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J. Biol. Chem., Vol. 283, Issue 34, 22895-22906, August 22, 2008

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Motion of a DNA Sliding Clamp Observed by Single Molecule Fluorescence Spectroscopy^*

Ted A. Laurence¹, Youngeun Kwon, Aaron Johnson, Christopher W. Hollars, Mike O'Donnell, Julio A. Camarero, and Daniel Barsky²

From the Chemistry, Materials, Earth, and Life Sciences, Lawrence Livermore National Laboratory, California 94550 and Rockefeller University and Howard Hughes Medical Institute, New York, New York 10065

DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III β subunit or β clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the β clamp diffusing along DNA is on the order of 10^–14 m²/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the β clamp remains at the 3' end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA.

Received for publication, January 8, 2008 , and in revised form, June 10, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM38839 (to M. O. D.). This work was also supported by the laboratory-directed research and development program at Lawrence Livermore National Laboratory (to T. A. L., J. A. C., and D. B.). 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 supplemental Figs. S1–S9.

¹ To whom correspondence may be addressed. E-mail: laurence2{at}llnl.gov. ² To whom correspondence may be addressed. E-mail: barsky{at}llnl.gov.

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