Abstract
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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Acknowledgements
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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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). https://doi.org/10.1038/nature02813
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DOI: https://doi.org/10.1038/nature02813
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