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J. Biol. Chem., Vol. 279, Issue 6, 4386-4393, February 6, 2004

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Mechanism of Loading the Escherichia coli DNA Polymerase III Sliding Clamp

II. UNCOUPLING THE AND DNA BINDING ACTIVITIES OF THE COMPLEX^*

Anita K. Snyder, Christopher R. Williams, Aaron Johnson, Mike O'Donnell, and Linda B. Bloom¶

From the Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245 and the Rockefeller University and Howard Hughes Medical Institute, New York, New York 10021

Sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. The Escherichia coli complex loads the sliding clamp onto DNA in an ATP-dependent reaction in which ATP binding and hydrolysis modulate the affinity of the complex for and DNA. This is the second of two reports (Williams, C. R., Snyder, A. K., Kuzmi, P., O'Donnell, M., and Bloom, L. B. (2004) J. Biol. Chem. 279, 4376–4385) addressing the question of how ATP binding and hydrolysis regulate specific interactions with DNA and . Mutations were made to an Arg residue in a conserved SRC motif in the ' and subunits that interacts with the ATP site of the neighboring subunit. Mutation of the ' subunit reduced the ATP-dependent binding activity, whereas mutation of the subunits reduced the DNA binding activity of the complex. The complex containing the ' mutation gave a pre-steady-state burst of ATP hydrolysis, but at a reduced rate and amplitude relative to the wild-type complex. A pre-steady-state burst of ATP hydrolysis was not observed for the complex containing the mutations, consistent with the reduced DNA binding activity of this complex. The differential effects of these mutations suggest that ATP binding at the ₁ site may be coupled to conformational changes that largely modulate interactions with , whereas ATP binding at the ₂ and/or ₃ site may be coupled to conformational changes that have a major role in interactions with DNA. Additionally, these results show that the "arginine fingers" play a structural role in facilitating the formation of a conformation that has high affinity for and DNA.

Received for publication, September 22, 2003 , and in revised form, November 3, 2003.

^* This work was supported by National Institutes of Health Grants GM55596 (to L. B. B.) and GM38839 (to M. O.). 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.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Florida, P. O. Box 100245, Gainesville, FL 32610-0245. Tel.: 352-392-8708; Fax: 352-392-6511; E-mail: lbloom{at}ufl.edu.

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