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Structural basis for inhibition of the replication licensing factor Cdt1 by geminin


To maintain chromosome stability in eukaryotic cells, replication origins must be licensed by loading mini-chromosome maintenance (MCM2–7) complexes once and only once per cell cycle1,2,3,4,5,6,7,8,9. This licensing control is achieved through the activities of geminin10,11,12 and cyclin-dependent kinases9,13,14. Geminin binds tightly to Cdt1, an essential component of the replication licensing system6,15,16,17,18, and prevents the inappropriate reinitiation of replication on an already fired origin. The inhibitory effect of geminin is thought to prevent the interaction between Cdt1 and the MCM helicase19,20. Here we describe the crystal structure of the mouse geminin–Cdt1 complex using tGeminin (residues 79–157, truncated geminin) and tCdt1 (residues 172–368, truncated Cdt1). The amino-terminal region of a coiled-coil dimer of tGeminin interacts with both N-terminal and carboxy-terminal parts of tCdt1. The primary interface relies on the steric complementarity between the tGeminin dimer and the hydrophobic face of the two short N-terminal helices of tCdt1 and, in particular, Pro 181, Ala 182, Tyr 183, Phe 186 and Leu 189. The crystal structure, in conjunction with our biochemical data, indicates that the N-terminal region of tGeminin might be required to anchor tCdt1, and the C-terminal region of tGeminin prevents access of the MCM complex to tCdt1 through steric hindrance.

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Figure 1: Structure of the tGeminin–tCdt1 complex.
Figure 2: Bipartite tGeminin–tCdt1 interface.
Figure 3: The C-terminal part of tGeminin is important for inhibition of replication and MCM-binding.


  1. Bell, S. P. & Dutta, A. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 (2002)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Diffley, J. F. DNA replication: building the perfect switch. Curr. Biol. 11, R367–R370 (2001)

    Article  CAS  Google Scholar 

  4. Bell, S. P. & Stillman, B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature 357, 128–134 (1992)

    Article  ADS  CAS  Google Scholar 

  5. 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)

    Article  CAS  Google Scholar 

  6. 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)

    Article  ADS  CAS  Google Scholar 

  7. Shreeram, S. & Blow, J. J. The role of the replication licensing system in cell proliferation and cancer. Prog. Cell Cycle Res. 5, 287–293 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Vaziri, C. et al. A p53-dependent checkpoint pathway prevents rereplication. Mol. Cell 11, 997–1008 (2003)

    Article  CAS  Google Scholar 

  9. Kearsey, S. E. & Cotterill, S. Enigmatic variations: divergent modes of regulating eukaryotic DNA replication. Mol. Cell 12, 1067–1075 (2003)

    Article  CAS  Google Scholar 

  10. McGarry, T. J. & Kirschner, M. W. Geminin, an inhibitor of DNA replication, is degraded during mitosis. Cell 93, 1043–1053 (1998)

    Article  CAS  Google Scholar 

  11. 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)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  13. Yamaguchi, R. & Newport, J. A role for Ran-GTP and Crm1 in blocking re-replication. Cell 113, 115–125 (2003)

    Article  CAS  Google Scholar 

  14. Li, A. & Blow, J. J. Non-proteolytic inactivation of geminin requires CDK-dependent ubiquitination. Nature Cell Biol. 6, 260–267 (2004)

    Article  CAS  Google Scholar 

  15. Hofmann, J. F. & 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)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  17. 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 

  18. Arentson, E. et al. Oncogenic potential of the DNA replication licensing protein CDT1. Oncogene 21, 1150–1158 (2002)

    Article  CAS  Google Scholar 

  19. Yanagi, K., Mizuno, T., You, Z. & Hanaoka, F. Mouse geminin inhibits not only Cdt1–MCM6 interactions but also a novel intrinsic Cdt1 DNA binding activity. J. Biol. Chem. 277, 40871–40880 (2002)

    Article  CAS  Google Scholar 

  20. Cook, J. G., Chasse, D. A. & Nevins, J. R. The regulated association of Cdt1 with minichromosome maintenance proteins and Cdc6 in mammalian cells. J. Biol. Chem. 279, 9625–9633 (2004)

    Article  CAS  Google Scholar 

  21. Bussiere, D. E., Bastia, D. & White, S. W. Crystal structure of the replication terminator protein from B. subtilis at 2.6 Å. Cell 80, 651–660 (1995)

    Article  CAS  Google Scholar 

  22. Gautam, A., Mulugu, S., Alexander, K. & Bastia, D. A single domain of the replication termination protein of Bacillus subtilis is involved in arresting both DnaB helicase and RNA polymerase. J. Biol. Chem. 276, 23471–23479 (2001)

    Article  CAS  Google Scholar 

  23. 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)

    Article  CAS  Google Scholar 

  24. O'Shea, E. K., Klemm, J. D., Kim, P. S. & Alber, T. X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science 254, 539–544 (1991)

    Article  ADS  CAS  Google Scholar 

  25. Shreeram, S., Sparks, A., Lane, D. P. & Blow, J. J. Cell type-specific responses of human cells to inhibition of replication licensing. Oncogene 21, 6624–6632 (2002)

    Article  CAS  Google Scholar 

  26. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  27. Brünger, A. T. et al. Crystallography and NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

  28. Kleywegt, G. J. & Jones, T. A. Efficient rebuilding of protein structures. Acta Crystallogr. D 50, 829–832 (1996)

    Article  Google Scholar 

  29. You, Z., Komamura, Y. & Ishimi, Y. Biochemical analysis of the intrinsic Mcm4–Mcm6–Mcm7 DNA helicase activity. Mol. Cell. Biol. 12, 8003–8015 (1999)

    Article  Google Scholar 

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We thank S. Son and A. Jeon for help in initial protein purification, Y. Kong for MEF cells, J. Lee for anti-MCM6 antibody, and P. A. Karplus, S. H. Kim, Y. Kong and J. Bradbury for critical readings of the manuscript. This work was supported by the funds from the National Creative Research Initiatives (Ministry of Science and Technology).

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Correspondence to Yunje Cho.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure 1

Heptad repeat and intermolecular interactions within tGeminin dimer. (PDF 564 kb)

Supplementary Figure 2

Structural alignment and conservation in geminin and Cdt1. (PDF 57 kb)

Supplementary Figure 3

Interaction between tCdt1 and naked DNA. (PDF 444 kb)

Supplementary Figure 4

Structure and stability of various tGeminin and tCdt1 mutant proteins. (PDF 93 kb)

Supplementary Methods and Figure Legends

Structure and stability of various tGeminin and tCdt1 mutant proteins. (DOC 50 kb)

Supplementary Table 1

Statistics from crystallographic analysis. (DOC 48 kb)

Supplementary Table 2

Dissociation constants (nM) of Cdt1 to geminin. (DOC 34 kb)

Supplementary Table 3

Dissociation constants (nM) of Cdt1 to geminin. (DOC 35 kb)

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Lee, C., Hong, B., Choi, J. et al. Structural basis for inhibition of the replication licensing factor Cdt1 by geminin. Nature 430, 913–917 (2004).

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