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J. Biol. Chem., Vol. 281, Issue 38, 27855-27861, September 22, 2006

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Replication Protein A Directs Loading of the DNA Damage Checkpoint Clamp to 5'-DNA Junctions^*

Jerzy Majka, Sara K. Binz, Marc S. Wold, and Peter M. J. Burgers¹

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110 and Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242

The heterotrimeric checkpoint clamp comprises the Saccharomyces cerevisiae Rad17, Mec3, and Ddc1 subunits (Rad17/3/1, the 9-1-1 complex in humans). This DNA damage response factor is loaded onto DNA by the Rad24-RFC (replication factor C-like complex with Rad24) clamp loader and ATP. Although Rad24-RFC alone does not bind to naked partial double-stranded DNA, coating of the single strand with single-stranded DNA-binding protein RPA (replication protein A) causes binding of Rad24-RFC via interactions with RPA. However, RPA-mediated binding is abrogated when the DNA is coated with RPA containing a rpa1-K45E (rfa1-t11) mutation. These properties allowed us to determine the role of RPA in clamp-loading specificity. The Rad17/3/1 clamp is loaded with comparable efficiency onto naked primer/template DNA with either a 3'-junction or a 5'-junction. Remarkably, when the DNA was coated with RPA, loading of Rad17/3/1 at 3'-junctions was completely inhibited, thereby providing specificity to loading at 5'-junctions. However, Rad17/3/1 loaded at 5'-junctions can slide across double-stranded DNA to nearby 3'-junctions and thereby affect the activity of proteins that act at 3'-termini. These studies show a unique specificity of the checkpoint loader for 5'-junctions of RPA-coated DNA. The implications of this specificity for checkpoint function are discussed.

Received for publication, May 30, 2006 , and in revised form, July 12, 2006.

^* This work was supported in part by Grants GM32431 (to P. M. J. B.) and GM44721 (to M. S. W.) from the National Institutes of Health. 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.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-3872; E-mail: burgers{at}biochem.wustl.edu.

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