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Molecular and Cellular Biology, December 2008, p. 7345-7353, Vol. 28, No. 24
0270-7306/08/$08.00+0     doi:10.1128/MCB.01079-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Basic Cleft of RPA70N Binds Multiple Checkpoint Proteins, Including RAD9, To Regulate ATR Signaling{triangledown}

Xin Xu, Sivaraja Vaithiyalingam, Gloria G. Glick, Daniel A. Mordes, Walter J. Chazin, and David Cortez*

Department of Biochemistry and Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Received 9 July 2008/ Returned for modification 31 July 2008/ Accepted 7 October 2008

ATR kinase activation requires the recruitment of the ATR-ATRIP and RAD9-HUS1-RAD1 (9-1-1) checkpoint complexes to sites of DNA damage or replication stress. Replication protein A (RPA) bound to single-stranded DNA is at least part of the molecular recognition element that recruits these checkpoint complexes. We have found that the basic cleft of the RPA70 N-terminal oligonucleotide-oligosaccharide fold (OB-fold) domain is a key determinant of checkpoint activation. This protein-protein interaction surface is able to bind several checkpoint proteins, including ATRIP, RAD9, and MRE11. RAD9 binding to RPA is mediated by an acidic peptide within the C-terminal RAD9 tail that has sequence similarity to the primary RPA-binding surface in the checkpoint recruitment domain (CRD) of ATRIP. Mutation of the RAD9 CRD impairs its localization to sites of DNA damage or replication stress without perturbing its ability to form the 9-1-1 complex or bind the ATR activator TopBP1. Disruption of the RAD9-RPA interaction also impairs ATR signaling to CHK1 and causes hypersensitivity to both DNA damage and replication stress. Thus, the basic cleft of the RPA70 N-terminal OB-fold domain binds multiple checkpoint proteins, including RAD9, to promote ATR signaling.

* Corresponding author. Mailing address: Department of Biochemistry, Vanderbilt University School of Medicine, 613 Light Hall, 23rd at Pierce Avenue, Nashville, TN 37232. Phone: (615) 322-8547. Fax: (615) 343-0704. E-mail: david.cortez{at}vanderbilt.edu

{triangledown} Published ahead of print on 20 October 2008.

Molecular and Cellular Biology, December 2008, p. 7345-7353, Vol. 28, No. 24
0270-7306/08/$08.00+0     doi:10.1128/MCB.01079-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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