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 E. 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 (koff) of Fis from DNA shows an initially linear dependence on solution DNA concentration, characterized by an exchange rate of kex ≈ 9×10−4 s−1 (ng/μl)−1 for 100 mM univalent salt buffer, with a very small off-rate at zero DNA concentration. The off-rate saturates at approximately koff,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 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.

Single-Molecule Interactions of a Monoclonal Anti-DNA Antibody with DNA

“…However, the off-rate obtained is within the range reported for DNA-antibody or DNA-protein interactions, for example, the k off of D17.4 DNA aptamer/IgE complex was calculated to be (2.92 ± 0.18) × 10 −3 s −1 or (2.85 ± 0.14) × 10 −3 s −1 depending on the environmental conditions [57, 58]. Giuntoli et al [59] showed that the rate of dissociation from DNA of the Escherichia coli nucleoid protein Fis was approximately k off ~ 8 × 10 −3 s −1 .…”

Effects of electrostatic interactions on ligand dissociation kinetics

“…3 at low salt concentrations are larger due to the computational cost of producing data when the electrostatic interactions are stronger. In experiments where protein unbinding from single binding sites was studied, a high concentration of free ligands (proteins) weakened the salt-dependence of the offrates [20,22,27]. In our simulations, we also investigated the effects of the electrostatic scheme on FD by introducing a fixed concentration of unbound ligands in the solution (see Fig.…”

“…Besides univalent salt ions, an excess amount of free ligands in solution can also facilitate dissociation of a bound ligand by decreasing the lifetime of the ligand on the binding site [19][20][21][22][23][24][25][26][27]. The free ligands can be identical to the initially bound ligand [21,22,[27][28][29][30].…”

“…The free ligands can be identical to the initially bound ligand [21,22,[27][28][29][30]. However, different ligand molecules [27,30,31] or even nucleic acid fragments [20] can lead to the facilitated dissociation (FD). According to the proposed molecular mechanism for FD, a free ligand binds to an already occupied binding site and destabilizes the complex by forming a ternary complex (i.e., two ligands on the same site) [19,23,27].…”

Molecular Mechanism of Facilitated Dissociation of Fis Protein from DNA

“…6 Dissociation can be facilitated not only by the Fis molecule itself but also by the action of the bacterial protein HU, the yeast HMGB protein NHP6A, 6 as well as other DNA segments. 7 Facilitated dissociation has been observed experimentally for a wide variety of genetic systems, including DNA polymerase exchange in phage T7 replisome during DNA replication, 8 dissociation of metal-dependent transcription factor CueR by apo-CueR, 9 and the facilitated dissociation of eukaryotic High-Mobility Group B (HMGB) protein by itself. 10 In addition, facilitated dissociation has also be seen in single-stranded DNA binding, 11 highlighting its widespread importance across all types of regulatory processes in cells.…”