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Phosphorylation of Chk1 by ATM- and Rad3-related (ATR) in Xenopus Egg Extracts Requires Binding of ATRIP to ATR but Not the Stable DNA-binding or Coiled-coil Domains of ATRIP

Kim, Soon-Mi and Kumagai, Akiko and Lee, Joon and Dunphy, William G. (2005) Phosphorylation of Chk1 by ATM- and Rad3-related (ATR) in Xenopus Egg Extracts Requires Binding of ATRIP to ATR but Not the Stable DNA-binding or Coiled-coil Domains of ATRIP. Journal of Biological Chemistry, 280 (46). pp. 38355-38364. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:KIMjbc05

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Abstract

ATR, a critical regulator of DNA replication and damage checkpoint responses, possesses a binding partner called ATRIP. We have studied the functional properties of Xenopus ATR and ATRIP in incubations with purified components and in frog egg extracts. In purified systems, ATRIP associates with DNA in both RPA-dependent and RPA-independent manners, depending on the composition of the template. However, in egg extracts, only the RPA-dependent mode of binding to DNA can be detected. ATRIP adopts an oligomeric state in egg extracts that depends upon binding to ATR. In addition, ATR and ATRIP are mutually dependent on one another for stable binding to DNA in egg extracts. The ATR-dependent oligomerization of ATRIP does not require an intact coiled-coil domain in ATRIP and does not change in the presence of checkpoint-inducing DNA templates. Egg extracts containing a mutant of ATRIP that cannot bind to ATR are defective in the phosphorylation of Chk1. However, extracts containing mutants of ATRIP lacking stable DNA-binding and coiled-coil domains show no reduction in the phosphorylation of Chk1 in response to defined DNA templates. Furthermore, activation of Chk1 does not depend upon RPA under these conditions. These results suggest that ATRIP must associate with ATR in order for ATR to carry out the phosphorylation of Chk1 effectively. However, this function of ATRIP does not involve its ability to mediate the stable binding of ATR to defined checkpoint-inducing DNA templates in egg extracts, does not require an intact coiled-coil domain, and does not depend on RPA.


Item Type:Article
Additional Information:Copyright © 2005 by the American Society for Biochemistry and Molecular Biology. Received for publication, August 5, 2005 , and in revised form, September 22, 2005. Originally published In Press as doi:10.1074/jbc.M508673200 on September 25, 2005 We are grateful to Marc Wold for generously providing human mutant RPA protein constructs. We thankfully acknowledge the kind assistance of Cheol-Sang Hwang with the gel filtration experiments. We are grateful to our colleagues in the laboratory for helpful advice on this project and to Karen Wawrousek for comments on the manuscript. This work was supported by National Institutes of Health Grants GM070891 and GM043974 (to W. G. D.) and a Donald E. and Deila B. Baxter Postdoctoral Fellowship (to J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Record Number:CaltechAUTHORS:KIMjbc05
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:KIMjbc05
Alternative URL:http://dx.doi.org/10.1074/jbc.M508673200
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:5599
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:25 Oct 2006
Last Modified:26 Dec 2012 09:13

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