Abstract
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 p21
Cip1
/p27
Kip1
/p57
Kip2
family of CDK (cyclin-dependent kinase) inhibitors in humans and by the related p27
Xic1
protein in
Xenopus
. Activation of cyclin E–Cdk2 is linked to the ubiquitination of human p27
Kip1
or
Xenopus
p27
Xic1
by SCF (for Skp1–Cullin–F-box protein) ubiquitin ligases. For human p27
Kip1
, 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.