title: DNA-Segment-Facilitated Dissociation of Fis and NHP6A from DNA Detected via Single-Molecule Mechanical Response.
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.
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA.
authors.forename: Rebecca D
authors.initials: RD
authors.lastname: Giuntoli
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA.
authors.forename: Nora B
authors.initials: NB
authors.lastname: Linzer
authors.affiliation: Department of Physics and Astronomy, Northwestern University, Evanston, IL 60208, USA.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Banigan
authors.affiliation: 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.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Sing
authors.affiliation: Department of Materials Science and Engineering, Northwestern University, Evanston, IL 60208, USA.
authors.forename: Monica Olvera
authors.initials: MO
authors.lastname: de la Cruz
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: John S
authors.initials: JS
authors.lastname: Graham
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095-1737, USA.
authors.forename: Reid C
authors.initials: RC
authors.lastname: Johnson
authors.affiliation: 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.
authors.forename: John F
authors.initials: JF
authors.lastname: Marko
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D035021:Factor For Inversion Stimulation Protein / Q000378:metabolism*; D024001:HMGN Proteins / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D012492:Salts / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; C501034:Fis protein, E coli; D024001:HMGN Proteins; C086471:NHP6A protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012492:Salts; D004247:DNA
keywords: affinity; binding kinetics; biomolecule interactions; off-rate; unbinding