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J. Biol. Chem., Vol. 278, Issue 42, 41077-41082, October 17, 2003

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Independent and Coordinated Functions of Replication Protein A Tandem High Affinity Single-stranded DNA Binding Domains^*,

Alphonse I. Arunkumar , Melissa E. Stauffer , Elena Bochkareva , Alexey Bochkarev  and Walter J. Chazin  ¶

From the Departments of Biochemistry and Physics and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232-8725 and the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190

The initial high affinity binding of single-stranded DNA (ssDNA) by replication protein A (RPA) is involved in the tandem domains in the central region of the RPA70 subunit (RPA70AB). However, it was not clear whether the two domains, RPA70A and RPA70B, bind DNA simultaneously or sequentially. Here, using primarily heteronuclear NMR complemented by fluorescence spectroscopy, we have analyzed the binding characteristics of the individual RPA70A and RPA70B domains and compared them with the intact RPA70AB. NMR chemical shift comparisons confirmed that RPA70A and RPA70B tumble independently in solution in the absence of ssDNA. NMR chemical shift perturbations showed that all ssDNA oligomers bind to the same sites as observed in the x-ray crystal structure of RPA70AB complexed to d(C)₈. Titrations using a variety of 5'-mer ssDNA oligomers showed that RPA70A has a 5–10-fold higher affinity for ssDNA than RPA70B. Detailed analysis of ssDNA binding to RPA70A revealed that all DNA sequences interact in a similar mode. Fluorescence binding measurements with a variety of 8–10'-mer DNA sequences showed that RPA70AB interacts with DNA with 100-fold higher affinity than the isolated domains. Calculation of the theoretical "linkage effect" from the structure of RPA70AB suggests that the high overall affinity for ssDNA is a byproduct of the covalent attachment of the two domains via a short flexible tether, which increases the effective local concentration. Taken together, our data are consistent with a sequential model of DNA binding by RPA according to which RPA70A binds the majority of DNA first and subsequent loading of RPA70B domain is facilitated by the linkage effect.

Received for publication, June 4, 2003 , and in revised form, July 24, 2003.

^* This work was supported by U. S. National Institutes of Health Grant RO1 GM65484 (to W. J. C.) and Grant RO1 GM61192 (to A. B.). Use of core facilities was supported in part by the Vanderbilt Center for Molecular Toxicology (NIH P30 ES00267) and the Vanderbilt Ingram Cancer Center (NIH P30 CA68485). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplementary figure S1 showing the ¹H-¹⁵N HSQC spectrum of RPA70AB with the backbone assignments.

^¶ To whom correspondence should be addressed: Depts. of Biochemistry and Physics, Center for Structural Biology, 5140 BIOSCI/MRBIII, 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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