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Title: Single molecule study of a processivity clamp sliding on DNA

Using solution based single molecule spectroscopy, we study the motion of the polIII {beta}-subunit DNA sliding clamp ('{beta}-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 {beta}-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 {beta}-clamp on DNA satisfies D {le} 10{sup -14} cm{sup 2}/s, much slower than the frictionless limit of D = 10{sup -10} cm{sup 2}/s. We find that the {beta} 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 themore » junction allows sliding clamp to accumulate on DNA. This result implies that the clamp not only acts as a tether, but also a placeholder.« less
Authors:
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Publication Date:
OSTI Identifier:
940858
Report Number(s):
UCRL-JRNL-232518
Journal ID: ISSN 0021-9258; JBCHA3; TRN: US200824%%355
DOE Contract Number:
W-7405-ENG-48
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry, vol. 283, no. 34, August 22, 2008, pp. 22895?22906,; Journal Volume: 283; Journal Issue: 34
Research Org:
Lawrence Livermore National Laboratory (LLNL), Livermore, CA
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ABUNDANCE; DIFFUSION; DNA; DNA POLYMERASES; ELECTROSTATICS; LOADERS; PLASMIDS; POLYMERASES; PROTEINS; RESIDUES; SPECTROSCOPY