Active cyclin B-cdc2 kinase does not inhibit DNA replication and cannot drive prematurely fertilized sea urchin eggs into mitosis

Anne-Marie Genevire-Garrigues 1 Abdelhamid Barakat 1 Marcel Dore 0 Jean-Luc Moreau 1 Andr Picard 1 0 CRBM , route de Mende, BP 5051, F34033, Montpellier , France 1 Laboratoire Arago , F66650, Banyuls-sur Mer , France *Author for correspondence - 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 Bdependent 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 Progression from G2 to M phase of the eukaryotic cell cycle depends on activation of a protein kinase composed of cdc2 associated with cyclin B (reviewed by Nurse, 1990, 1994; Masui, 1992; King et al., 1994). Dephosphorylation of cdc2 on tyrosine 15 is required for activation of the cyclin B-cdc2 complex (Solomon et al.,1990; Gould and Nurse, 1989; Dunphy and Newport, 1989; Morla et al., 1989) and is brought about by the tyrosine phosphatase cdc25 (Strausfeld et al., 1991; Dunphy and Kumagai, 1991; Gautier et al., 1991), which antagonizes the activity of the inhibitory tyrosine kinases wee1 and mik1 in fission yeast, and their homologs in higher eukaryotes (Russell and Nurse, 1987; Featherstone and Russell, 1991; Parker et al., 1992; McGowan and Russell, 1993). In somatic cells, feedback controls prevent M phase from occurring before S phase is completed (see Hartwell and Weinert, 1989, for review). In fission yeast these controls are lost in mutants defective in undergoing inhibitory phosphorylation on tyrosine 15, due to either overproduction of cdc25 or suppression of both mik1 and wee1 activities (Enoch and Nurse, 1990; Lundgren et al., 1991). This strongly suggests that feedback mechanisms that couple mitosis to completion of S phase act through regulation of tyrosine 15 phosphorylation of cdc2. Nonetheless, although mitotic cytoskeletal events 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. occur, chromosome condensation is not observed in these mutants (Enoch and Nurse, 1990). Thus even in fission yeast premature activation of cyclin B-cdc2 kinase does not appear to be sufficient to completely bypass mechanisms that prevent cells from entering mitosis before S phase is completed. In budding yeast, mutations in the cdc2 homolog CDC28 that prevent phosphorylation of tyrosine 19 (the equivalent of tyrosine 15 in cdc2) do not alter the timing of the cell cycle and do not lead to any premature mitotic event, showing that other mechanisms than regulated phosphorylation of tyrosine 15/19 may prevent M phase from occurring before completion of S phase (Sorger and Murray, 1992; Amon et al., 1992). In higher eukaryotes it is generally accepted that the mechanisms which couple M phase entry to completion of DNA replication ultimately regulate cyclin B-dependent kinase activity. Premature chromosome condensation is observed in RCC1mutated tsBN2 hamster cells grown at restrictive temperatures, even in S or G1 phase cells. This phenotype has been shown to depend on cdc25 translocation into the nucleus (Seki et al., 1992), suggesting a link between RCC1-dependent control and the regulation of tyrosine 15 phosphorylation of cdc2. In early frog or sea urchin embryos inhibition of DNA replication by aphidicolin or hydroxyurea leads to accumulation of cdc2 in a tyrosine-phosphorylated and inactive form (Dasso and Newport, 1990; Kumagai and Dunphy, 1991; Meijer et al., 1991). Expression in human somatic cells of cdc2 carrying mutations at tyrosine 15 and threonine 14 also induces premature chromosome condensation and lamina disassembly but it fails to induce spindle formation (Krek and Nigg, 1991), suggesting that control mechanisms other than inhibitory phosphorylations on cdc2 are necessary to ensure the correct timing of mitosis. Moreover, it is commonly assumed that a desynchronized activation of the mitotic kinase would induce a reentry into mitosis and inhibit genome duplication. Conversely, in fission yeast the experimental destruction of cyclin B-cdc2 complex in late G2 leads to DNA re-replication (Broeck et al., 1991; Hayles et al., 1994). This suggests that the active complex exerts a negative control on DNA replication, ensuring that the cell replicates DNA only once during the cell cycle. Taken together these results support the view that the mutual exclusion of the different phases governs progression through the cell cycle: activities which allow a cell to be in M phase repress activities that would allow the cell to enter S phase, and conversely. In the present work we used sea urchin eggs to further analyse the relationships between cyclin B-cdc2 kinase activation and cell cycle progression. Unfertilized sea urchin eggs are arrested in G1 of the cell cycle. After fertilization sperm chromatin decondenses, forms a male pronucleus which fuses with the female pronucleus, and then S phase occurs in the zygotic nucleus. The fertilized egg then divides and subsequent cell cycles proceed without a significant increase in mass as rapidly alternating rounds of highly synchronous S and M phases. We found that fertilization already induces cyclin B-cdc2 kinase activation at late G1 phase, and that cyclin B-cdc2 kinase activity is higher at S phase than the threshold required for entry into M phase, at least as measured in egg extracts. However, nuclear events characteristic of M phase do not occur as long as DNA replication is not complete. This suggests that an unknown mechanism prevents active cyclin B-cdc2 kinase to drive sea urchin embryos into M phase before completion of S phase. MATERIALS AND METHODS The sea urchins Sphaerechinus granularis were collected over the year, and Paracentrotus lividus from January to June in the Mediterranean sea near Banyuls (France). Animals were kept for up to two months in running sea water. Handling of gametes Gametes were collected after injection of 0.1 ml of 0.2 M acetylcholine through the perior (...truncated)