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Regulation of mitotic inhibitor Mik1 helps to enforce the DNA damage checkpoint
B A Baber-Furnari1, N Rhind, M N Boddy
1Departments of Molecular Biology and Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Abstract:
The protein kinase Chk1 enforces the DNA damage checkpoint. This checkpoint delays mitosis until damaged DNA is repaired. Chk1 regulates the activity and localization of Cdc25, the tyrosine phosphatase that activates the cdk Cdc2. Here we report that Mik1, a tyrosine kinase that inhibits Cdc2, is positively regulated by the DNA damage checkpoint. Mik1 is required for checkpoint response in strains that lack Cdc25. Long-term DNA damage checkpoint arrest fails in Deltamik1 cells. DNA damage increases Mik1 abundance in a Chk1-dependent manner. Ubiquitinated Mik1 accumulates in a proteasome mutant, which indicates that Mik1 normally has a short half-life. Thus, the DNA damage checkpoint might regulate Mik1 degradation. Mik1 protein and mRNA oscillate during the unperturbed cell cycle, with peak amounts detected around S phase. These data indicate that regulation of Mik1 abundance helps to couple mitotic onset to the completion of DNA replication and repair. Coordinated negative regulation of Cdc25 and positive regulation of Mik1 ensure the effective operation of the DNA damage checkpoint.
Insights
The DNA damage checkpoint uses protein kinase Chk1 to regulate Mik1, a tyrosine kinase that inhibits Cdc2. Mik1 abundance increases with DNA damage, ensuring cell cycle arrest until DNA repair is complete.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The DNA damage checkpoint is crucial for genomic stability, preventing mitosis until DNA repair.
- Protein kinase Chk1 is a key regulator of this checkpoint, controlling Cdc25 phosphatase activity.
- Cdc25 activates cyclin-dependent kinase 2 (cdkCdc2), driving cell cycle progression.
Purpose of the Study:
- To investigate the role of Mik1, a tyrosine kinase inhibiting cdkCdc2, in the DNA damage checkpoint.
- To elucidate how Mik1 is regulated by the DNA damage checkpoint and its contribution to cell cycle arrest.
Main Methods:
- Analysis of Mik1 abundance and localization in response to DNA damage.
- Investigation of Mik1 regulation in cells lacking Cdc25 or with proteasome mutations.
- Examination of Mik1 protein and mRNA oscillations during the cell cycle.
Main Results:
- Mik1 is positively regulated by the DNA damage checkpoint in a Chk1-dependent manner.
- Mik1 is essential for checkpoint arrest in cells lacking Cdc25 and for long-term arrest.
- DNA damage increases Mik1 abundance, and Mik1 has a short half-life, suggesting regulation of its degradation.
- Mik1 levels oscillate during the cell cycle, peaking around S phase.
Conclusions:
- The DNA damage checkpoint regulates Mik1 abundance, potentially through degradation, to ensure proper cell cycle arrest.
- Mik1's regulation couples mitotic entry to DNA replication and repair completion.
- Coordinated regulation of Cdc25 and Mik1 by the DNA damage checkpoint is vital for effective DNA repair and genomic integrity.