Lee, Joon and Gold, Daniel A. and Shevchenko, Anna and Shevchenko, Andrej and Dunphy, William G. (2005) Roles of replication fork-interacting and Chk1-activating domains from claspin in a DNA replication checkpoint response. Molecular Biology of the Cell, 16 (11). pp. 5269-5282. ISSN 1059-1524 http://resolver.caltech.edu/CaltechAUTHORS:LEEmbc05
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Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. Claspin associates with replication forks upon origin unwinding. We show that Claspin contains a replication fork-interacting domain (RFID, residues 265–605) that associates with Cdc45, DNA polymerase ε, replication protein A, and two replication factor C complexes on chromatin. The RFID contains two basic patches (BP1 and BP2) at amino acids 265–331 and 470–600, respectively. Deletion of either BP1 or BP2 compromises optimal binding of Claspin to chromatin. Absence of BP1 has no effect on the ability of Claspin to mediate activation of Chk1. By contrast, removal of BP2 causes a large reduction in the Chk1-activating potency of Claspin. We also find that Claspin contains a small Chk1-activating domain (residues 776–905) that does not bind stably to chromatin, but it is fully effective at high concentrations for mediating activation of Chk1. These results indicate that stable retention of Claspin on chromatin is not necessary for activation of Chk1. Instead, our findings suggest that only transient interaction of Claspin with replication forks potentiates its Chk1-activating function. Another implication of this work is that stable binding of Claspin to chromatin may play a role in other functions besides the activation of Chk1.
|Additional Information:||Copyright © 2005 by The American Society for Cell Biology. Submitted July 25, 2005; revised August 25, 2005; accepted August 29, 2005. Originally published as MBC in Press, 10.1091/mbc.E05-07-0671 on September 7, 2005 We are grateful to S. Zhou, D. King, and R. Tjian (Howard Hughes Medical Institute, University of California, Berkeley) for identification of importins as Claspin-associated proteins. We thank S. Waga, H. Takisawa, P. Jackson, J. Blow, J. Hurwitz, D. Görlich, and T. Hunter for generosity in providing valuable reagents. We are indebted to A. Kumagai, E. Bae, and S.-Y. Jeong for supplying the constructs for full-length His6-Claspin-FLAG and His6-Claspin(774-1285)-FLAG and to S.-M. Kim for providing the 30d-40s DNA. We are also grateful to our colleagues in the laboratory for helpful comments throughout the course of this work. J.L. is a recipient of the Donald E. and Deila B. Baxter Postdoctoral Fellowship. This work was supported by National Institutes of Health Grants GM-043974 and GM-070891 (to W.G.D.).|
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|Deposited By:||Lindsay Cleary|
|Deposited On:||08 Nov 2006|
|Last Modified:||26 Dec 2012 09:16|
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