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Molecular Cell, Volume 11, Issue 2, 1 February 2003, Pages 329-340
Joon Lee, Akiko Kumagai and William G. Dunphy
SummaryClaspin is required for the ATR-dependent activation of Chk1 in egg extracts containing incompletely replicated DNA. We show here that Claspin associates with chromatin in a regulated manner during S phase. Binding of Claspin to chromatin depends on the pre-replication complex (pre-RC) and Cdc45 but not on replication protein A (RPA). These dependencies suggest that binding of Claspin occurs around the time of initial DNA unwinding at replication origins. By contrast, both ATR and Rad17 require RPA for association with DNA. Claspin, ATR, and Rad17 all bind to chromatin independently. These findings suggest that Claspin plays a role in monitoring DNA replication during S phase. Claspin, ATR, and Rad17 may collaborate in checkpoint regulation by detecting different aspects of a DNA replication fork.
Summary | Full Text | PDF (512 kb) - Adaptation of a DNA Replication Checkpoint Response Depends ...Adaptation of a DNA Replication Checkpoint Response Depends upon Inactivation of Claspin by the Polo-like Kinase
Cell, Volume 117, Issue 5, 28 May 2004, Pages 575-588
Hae Yong Yoo, Akiko Kumagai, Anna Shevchenko, Andrej Shevchenko and William G. Dunphy
SummaryThe checkpoint mediator protein Claspin is essential for the ATR-dependent activation of Chk1 in egg extracts containing aphidicolin-induced DNA replication blocks. We show that, during this checkpoint response, Claspin becomes phosphorylated on threonine 906 (T906), which creates a docking site for Plx1, the Polo-like kinase. This interaction promotes the phosphorylation of Claspin on a nearby serine (S934) by Plx1. After a prolonged interphase arrest, aphidicolin-treated egg extracts typically undergo adaptation and enter into mitosis despite the presence of incompletely replicated DNA. In this process, Claspin dissociates from chromatin, and Chk1 undergoes inactivation. By contrast, aphidicolin-treated extracts containing mutants of Claspin with alanine substitutions at positions 906 or 934 (T906A or S934A) are unable to undergo adaptation. Under such adaptation-defective conditions, Claspin accumulates on chromatin at high levels, and Chk1 does not decrease in activity. These results indicate that the Plx1-dependent inactivation of Claspin results in termination of a DNA replication checkpoint response.
Summary | Full Text | PDF (1202 kb) - TopBP1 Activates the ATR-ATRIP ComplexTopBP1 Activates the ATR-ATRIP Complex
Cell, Volume 124, Issue 5, 10 March 2006, Pages 943-955
Akiko Kumagai, Joon Lee, Hae Yong Yoo and William G. Dunphy
SummaryATR is a key regulator of checkpoint responses to incompletely replicated and damaged DNA, but the mechanisms underlying control of its kinase activity are unknown. TopBP1, the vertebrate homolog of yeast Cut5/Dbp11, has dual roles in initiation of DNA replication and regulation of checkpoint responses. We show that recombinant TopBP1 induces a large increase in the kinase activity of both and human ATR. The ATR-activating domain resides in a conserved segment of TopBP1 that is distinct from its numerous BRCT repeats. The isolated ATR-activating domain from TopBP1 induces ectopic activation of ATR-dependent signaling in both egg extracts and human cells. Furthermore, egg extracts containing a version of TopBP1 with an inactivating point mutation in the ATR-activating domain are defective in checkpoint regulation. These studies establish that activation of ATR by TopBP1 is a crucial step in the initiation of ATR-dependent signaling processes.
Summary | Full Text | PDF (737 kb) - …more
Copyright © 2000 Cell Press. All rights reserved.
Molecular Cell, Volume 6, Issue 4, 839-849, 1 October 2000
doi:10.1016/S1097-2765(05)00092-4
Article
Claspin, a Novel Protein Required for the Activation of Chk1 during a DNA Replication Checkpoint Response in Xenopus Egg Extracts
Akiko Kumagai1 and William G. Dunphy1, *, *, 
Corresponding author: William G. Dunphy, (626) 395-8433 (phone), (626) 795-7563 (fax)Summary
We have identified Claspin, a novel protein that binds to Xenopus Chk1 (Xchk1). Binding of Claspin to Xchk1 is highly elevated in the presence of DNA templates that trigger a checkpoint arrest of the cell cycle in Xenopus egg extracts. Xchk1 becomes phosphorylated during a checkpoint response, and we demonstrate directly that this phosphorylation results in the activation of Xchk1. Immunodepletion of Claspin from egg extracts abolishes both the phosphorylation and activation of Xchk1. Furthermore, Claspin-depleted extracts are unable to arrest the cell cycle in response to DNA replication blocks. Taken together, these findings indicate that Claspin is an essential upstream regulator of Xchk1.
