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Cdc2 tyrosine phosphorylation is required for the DNA damage checkpoint in fission yeast
N Rhind1, B Furnari, P Russell
1Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Abstract:
A common cellular response to DNA damage is cell cycle arrest. This checkpoint control has been the subject of intensive genetic investigation, but the biochemical mechanism that prevents mitosis following DNA damage is unknown. In Schizosaccharomyces pombe, as well as vertebrates, the timing of mitosis under normal circumstances is determined by the balance of kinases and phosphatases that regulate inhibitory phosphorylation of Cdc2. In S. pombe, the phosphorylation occurs on tyrosine-15. This method of mitotic control is also used in S. pombe to couple mitosis with completion of DNA replication, but the role of Cdc2 tyrosine phosphorylation in the Chk1 kinase-mediated DNA damage checkpoint has remained uncertain. We show that, in contrast to recent speculation, the G2 DNA damage checkpoint arrest in S. pombe depends on the inhibitory tyrosine phosphorylation of Cdc2 carried out by the Wee1 and Mik1 kinases. Furthermore, the rate of Cdc2 tyrosine dephosphorylation is reduced by irradiation. This result implicates regulation of Cdc2 tyrosine dephosphorylation, mainly carried out by the Cdc25 tyrosine phosphatase, as an important part of the mechanism by which the DNA damage checkpoint induces Cdc2 inhibition and G2 arrest.
Insights
DNA damage triggers cell cycle arrest via Cdc2 tyrosine phosphorylation. This study reveals that Wee1 and Mik1 kinases maintain this phosphorylation, while Cdc25 phosphatase activity is reduced, ensuring G2 arrest.
Area of Science:
- Cellular Biology
- Molecular Biology
- Genetics
Background:
- Cell cycle arrest is a common response to DNA damage.
- The precise biochemical mechanisms underlying DNA damage-induced mitotic inhibition remain unclear.
- Cdc2 phosphorylation, particularly at tyrosine-15, regulates mitosis timing in Schizosaccharomyces pombe.
Purpose of the Study:
- To elucidate the role of Cdc2 tyrosine phosphorylation in the Chk1 kinase-mediated DNA damage checkpoint.
- To investigate the biochemical mechanisms preventing mitosis following DNA damage in S. pombe.
Main Methods:
- Genetic investigation of checkpoint control.
- Biochemical analysis of kinase and phosphatase activity.
- Study of Cdc2 phosphorylation and dephosphorylation dynamics.
Main Results:
- G2 DNA damage checkpoint arrest in S. pombe relies on inhibitory tyrosine phosphorylation of Cdc2 by Wee1 and Mik1 kinases.
- Irradiation reduces the rate of Cdc2 tyrosine dephosphorylation.
- Cdc25 tyrosine phosphatase activity is implicated in maintaining Cdc2 inhibition.
Conclusions:
- The DNA damage checkpoint utilizes Wee1 and Mik1 kinases to maintain inhibitory Cdc2 phosphorylation.
- Regulation of Cdc25 phosphatase activity is crucial for DNA damage-induced G2 arrest.
- These findings clarify the biochemical basis of DNA damage checkpoint control.