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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/?l)(-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/?l. 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.

SUBMITTER: Giuntoli RD

PROVIDER: S-EPMC4579036 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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

Giuntoli Rebecca D RD Linzer Nora B NB Banigan Edward J EJ Sing Charles E CE de la Cruz Monica Olvera MO Graham John S JS Johnson Reid C RC Marko John F JF

Journal of molecular biology 20150726 19

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 concen ...[more]

PMID: 26220077

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Project description:Cellular structures are continually subjected to forces, which may serve as mechanical signals for cells through their effects on biomolecule interaction kinetics. Typically, molecular complexes interact via "slip bonds," so applied forces accelerate off rates by reducing transition energy barriers. However, biomolecules with multiple dissociation pathways may have considerably more complicated force dependencies. This is the case for DNA-binding proteins that undergo "facilitated dissociation," in which competitor biomolecules from solution enhance molecular dissociation in a concentration-dependent manner. Using simulations and theory, we develop a generic model that shows that proteins undergoing facilitated dissociation can form an alternative type of molecular bond, known as a "catch bond," for which applied forces suppress protein dissociation. This occurs because the binding by protein competitors responsible for the facilitated dissociation pathway can be inhibited by applied forces. Within the model, we explore how the force dependence of dissociation is regulated by intrinsic factors, including molecular sensitivity to force and binding geometry and the extrinsic factor of competitor protein concentration. We find that catch bonds generically emerge when the force dependence of the facilitated unbinding pathway is stronger than that of the spontaneous unbinding pathway. The sharpness of the transition between slip- and catch-bond kinetics depends on the degree to which the protein bends its DNA substrate. This force-dependent kinetics is broadly regulated by the concentration of competitor biomolecules in solution. Thus, the observed catch bond is mechanistically distinct from other known physiological catch bonds because it requires an extrinsic factor-competitor proteins-rather than a specific intrinsic molecular structure. We hypothesize that this mechanism for regulating force-dependent protein dissociation may be used by cells to modulate protein exchange, regulate transcription, and facilitate diffusive search processes.

Dataset Information

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

Publications

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

Similar Datasets

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