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Vol. 17, Issue 4, 1559-1569, April 2006
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Department of Molecular Pharmacology, Stanford University, Stanford, CA 94305-5441
Submitted September 18, 2005;
Revised December 20, 2005;
Accepted January 13, 2006
Monitoring Editor: John York
The DNA damage checkpoint pathways sense and respond to DNA damage to ensure genomic stability. The ATR kinase is a central regulator of one such pathway and phosphorylates a number of proteins that have roles in cell cycle progression and DNA repair. Using the Xenopus egg extract system, we have investigated regulation of the Rad1/Hus1/Rad9 complex. We show here that phosphorylation of Rad1 and Hus1 occurs in an ATR- and TopBP1-dependent manner on T5 of Rad1 and S219 and T223 of Hus1. Mutation of these sites has no effect on the phosphorylation of Chk1 by ATR. Interestingly, phosphorylation of Rad1 is independent of Claspin and the Rad9 carboxy terminus, both of which are required for Chk1 phosphorylation. These data suggest that an active ATR signaling complex exists in the absence of the carboxy terminus of Rad9 and that this carboxy-terminal domain may be a specific requirement for Chk1 phosphorylation and not necessary for all ATR-mediated signaling events. Thus, Rad1 phosphorylation provides an alternate and early readout for the study of ATR activation.
Abbreviations used: ATR, ATM and Rad3-related; ATRIP, ATR-interacting protein; exo, exonuclease; RHR, rad9, hus1, rad1; RPA, replication protein A; TopBP1, topoisomerase II binding protein 1; UV, ultraviolet radiation.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
Address correspondence to: Karlene A. Cimprich (cimprich{at}stanford.edu).
This article has been cited by other articles:
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  A. E. Burrows and S. J. Elledge How ATR turns on: TopBP1 goes on ATRIP with ATR Genes & Dev., June 1, 2008; 22(11): 1416 - 1421. [Abstract] [Full Text] [PDF]
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 J. Lee, A. Kumagai, and W. G. Dunphy The Rad9-Hus1-Rad1 Checkpoint Clamp Regulates Interaction of TopBP1 with ATR J. Biol. Chem., September 21, 2007; 282(38): 28036 - 28044. [Abstract] [Full Text] [PDF]
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S. Delacroix, J. M. Wagner, M. Kobayashi, K.-i. Yamamoto, and L. M. Karnitz The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1 Genes & Dev., June 15, 2007; 21(12): 1472 - 1477. [Abstract] [Full Text] [PDF]
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L. Zou Single- and double-stranded DNA: building a trigger of ATR-mediated DNA damage response Genes & Dev., April 15, 2007; 21(8): 879 - 885. [Full Text] [PDF]
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C. A. MacDougall, T. S. Byun, C. Van, M.-c. Yee, and K. A. Cimprich The structural determinants of checkpoint activation Genes & Dev., April 15, 2007; 21(8): 898 - 903. [Abstract] [Full Text] [PDF]
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D. J. Chang, P. J. Lupardus, and K. A. Cimprich Monoubiquitination of Proliferating Cell Nuclear Antigen Induced by Stalled Replication Requires Uncoupling of DNA Polymerase and Mini-chromosome Maintenance Helicase Activities J. Biol. Chem., October 27, 2006; 281(43): 32081 - 32088. [Abstract] [Full Text] [PDF]
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S. Liu, S. Bekker-Jensen, N. Mailand, C. Lukas, J. Bartek, and J. Lukas Claspin Operates Downstream of TopBP1 To Direct ATR Signaling towards Chk1 Activation. Mol. Cell. Biol., August 1, 2006; 26(16): 6056 - 6064. [Abstract] [Full Text] [PDF]
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