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J. Biol. Chem., Vol. 278, Issue 37, 35584-35591, September 12, 2003
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The Phosphorylation Domain of the 32-kDa Subunit of Replication Protein A (RPA) Modulates RPA-DNA Interactions
EVIDENCE FOR AN INTERSUBUNIT INTERACTION*
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From the
Department of Biochemistry, University of
Iowa College of Medicine, Iowa City, Iowa 52242-1109 and
¶Pacific Northwest National Laboratory, Richland,
Washington 99352
Replication protein A (RPA) is a heterotrimeric (subunits of 70, 32, and 14 kDa) single-stranded DNA-binding protein that is required for DNA replication, recombination, and repair. The 40-residue N-terminal domain of the 32-kDa subunit of RPA (RPA32) becomes phosphorylated during S-phase and after DNA damage. Recently it has been shown that phosphorylation or the addition of negative charges to this N-terminal phosphorylation domain modulates RPA-protein interactions and increases cell sensitivity to DNA damage. We found that addition of multiple negative charges to the N-terminal phosphorylation domain also caused a significant decrease in the ability of a mutant form of RPA to destabilize double-stranded (ds) DNA. Kinetic studies suggested that the addition of negative charges to the N-terminal phosphorylation domain caused defects in both complex formation (nucleation) and subsequent destabilization of dsDNA by RPA. We conclude that the N-terminal phosphorylation domain modulates RPA interactions with dsDNA. Similar changes in DNA interactions were observed with a mutant form of RPA in which the N-terminal domain of the 70-kDa subunit was deleted. This suggested a functional link between the N-terminal domains of the 70- and 32-kDa subunits of RPA. NMR experiments provided evidence for a direct interaction between the N-terminal domain of the 70-kDa subunit and the negatively charged N-terminal phosphorylation domain of RPA32. These findings suggest that phosphorylation causes a conformational change in the RPA complex that regulates RPA function.
Received for publication, May 22, 2003
* This work was supported in part by NIGMS, National Institutes of Health Grant GM4471 (to M. S. W.) and in part by the Office of Science (Biological and Environmental Research), United States Department of Energy Contract DE-AC06-76RL01830 (to D. F. 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.
Present address: Lexicon, Inc., Houston, TX.
|| To whom correspondence should be addressed: Dept. of Biochemistry, University of Iowa College of Medicine, 51 Newton Rd., Iowa City, IA 52242-1109. Tel.: 319-335-6784; Fax: 319-384-4770; E-mail: marc-wold{at}uiowa.edu.
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