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Control of the Cdc2/cyclin B complex in Xenopus egg extracts arrested at a G2/M checkpoint with DNA synthesis inhibitors

Kumagai, Akiko and Dunphy, William G. (1995) Control of the Cdc2/cyclin B complex in Xenopus egg extracts arrested at a G2/M checkpoint with DNA synthesis inhibitors. Molecular Biology of the Cell, 6 (2). pp. 199-213. ISSN 1059-1524. http://resolver.caltech.edu/CaltechAUTHORS:KUMmbc95

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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.


Item Type:Article
Additional Information:Copyright © 1995 by The American Society for Cell Biology Submitted November 18, 1994; Accepted January 11, 1995. Monitoring Editor: Tim Hunt We thank our colleagues for comments on the manuscript. We are grateful to D. Morgan (University of California, San Francisco) for providing a baculovirus vector for histidine tagging. M. Solomon (Yale University) kindly provided the Cdc2 mutant plasmids. This work was supported by grants from the National Institutes of Health, Lucille P. Markey Charitable Trust, National Science Foundation, and Gustavus and Louise Pfeiffer Foundation. W.G.D. is an investigator of the Howard Hughes Medical Institute.
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ID Code:3895
Collection:CaltechAUTHORS
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Deposited On:18 Jul 2006
Last Modified:26 Dec 2012 08:56

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