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Active cyclin B-cdc2 kinase does not inhibit DNA replication and cannot drive prematurely fertilized sea urchin eggs
A M Genevière-Garrigues1, A Barakat, M Dorée
1Laboratoire Arago, Banyuls-sur Mer, France.
Abstract:
Feedback mechanisms preventing M phase occurrence before S phase completion are assumed to depend on inhibition of cyclin B-cdc2 kinase activation by unreplicated DNA. In sea urchin, fertilization stimulates protein synthesis and releases eggs from G1 arrest. We found that in the one-cell sea urchin embryo cyclin B-cdc2 kinase undergoes partial activation before S phase, reaching in S phase a level that is sufficient for G2-M phase transition. S phase entry is not inhibited by this level of cyclin B-dependent kinase activity. Inhibition of DNA replication by aphidicolin suppresses nuclear envelope breakdown, yet it does not prevent the microtubule array from being converted from its interphasic to its mitotic state. Moreover, mitotic cytoplasmic events occur at the same time in control and aphidicolin-treated embryos. Thus unreplicated DNA only prevents mitotic nuclear, not cytoplasmic, events from occurring prematurely. These results together show that the inhibition of cyclin B-cdc2 kinase activation is probably not the only mechanism that prevents mitotic nuclear events from occurring as long as DNA replication has not been completed. In contrast, cytoplasmic mitotic events seem to be controlled by a timing mechanism independent of DNA replication, set up at fertilization, that prevents premature opening of a window for mitotic events.
Insights
In sea urchin embryos, unreplicated DNA prevents premature nuclear events but not cytoplasmic ones. A timing mechanism independent of DNA replication controls cytoplasmic mitotic events after fertilization.
Area of Science:
- Cell Biology
- Developmental Biology
- Molecular Biology
Background:
- Cell cycle progression is regulated by feedback mechanisms ensuring proper order of events.
- Cyclin B-cdc2 kinase activation is a key regulator of the G2-M phase transition.
- Unreplicated DNA is generally thought to inhibit cyclin B-cdc2 kinase activation, preventing entry into M phase.
Purpose of the Study:
- To investigate the role of unreplicated DNA in regulating M phase entry in early sea urchin embryos.
- To determine whether cyclin B-cdc2 kinase activation is solely responsible for preventing premature mitosis.
- To differentiate the regulation of nuclear versus cytoplasmic events during mitosis.
Main Methods:
- Studied one-cell sea urchin embryos.
- Assessed cyclin B-cdc2 kinase activity during the cell cycle.
- Inhibited DNA replication using aphidicolin.
- Observed nuclear envelope breakdown and microtubule array organization.
- Monitored cytoplasmic mitotic events.
Main Results:
- Cyclin B-cdc2 kinase is partially activated before S phase and sufficiently activated during S phase for G2-M transition.
- S phase entry is not inhibited by this kinase activity.
- Aphidicolin treatment prevents nuclear envelope breakdown but not the conversion of the microtubule array to the mitotic state.
- Mitotic cytoplasmic events occur concurrently in control and aphidicolin-treated embryos.
Conclusions:
- Unreplicated DNA specifically inhibits premature nuclear mitotic events, not cytoplasmic ones.
- Mechanisms beyond cyclin B-cdc2 kinase inhibition are involved in preventing premature nuclear events.
- Cytoplasmic mitotic events are regulated by a fertilization-dependent timing mechanism independent of DNA replication.