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Single molecule study of a processivity clamp sliding on DNA [electronic resource].

Published
Washington, D.C. : United States. Dept. of Energy, 2007.
Oak Ridge, Tenn. : Distributed by the Office of Scientific and Technical Information, U.S. Dept. of Energy.
Physical Description
PDF-file: 52 pages; size: 1.3 Mbytes
Additional Creators
Lawrence Berkeley National Laboratory, United States. Department of Energy, and United States. Department of Energy. Office of Scientific and Technical Information
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Summary
Using solution based single molecule spectroscopy, we study the motion of the polIII β-subunit DNA sliding clamp ('β-clamp') on DNA. Present in all cellular (and some viral) forms of life, DNA sliding clamps attach to polymerases and allow rapid, processive replication of DNA. In the absence of other proteins, the DNA sliding clamps are thought to 'freely slide' along the DNA; however, the abundance of positively charged residues along the inner surface may create favorable electrostatic contact with the highly negatively charged DNA. We have performed single-molecule measurements on a fluorescently labeled β-clamp loaded onto freely diffusing plasmids annealed with fluorescently labeled primers of up to 90 bases. We find that the diffusion constant for 1D diffusion of the β-clamp on DNA satisfies D ≤ 10⁻¹⁴ cm²/s, much slower than the frictionless limit of D = 10⁻¹° cm²/s. We find that the β clamp remains at the 3-foot end in the presence of E. coli single-stranded binding protein (SSB), which would allow for a sliding clamp to wait for binding of the DNA polymerase. Replacement of SSB with Human RP-A eliminates this interaction; free movement of sliding clamp and poor binding of clamp loader to the junction allows sliding clamp to accumulate on DNA. This result implies that the clamp not only acts as a tether, but also a placeholder.
Report Numbers
E 1.99:ucrl-jrnl-232518
ucrl-jrnl-232518
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Note
Published through SciTech Connect.
07/05/2007.
"ucrl-jrnl-232518"
Journal of Biological Chemistry, vol. 283, no. 34, August 22, 2008, pp. 22895?22906, 283 34 ISSN 0021-9258; JBCHA3 FT
Johnson, A; Kwon, Y; Camarero, J A; Laurence, T A; Barsky, D; Hollars, C; O?Donnell, M.
Funding Information
W-7405-ENG-48
View MARC record | catkey: 13809563