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Triggering ubiquitination of a CDK inhibitor at origins of DNA replication

L Furstenthal1, C Swanson, B K Kaiser

  • 1Departments of Pathology and Microbiology & Immunology, Stanford University School of Medicine, 300 Pasteur Drive, MC 5324, Palo Alto, California 94305-5324, USA.

Nature Cell Biology
|August 3, 2001
PubMed

Insights

The cell cycle

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The G1-S transition is tightly regulated to ensure proper cell cycle progression.
  • Cyclin E-Cdk2 complex initiates DNA replication but is inhibited by CDK inhibitors like p27(Kip1) and p27(Xic1).
  • Ubiquitination by SCF ubiquitin ligases targets these inhibitors for degradation, activating Cyclin E-Cdk2.

Purpose of the Study:

  • To elucidate the mechanism of Xenopus p27(Xic1) ubiquitination and degradation.
  • To compare the regulation of p27(Xic1) with human p27(Kip1) in cell cycle control.
  • To understand the spatial and temporal control of Cyclin E-Cdk2 activation at replication origins.

Main Methods:

  • Biochemical assays to study protein-protein interactions and enzymatic activities.
  • In vitro ubiquitination assays using purified proteins and cell extracts.
  • Analysis of protein localization and recruitment to chromatin using Xenopus egg extracts and replication systems.

Main Results:

  • Xenopus p27(Xic1) ubiquitination by SCF ligases does not require direct phosphorylation by Cyclin E-Cdk2, unlike human p27(Kip1).
  • Xic1 ubiquitination requires the assembly of a preinitiation complex at chromatin, involving ORC and Cdc6.
  • SCF components (Skp1, Cul1) are recruited to chromatin via Cyclin E-Cdk2 and the preinitiation complex, and geminin inactivation is necessary.

Conclusions:

  • The activation of Cyclin E-Cdk2 and degradation of its inhibitor p27(Xic1) are spatially restricted to replication origins.
  • This localization ensures that DNA replication initiation is precisely coordinated with preinitiation complex assembly.
  • The findings reveal a novel mechanism for cell cycle control, linking replication initiation to inhibitor degradation.

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