Separate SCF(CDC4) recognition elements target Cdc6 for proteolysis in S phase and mitosis.

Area of Science:

• Molecular Biology
• Cell Cycle Regulation
• Yeast Genetics

Background:

• Cdc6 is a key DNA replication initiation factor in Saccharomyces cerevisiae.
• Cdc6 is regulated by ubiquitin-mediated proteolysis via the SCF(CDC4) E3 ubiquitin ligase.
• SCF(CDC4)-mediated proteolysis targets Cdc6 from late G1 phase through mitosis.

Purpose of the Study:

• To characterize a dominant-negative CDC6 mutant.
• To investigate the mechanism by which this mutant inhibits cell cycle progression.
• To identify novel regulatory sites on Cdc6 involved in proteolysis and cell cycle control.

Main Methods:

• Overexpression of a dominant-negative CDC6 mutant.
• Cell cycle arrest analysis.
• Analysis of cyclin-dependent kinase (CDK) inhibition.
• Investigation of SCF(CDC4)-mediated proteolysis.
• Site-directed mutagenesis to identify phosphorylation sites and interaction domains.

Main Results:

• The dominant-negative CDC6 mutant arrests the cell cycle by inhibiting CDKs.
• The mutant protein is refractory to SCF(CDC4)-mediated proteolysis, leading to its accumulation.
• A mutation destroying a putative CDK phosphorylation site confers the dominant-negative phenotype.
• This mutation affects a novel Cdc4-interacting domain, distinct from a previously identified N-terminal site.
• Both identified sites regulate Cdc6 proteolysis, but the novel site is crucial during mitosis.

Conclusions:

• A novel dominant-negative CDC6 mutant inhibits cell cycle progression by interfering with CDK activity.
• The resistance of this mutant to proteolysis is key to its accumulation and cell cycle arrest.
• A newly identified Cdc4-interacting domain and CDK phosphorylation site are critical for Cdc6 regulation, particularly during mitosis.