Motion of a DNA sliding clamp observed by single molecule fluorescence spectroscopy.
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 beta subunit or beta 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 beta clamp diffusing along DNA is on the order of 10(-14) m(2)/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the beta 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.
Laurence
Ted A
TA
Lawrence Livermore National Laboratory, California 94550, USA. laurence2@llnl.gov
Kwon
Youngeun
Y
Johnson
Aaron
A
Hollars
Christopher W
CW
O'Donnell
Mike
M
Camarero
Julio A
JA
Barsky
Daniel
D
D017104:Bacteriophage M13 / Q000235:genetics; D001671:Biochemistry / Q000379:methods; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004249:DNA Damage; D004261:DNA Replication; D004058:Diffusion; D004926:Escherichia coli / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007834:Lasers; D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D017785:Photons; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
D004247:DNA