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J Mol Biol. 2015 Sep 25;427(19):3123-36. doi: 10.1016/j.jmb.2015.07.015. Epub 2015 Jul 26.

DNA-Segment-Facilitated Dissociation of Fis and NHP6A from DNA Detected via Single-Molecule Mechanical Response.

Author information

1
Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA.
2
Department of Physics and Astronomy, Northwestern University, Evanston, IL 60208, USA.
3
Department of Materials Science and Engineering, Northwestern University, Evanston, IL 60208, USA; Department of Chemical and Biomolecular Engineering, University of Illinois, Urbana, IL 61801, USA.
4
Department of Materials Science and Engineering, Northwestern University, Evanston, IL 60208, USA.
5
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
6
Department of Biological Chemistry, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095-1737, USA.
7
Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA; Department of Physics and Astronomy, Northwestern University, Evanston, IL 60208, USA. Electronic address: john-marko@northwestern.edu.

Abstract

The rate of dissociation of a DNA-protein complex is often considered to be a property of that complex, without dependence on other nearby molecules in solution. We study the kinetics of dissociation of the abundant Escherichia coli nucleoid protein Fis from DNA, using a single-molecule mechanics assay. The rate of Fis dissociation from DNA is strongly dependent on the solution concentration of DNA. The off-rate (k(off)) of Fis from DNA shows an initially linear dependence on solution DNA concentration, characterized by an exchange rate of k(ex)≈9×10(-4) (ng/μl)(-1) s(-1) for 100 mM univalent salt buffer, with a very small off-rate at zero DNA concentration. The off-rate saturates at approximately k(off,max)≈8×10(-3) s(-1) for DNA concentrations above ≈20 ng/μl. This exchange reaction depends mainly on DNA concentration with little dependence on the length of the DNA molecules in solution or on binding affinity, but this does increase with increasing salt concentration. We also show data for the yeast HMGB protein NHP6A showing a similar DNA-concentration-dependent dissociation effect, with faster rates suggesting generally weaker DNA binding by NHP6A relative to Fis. Our results are well described by a model with an intermediate partially dissociated state where the protein is susceptible to being captured by a second DNA segment, in the manner of "direct transfer" reactions studied for other DNA-binding proteins. This type of dissociation pathway may be important to protein-DNA binding kinetics in vivo where DNA concentrations are large.

KEYWORDS:

affinity; binding kinetics; biomolecule interactions; off-rate; unbinding

PMID:
26220077
PMCID:
PMC4579036
DOI:
10.1016/j.jmb.2015.07.015
[Indexed for MEDLINE]
Free PMC Article

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