Motion of a DNA sliding clamp observed by single molecule fluorescence spectroscopy

Ted A. Laurence, Youngeun Kwon, Aaron Johnson, Christopher W. Hollars, Mike O'Donnell, Julio A. Camarero, Daniel Barsky

Research output: Contribution to journalArticlepeer-review

42 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)22895-22906
Number of pages12
JournalJournal of Biological Chemistry
Volume283
Issue number34
DOIs
StatePublished - 22 Aug 2008

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