To ensure proper timing of the G1–S transition in the cell cycle, the cyclin E–Cdk2 complex, which is responsible for the initiation of DNA replication, is restrained by the p21Cip1/p27Kip1/p57Kip2 family of CDK (cyclin-dependent kinase) inhibitors in humans and by the related p27Xic1 protein in Xenopus. Activation of cyclin E–Cdk2 is linked to the ubiquitination of human p27Kip1 or Xenopus p27Xic1 by SCF (for Skp1–Cullin–F-box protein) ubiquitin ligases. For human p27Kip1, ubiquitination requires direct phosphorylation by cyclin E–Cdk2. We show here that Xic1 ubiquitination does not require phosphorylation by cyclin E–Cdk2, but it does require nuclear accumulation of the Xic1–cyclin E–Cdk2 complex and recruitment of this complex to chromatin by the origin-recognition complex together with Cdc6 replication preinitiation factors; it also requires an activation step necessitating cyclin E–Cdk2-kinase and SCF ubiquitin-ligase activity, and additional factors associated with mini-chromosome maintenance proteins, including the inactivation of geminin. Components of the SCF ubiquitin-ligase complex, including Skp1 and Cul1, are also recruited to chromatin through cyclin E–Cdk2 and the preinitiation complex. Thus, activation of the cyclin E–Cdk2 kinase and ubiquitin-dependent destruction of its inhibitor are spatially constrained to the site of a properly assembled preinitiation complex.
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We would like to thank A. Dutta, P. R. Yew, J. Maller, M. Rexach and R. Laskey for their generous gifts of reagents, and T. Coleman, P. Carpenter and D. Wolf for critical reading of the manuscript. This work was supported by grants from the National Institutes of Health (to P.K.J.), the National Cancer Institute (to L.F. and B.K.K.), Howard Hughes Medical Institute (to A.G.E.) and a Lieberman Fellowship (to B.K.K.).
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