Yoo, Hae Yong and Shevchenko, Anna and Shevchenko, Andrej and Dunphy, William G. (2004) Mcm2 is a direct substrate of ATM and ATR during DNA damage and DNA replication checkpoint responses. Journal of Biological Chemistry, 279 (51). pp. 53353-53364. ISSN 0021-9258 http://resolver.caltech.edu/CaltechAUTHORS:YOOjbc04
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In vertebrates, ATM and ATR are critical regulators of checkpoint responses to damaged and incompletely replicated DNA. These checkpoint responses involve the activation of signaling pathways that inhibit the replication of chromosomes with DNA lesions. In this study, we describe the isolation of a cDNA encoding a full-length version of Xenopus ATM. Using antibodies against the regulatory domain of ATM, we have identified the essential replication protein Mcm2 as an ATM-binding protein in Xenopus egg extracts. Xenopus Mcm2 underwent phosphorylation at Ser92 in response to the presence of double-stranded DNA breaks or DNA replication blocks in egg extracts. This phosphorylation involved both ATM and ATR, but the relative contribution of each kinase depended upon the checkpoint-inducing DNA signal. Furthermore, both ATM and ATR phosphorylated Mcm2 directly at Ser92 in cell-free kinase assays. Immunodepletion of both ATM and ATR abrogated the checkpoint response that blocks chromosomal DNA replication in egg extracts containing double-stranded DNA breaks. These experiments indicate that ATM and ATR phosphorylate the functionally critical replication protein Mcm2 during both DNA damage and replication checkpoint responses in Xenopus egg extracts.
|Additional Information:||© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Received for publication, July 15, 2004, and in revised form, August 30, 2004. Published, JBC Papers in Press, September 22, 2004, DOI 10.1074/jbc.M408026200 We are grateful to our colleagues in the laboratory for helpful comments on the manuscript, to Drs. I. M. Ward and J. Chen (Mayo Clinic) for technical advice on the UV filter assay, and to Dr. J. J. Blow for anti-Xmcm4 and anti-Xmcm7 antibodies. Supplemental Figure S1. Sequence alignment of N-terminal halves of Xatm and human ATM.|
|Subject Keywords:||XENOPUS EGG EXTRACTS; CELL-FREE-EXTRACTS; EARLY EMBRYONIC LETHALITY; S-PHASE; ATAXIA-TELANGIECTASIA; DEPENDENT CHECKPOINT; PROTEINS; KINASE; CHK1; ACTIVATION|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Lindsay Cleary|
|Deposited On:||10 Oct 2006|
|Last Modified:||26 Dec 2012 09:05|
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