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A novel chk1-dependent G1/M checkpoint in fission yeast

Marianne Synnes1, Esben A Nilssen, Erik Boye

  • 1Department of Cell Biology, Institute for Cancer Research, Montebello, 0310 Oslo, Norway.

Journal of Cell Science
|August 21, 2002
PubMed

Insights

Fission yeast DNA replication is blocked by Orp1 protein deficiency. Checkpoint proteins, including Chk1, unexpectedly activate early to halt cell division, preventing DNA replication.

Area of Science:

  • Cell cycle regulation
  • DNA replication
  • Molecular biology

Background:

  • Orp1 is a crucial component of the origin recognition complex, essential for initiating DNA replication.
  • Temperature-sensitive orp1-4 mutants in fission yeast fail DNA replication at restrictive temperatures.
  • Cell cycle arrest in these mutants involves checkpoint Rad proteins and Chk1, with a portion undergoing aberrant mitosis ('cut').

Purpose of the Study:

  • To investigate the role of Chk1 in the G1/S DNA replication checkpoint.
  • To elucidate the molecular mechanisms underlying cell cycle arrest and 'cut' phenotypes in orp1-4 mutants.
  • To determine the timing of Chk1 activation and Cdc2 inactivation during checkpoint activation.

Main Methods:

  • Fission yeast genetics and cell cycle analysis.
  • Flow cytometry for DNA content analysis.
  • 2D gel electrophoresis to assess DNA replication origin initiation.
  • Western blotting or kinase assays to monitor protein phosphorylation and activity.

Main Results:

  • orp1-4 cells arrest with 1C DNA content, indicating failed DNA replication.
  • Chk1 activation precedes Cdc2 phosphorylation and inactivation by Wee1 in G1-arrested cells.
  • Early origin firing (ars3001) is inhibited in arrested cells.
  • Commitment to the 'cut' phenotype occurs as early as the G1 phase.

Conclusions:

  • Chk1 plays a novel role in inducing and maintaining Cdc2 phosphorylation during G1 checkpoint activation.
  • The DNA replication checkpoint is active earlier than previously thought, involving Chk1 in G1.
  • Aberrant cell division ('cut') can be initiated in early G1 phase in response to replication stress.

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