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Molecular and Cellular Biology, May 2007, p. 3367-3377, Vol. 27, No. 9
0270-7306/07/$08.00+0     doi:10.1128/MCB.02238-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Function of a Conserved Checkpoint Recruitment Domain in ATRIP Proteins{triangledown}

Heather L. Ball,1 Mark R. Ehrhardt,2 Daniel A. Mordes,1 Gloria G. Glick,1 Walter J. Chazin,2 and David Cortez1*

Department of Biochemistry,1 Departments of Biochemistry and Chemistry and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 372322

Received 29 November 2006/ Returned for modification 29 December 2006/ Accepted 16 February 2007

The ATR (ATM and Rad3-related) kinase is essential to maintain genomic integrity. ATR is recruited to DNA lesions in part through its association with ATR-interacting protein (ATRIP), which in turn interacts with the single-stranded DNA binding protein RPA (replication protein A). In this study, a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs was identified by biochemical mapping of the RPA binding site in combination with nuclear magnetic resonance, mutagenesis, and computational modeling. Mutations in the CRD of the Saccharomyces cerevisiae ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA-damaging agents, and partially compromise checkpoint signaling. These data demonstrate that the CRD is critical for localization and optimal DNA damage responses. However, the stimulation of ATR kinase activity by binding of topoisomerase binding protein 1 (TopBP1) to ATRIP-ATR can occur independently of the interaction of ATRIP with RPA. Our results support the idea of a multistep model for ATR activation that requires separable localization and activation functions of ATRIP.

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

{triangledown} Published ahead of print on 5 March 2007.

Molecular and Cellular Biology, May 2007, p. 3367-3377, Vol. 27, No. 9
0270-7306/07/$08.00+0     doi:10.1128/MCB.02238-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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