, and accumulates in the cell nucleus in G1.
Figure 3: Cdc21 associates with chromatin as cells prepare for S phase in a Cdc18-dependent manner.
Figure 4: Cdt1 depletion blocks the initiation of DNA replication by inhibiting the association of Cdc21 with chromatin.

References

  1. 1

    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).

  2. 2

    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).

  3. 3

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

  4. 4

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

  5. 5

    Rowles,A. et al. Interaction between the origin recognition complex and the replication licensing system in Xenopus. Cell 87, 287–296 (1996).

  6. 6

    Coleman,T. R., Carpenter,P. B. & Dunphy, W. G. The Xenopus Cdc6 protein is essential for the initiation of a single round of DNA replication in cell-free extracts. Cell 87, 53–63 (1996).

  7. 7

    Donovan,S., Harwood,J., Drury,L. S. & Diffley,J. F. Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc. Natl Acad. Sci. USA 94, 5611– 5616 (1997).

  8. 8

    Liang,C. & Stillman,B. Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants. Genes Dev. 11, 3375–3386 (1997).

  9. 9

    Aparicio,O. M., Weinstein,D. M. & Bell, S. P. Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell 91, 59–69 (1997).

  10. 10

    Tanaka,T., Knapp,D. & Nasmyth,K. Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs. Cell 90, 649–660 (1997).

  11. 11

    Kelly,T. J. et al. The fission yeast cdc18 gene product couples S-phase to start and mitosis. Cell 74, 371– 382 (1993).

  12. 12

    Nishitani,H. & Nurse,P. p65cdc18 plays a major role controlling the initiation of DNA replication in fission yeast. Cell 83, 397–405 (1995).

  13. 13

    Tada,S., Chong,J. P. J., Mahbubani, H. M. & Blow,J. J. The RLF-B component of the replication licensing system is distinct from Cdc6 and functions after Cdc6 binds to chromatin. Curr. Biol. 9, 211–214 (1999).

  14. 14

    Hofmann,J. F. X. & 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).

  15. 15

    Dutta,A. & Bell,S. P. Initiation of DNA replication in eukaryotic cells. Annu. Rev. Cell. Dev. Biol. 13, 293–332 (1997).

  16. 16

    Muzi-Falconi,M. & Kelly,T. J. Orp1, a member of the Cdc18/Cdc6 family of S-phase regulators, is homologous to a component of the origin recognition complex. Proc. Natl Acad. Sci. USA 92, 12475–12479 (1995).

  17. 17

    Grallert,B. & Nurse,P. The ORC1 homologue orp1 in fission yeast plays a key role in regulating onset of S phase. Genes Dev. 10, 2644–2654 ( 1996).

  18. 18

    Masai,H., Miyake,T. & Arai,K. hsk1+, a Schizosaccharomyces pombe gene related to Saccharomyces cerevisiae CDC7, is required for chromosomal replication. EMBO J. 14, 3094–3104 ( 1995).

  19. 19

    Fernandez-Sarabia,M., McInerny,C., Harris,P., Gordon,C. & Fantes,P. The cell cycle genes cdc22 and suc22 of the fission yeast Schizosaccharomyces pombe encode the large and small subunits of ribonucleotide reductase. Mol. Gen. Genet. 238, 241–251 (1993).

  20. 20

    Greenwood,E., Nishitani,H. & Nurse, P. Cdc18p can block mitosis by two independent mechanisms. J. Cell Sci. 111, 3101– 3108 (1998).

  21. 21

    Muzi-Falconi,M., Brown,G. W. & Kelly,T. J. cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe. Proc. Natl Acad. Sci. USA 93, 1566–1570 (1996).

  22. 22

    Maiorano,D., van Assendelft,G. B. & Kearsey, S. E. Fission yeast cdc21, a member of the MCM protein family, is required for onset of S phase and is located in the nucleus throughout the cell cycle. EMBO J. 15, 861– 872 (1996).

  23. 23

    Lygerou,Z. & Nurse,P. The fission yeast origin recognition complex is constitutively associated with chromatin and is differentially modified through the cell cycle. J. Cell Sci. 112, 3703–3712 (1999).

  24. 24

    Moreno,S., Klar,A. & Nurse,P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795– 823 (1991).

  25. 25

    Alfa,C., Fantes,P., Hyams,J., McLeod,M. & Warbrick, E. Experiments with Fission Yeast—A Laboratory Course Manual (CSHL Press, New York, 1993).

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Acknowledgements

We thank J. Bahler, J. Diffley, D. Griffiths, J. Hayles and H. Murakami for critical reading of the manuscript, T. Orr-Weaver for communicating results before publication, Y. Adachi, T. Carr, S. Kearsey, K. Nasmyth and T. Tanaka for clones and antibodies, and N. Peat for help with figure preparation. This work was supported by the ICRF, an EEC Marie Curie fellowship (Z.L.), and HFSPO and Uehara Foundation fellowships (H.N.).

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Correspondence to Zoi Lygerou.

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Nishitani, H., Lygerou, Z., Nishimoto, T. et al. The Cdt1 protein is required to license DNA for replication in fission yeast. Nature 404, 625–628 (2000) doi:10.1038/35007110

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