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DNA damage and replication checkpoints in fission yeast require nuclear exclusion of the Cdc25 phosphatase via 14-3-3

Y Zeng1, H Piwnica-Worms

  • 1Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.

Molecular and Cellular Biology
|October 19, 1999
PubMed

Insights

14-3-3 proteins binding to Cdc25 is crucial for cell cycle arrest in fission yeast when DNA is damaged or unreplicated. This binding also helps exclude Cdc25 from the nucleus, ensuring proper DNA damage and replication checkpoint responses.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • Cell cycle progression is regulated by checkpoints that respond to DNA damage or replication stress.
  • Cdc25 phosphatase activity is critical for mitotic entry and is regulated by phosphorylation and protein-protein interactions.
  • 14-3-3 proteins are known to bind phosphorylated Cdc25, influencing its localization and function.

Purpose of the Study:

  • To investigate the role of 14-3-3 protein binding in the function of fission yeast Cdc25.
  • To determine how 14-3-3 binding affects Cdc25 localization and its involvement in DNA damage and replication checkpoints.

Main Methods:

  • Generation of a fission yeast Cdc25 mutant impaired in 14-3-3 binding.
  • Analysis of DNA damage and replication checkpoint responses in cells expressing the mutant.
  • Microscopy to observe the subcellular localization of wild-type and mutant Cdc25.
  • Investigating the effect of Rad 24 (a 14-3-3 protein) overproduction on Cdc25 localization.

Main Results:

  • The 14-3-3 binding mutant of Cdc25 was unable to arrest the cell cycle in response to DNA damage or replication blocks.
  • Mutant Cdc25 predominantly localized to the nucleus, unlike wild-type Cdc25 which was found in both cytoplasm and nucleus.
  • Overproduction of Rad 24 led to nuclear exclusion of wild-type Cdc25, but not the 14-3-3 binding mutant.
  • Leptomycin B treatment caused nuclear accumulation of wild-type Cdc25, indicating active nuclear export.

Conclusions:

  • 14-3-3 binding to Cdc25 is essential for activating DNA damage and replication checkpoints in fission yeast.
  • 14-3-3 binding contributes to the nuclear exclusion of Cdc25, which is a critical step for checkpoint signaling.
  • Proper subcellular localization of Cdc25, regulated by 14-3-3 proteins, is vital for maintaining genomic integrity.

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