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The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast
L S Drury1, G Perkins, J F Diffley
1Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms EN6 3LD, UK.
The EMBO Journal
|October 6, 1997
Summary
Budding yeast Cdc6 protein (Cdc6p) levels are regulated by cell cycle-dependent degradation. This degradation, mediated by the Cdc4/34/53 pathway, ensures DNA replication occurs only once per cell cycle.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The budding yeast Cdc6 protein (Cdc6p) is crucial for initiating DNA replication by forming pre-replicative complexes (pre-RCs).
- Cell cycle regulation of pre-RC assembly ensures DNA replication occurs only once per cell cycle.
- Cdc6p levels naturally peak during the G1 phase due to gene transcription patterns.
Purpose of the Study:
- To investigate the role of Cdc6p degradation in regulating its cell cycle periodicity.
- To identify the mechanisms and regulatory pathways involved in Cdc6p degradation.
- To determine the functional consequences of preventing Cdc6p degradation.
Main Methods:
- Analysis of Cdc6p degradation rates throughout the cell cycle.
- Site-directed mutagenesis to remove a specific N-terminal domain of Cdc6p.
- Introduction of mutations in the Cdc4/34/53 ubiquitin-mediated degradation pathway.
- Genetic analysis to probe interactions between Cdc6p and the degradation machinery.
- Functional assays of wild-type and stable Cdc6p mutants, including overexpression studies.
Main Results:
- Cdc6p degradation rates are cell cycle-regulated, peaking in late G1/early S phase.
- A specific N-terminal domain (47 amino acids) is essential for Cdc6p degradation.
- The Cdc4/34/53 pathway mediates Cdc6p ubiquitination and degradation, with evidence of N-terminal interaction.
- A stable Cdc6p mutant, resistant to degradation, remains fully functional.
- Overexpression of either wild-type or stable Cdc6p does not cause re-replication or ectopic pre-RC assembly.
Conclusions:
- Rapid degradation of Cdc6p is a key mechanism contributing to its cell cycle periodicity, complementing transcriptional control.
- The N-terminus of Cdc6p is a critical regulatory domain for its degradation via the Cdc4/34/53 pathway.
- Functional Cdc6p can be stabilized without compromising its essential role in DNA replication initiation, and without inducing aberrant replication events.