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Control of the Cdc2/cyclin B complex in Xenopus egg extracts arrested at a G2/M checkpoint with DNA synthesis
1Division of Biology, Howard Hughes Medical Institute, California Institute of Technology, Pasadena 91125, USA.
Abstract:
Proliferating eukaryotic cells possess checkpoint mechanisms that block cell division in the presence of unreplicated or damaged DNA. Using cell-free extracts from Xenopus eggs, we have investigated the mechanisms underlying the inability of a recombinant Cdc2/cyclin B complex to induce mitosis in the presence of incompletely replicated DNA. We found that the activities of the kinases and phosphatases that regulate the major phosphorylation sites on Cdc2 (e.g., tyrosine 15, threonine 14, and threonine 161) are not altered significantly under conditions where Xenopus extracts remain stably arrested in interphase due to the presence of the replication inhibitor aphidicolin. However, at threshold concentrations, a Cdc2/cyclin B complex containing a mutant Cdc2 subunit that cannot be phosphorylated on either tyrosine 15 or threonine 14 displays a markedly reduced capacity to induce mitosis in the presence of aphidicolin. This observation indicates that the replication checkpoint in Xenopus egg extracts functions without the inhibitory tyrosine and threonine phosphorylation of Cdc2. We provide evidence that the checkpoint-dependent suppression of the Cdc2/cyclin B complex involves a titratable inhibitor that is regulated by the presence of unreplicated DNA.
Insights
The cell cycle checkpoint prevents cell division with unreplicated DNA. In Xenopus egg extracts, this checkpoint suppresses Cdc2/cyclin B activity via a titratable inhibitor, not by altering Cdc2 phosphorylation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Eukaryotic cells have checkpoints to halt division when DNA is damaged or unreplicated.
- The Cdc2/cyclin B complex is a key regulator of the cell cycle.
- Understanding checkpoint mechanisms is crucial for cell cycle regulation research.
Purpose of the Study:
- To investigate how the replication checkpoint prevents mitosis induction by Cdc2/cyclin B in Xenopus egg extracts.
- To determine the role of Cdc2 phosphorylation in replication checkpoint function.
Main Methods:
- Utilized cell-free extracts from Xenopus eggs.
- Employed the replication inhibitor aphidicolin to arrest cells in interphase.
- Assessed the activity of Cdc2/cyclin B complexes, including a mutant form of Cdc2, in the presence of aphidicolin.
Main Results:
- Kinase and phosphatase activities regulating Cdc2 phosphorylation (Tyr15, Thr14, Thr161) remained unchanged in aphidicolin-treated extracts.
- A mutant Cdc2/cyclin B complex, unable to be phosphorylated at Tyr15 or Thr14, showed reduced ability to induce mitosis.
- This suggests the replication checkpoint operates independently of inhibitory Cdc2 phosphorylation.
Conclusions:
- The replication checkpoint in Xenopus egg extracts functions without altering the inhibitory phosphorylation of Cdc2.
- Checkpoint-dependent suppression of Cdc2/cyclin B involves a titratable inhibitor regulated by unreplicated DNA.