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J. Biol. Chem., Vol. 279, Issue 24, 25638-25645, June 11, 2004

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Physical Interaction between Replication Protein A and Rad51 Promotes Exchange on Single-stranded DNA^*

Melissa E. Stauffer and Walter J. Chazin

From the Departments of Biochemistry and Physics and the Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232-8725

Replication protein A (RPA) is displaced from single-stranded DNA (ssDNA) by Rad51 during the initiation of homologous recombination. Interactions between these proteins have been reported, but the functional significance of the direct RPA-Rad51 interaction has yet to be elucidated. We have identified and characterized the interaction between DNA-binding domain A of RPA (RPA70A) and the N-terminal domain of Rad51 (Rad51N). NMR chemical shift mapping showed that Rad51N binds to the ssDNA-binding site of RPA70A, suggesting a competitive mechanism for the displacement of RPA from ssDNA by Rad51. A structure of the RPA70A-Rad51N complex was generated by experimentally guided modeling and then used to design mutations that disrupt the binding interface. Functional ATP hydrolysis assays were performed for wild-type Rad51 and a mutant defective in binding RPA. Rates of RPA displacement for the mutant were significantly below those of wild-type Rad51, suggesting that a direct RPA-Rad51 interaction is involved in displacing RPA in the initiation stage of genetic recombination.

Received for publication, January 5, 2004 , and in revised form, March 10, 2004.

^* This work was supported by National Institutes of Health Grant RO1 GM65484 (to W. J. C.). Access to core facilities was supported by National Institutes of Health Grants P30 CA68485 and P50 ES00267 (to the Vanderbilt-Ingram Cancer Center and the Vanderbilt Center in Molecular Toxicology, respectively). 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.

Supported by National Research Service Award (NRSA) Postdoctoral Fellowship F32 GM068352 and NRSA Institutional Research Training Grant T32 CA09582 from the National Institutes of Health.

To whom correspondence should be addressed: 5140 MRB III, Vanderbilt University, Nashville, TN 37232-8725. Tel.: 615-936-2210; Fax: 615-936-2211; E-mail: walter.chazin{at}vanderbilt.edu.

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