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CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing


To maintain genome stability, DNA replication is strictly regulated to occur only once per cell cycle. In eukaryotes, the presence of ‘licensing proteins’ at replication origins during the G1 cell-cycle phase allows the formation of the pre-replicative complex1. The removal of licensing proteins from chromatin during the S phase ensures that origins fire only once per cell cycle1. Here we show that the CUL-4 ubiquitin ligase temporally restricts DNA-replication licensing in Caenorhabditis elegans. Inactivation of CUL-4 causes massive DNA re-replication, producing cells with up to 100C DNA content. The C. elegans orthologue of the replication-licensing factor Cdt1 (refs 2, 3) is required for DNA replication. C. elegans CDT-1 is present in G1-phase nuclei but disappears as cells enter S phase. In cells lacking CUL-4, CDT-1 levels fail to decrease during S phase and instead remain constant in the re-replicating cells. Removal of one genomic copy of cdt-1 suppresses the cul-4 re-replication phenotype. We propose that CUL-4 prevents aberrant re-initiation of DNA replication, at least in part, by facilitating the degradation of CDT-1.

Figure 1: cul-4 mRNA levels.
Figure 2: cul-4 RNAi phenotype.
Figure 3: DNA re-replication in cul-4 RNAi seam cells.
Figure 4: CDT-1 expression in cul-4 RNAi animals.


  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

    Nishitani, H., Lygerou, Z., Nishimoto, T. & Nurse, P. The Cdt1 protein is required to license DNA for replication in fission yeast. Nature 404, 625–628 (2000)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Maiorano, D., Moreau, J. & Mechali, M. XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis. Nature 404, 622–625 (2000)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Nayak, S. et al. The Caenorhabditis elegans Skp1-related gene family: diverse functions in cell proliferation, morphogenesis, and meiosis. Curr. Biol. 12, 277–287 (2002)

    CAS  Article  Google Scholar 

  5. 5

    Tyers, M. & Jorgensen, P. Proteolysis and the cell cycle: with this RING I do thee destroy. Curr. Opin. Genet. Dev. 10, 54–64 (2000)

    CAS  Article  Google Scholar 

  6. 6

    Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Hedgecock, E. M., Culotti, J. G. & Hall, D. H. The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans. Neuron 4, 61–85 (1990)

    CAS  Article  Google Scholar 

  8. 8

    Sulston, J. E. & Horvitz, H. R. Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev. Biol. 56, 110–156 (1977)

    CAS  Article  Google Scholar 

  9. 9

    Seydoux, G. & Schedl, T. The germline in C. elegans: origins, proliferation, and silencing. Int. Rev. Cytol. 203, 139–185 (2001)

    CAS  Article  Google Scholar 

  10. 10

    Lorson, M. A., Horvitz, H. R. & van den Heuvel, S. LIN-5 is a novel component of the spindle apparatus required for chromosome segregation and cleavage plane specification in Caenorhabditis elegans. J. Cell Biol. 148, 73–86 (2000)

    CAS  Article  Google Scholar 

  11. 11

    Edgar, B. A. & Orr-Weaver, T. L. Endoreplication cell cycles: more for less. Cell 105, 297–306 (2001)

    CAS  Article  Google Scholar 

  12. 12

    Hendzel, M. J. et al. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348–360 (1997)

    CAS  Article  Google Scholar 

  13. 13

    Hong, Y., Roy, R. & Ambros, V. Developmental regulation of a cyclin-dependent kinase inhibitor controls postembryonic cell cycle progression in Caenorhabditis elegans. Development 125, 3585–3597 (1998)

    CAS  PubMed  Google Scholar 

  14. 14

    Hedgecock, E. M. & White, J. G. Polyploid tissues in the nematode Caenorhabditis elegans. Dev. Biol. 107, 128–133 (1985)

    CAS  Article  Google Scholar 

  15. 15

    Whittaker, A. J., Royzman, I. & Orr-Weaver, T. L. Drosophila double parked: a conserved, essential replication protein that colocalizes with the origin recognition complex and links DNA replication with mitosis and the down-regulation of S phase transcripts. Genes Dev. 14, 1765–1776 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Cimbora, D. M. & Groudine, M. The control of mammalian DNA replication: a brief history of space and timing. Cell 104, 643–646 (2001)

    CAS  PubMed  Google Scholar 

  17. 17

    Tanaka, S. & Diffley, J. F. Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2-7 during G1 phase. Nature Cell Biol. 4, 198–207 (2002)

    CAS  Article  Google Scholar 

  18. 18

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

    ADS  CAS  Article  Google Scholar 

  19. 19

    Edgar, L. G. & McGhee, J. D. DNA synthesis and the control of embryonic gene expression in C. elegans. Cell 53, 589–599 (1988)

    CAS  Article  Google Scholar 

  20. 20

    Nishitani, H., Taraviras, S., Lygerou, Z. & Nishimoto, T. The human licensing factor for DNA replication Cdt1 accumulates in G1 and is destabilized after initiation of S-phase. J. Biol. Chem. 276, 44905–44911 (2001)

    CAS  Article  Google Scholar 

  21. 21

    Praitis, V., Casey, E., Collar, D. & Austin, J. Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157, 1217–1226 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Todorov, I. T., Attaran, A. & Kearsey, S. E. BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication. J. Cell Biol. 129, 1433–1445 (1995)

    CAS  Article  Google Scholar 

  23. 23

    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)

    CAS  Article  Google Scholar 

  24. 24

    Nguyen, V. Q., Co, C. & Li, J. J. Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms. Nature 411, 1068–1073 (2001)

    ADS  CAS  Article  Google Scholar 

  25. 25

    Yanow, S. K., Lygerou, Z. & Nurse, P. Expression of Cdc18/Cdc6 and Cdt1 during G2 phase induces initiation of DNA replication. EMBO J. 20, 4648–4656 (2001)

    CAS  Article  Google Scholar 

  26. 26

    Timmons, L. & Fire, A. Specific interference by ingested dsRNA. Nature 395, 854 (1998)

    ADS  CAS  Article  Google Scholar 

  27. 27

    Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997)

    CAS  Article  Google Scholar 

  28. 28

    Koppen, M. et al. Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia. Nature Cell Biol. 3, 983–991 (2001)

    CAS  Article  Google Scholar 

  29. 29

    Miller, D. M. & Shakes, D. C. in Caenorhabditis elegans: Modern Biological Analysis of an Organism (eds Epstein, H. F. & Shakes, D. C.) 365–394 (Academic Press, San Diego, 1995)

    Google Scholar 

  30. 30

    Feng, H. et al. CUL-2 is required for the G1-to-S phase transition and mitotic chromosome condensation in Caenorhabditis elegans. Nature Cell Biol. 1, 486–492 (1999)

    CAS  Article  Google Scholar 

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We thank Y. Kohara, A. Fire, G. Seydoux, R. Roy and V. Ambros for reagents; the Caenorhabditis Genetics Center for nematode strains; R. Santurri for technical help; and H. Cai and P. Shen for critical reading of the manuscript. This work was supported by a grant from the National Institutes of Health to E.T.K.

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Correspondence to Edward T. Kipreos.

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Zhong, W., Feng, H., Santiago, F. et al. CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing. Nature 423, 885–889 (2003).

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