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Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe
13:22

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

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Transactivation of Schizosaccharomyces pombe cdt2+ stimulates a Pcu4-Ddb1-CSN ubiquitin ligase

Cong Liu1, Marius Poitelea, Adam Watson

  • 1Genome Damage and Stability Centre, University of Sussex, Brighton, UK.

The EMBO Journal
|October 28, 2005
PubMed

Insights

Cdt2 protein targets Spd1 for degradation, regulating ribonucleotide reductase (RNR) activity in fission yeast. This discovery clarifies the mechanism of RNR regulation and DNA damage response.

Area of Science:

  • Cellular Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Cullin-4 (Pcu4) forms ubiquitin ligase complexes with Ddb1 and the COP9 signalosome (CSN) in Schizosaccharomyces pombe.
  • Spd1, a target of this complex, regulates ribonucleotide reductase (RNR) activity.
  • Spd1 degradation is crucial for RNR subunit nuclear export during S phase and DNA damage response.

Purpose of the Study:

  • To identify the regulatory subunit of the Pcu4-Ddb1-CSN ubiquitin ligase complex.
  • To elucidate the role of Cdt2 in Spd1 regulation and RNR activity.
  • To understand the mechanism of Spd1 degradation and its impact on cellular processes.

Main Methods:

  • Co-immunoprecipitation assays to study protein interactions.
  • Gene deletion and overexpression studies to assess protein function.
  • Analysis of protein stability and localization.
  • Transcriptional analysis of cdt2+.

Main Results:

  • Cdt2, an unstable WD40 protein, was identified as a regulatory subunit of the Pcu4-Ddb1-CSN ubiquitin ligase.
  • cdt2 deletion stabilized Spd1 and prevented the nuclear export of the small RNR subunit.
  • Cdt2 transcription is regulated by Cdc10/DSC1 during S phase and DNA damage, mirroring Spd1 degradation patterns.
  • Cdt2 overexpression led to constitutive Spd1 degradation.

Conclusions:

  • Cdt2 acts as a WD40 repeat adaptor protein, mediating Spd1 targeting and degradation by the Cullin-4-based ubiquitin ligase.
  • Cdt2 incorporation into the Pcu4-Ddb1-CSN complex is a key step in Spd1 regulation.
  • This mechanism is essential for controlling RNR activity and responding to DNA damage in fission yeast.

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