HU is one of the most abundant proteins in bacterial chromosomes and participates in nucleoid compaction and gene regulation. We report experiments using DNA stretching that study the dependence of DNA condensation by HU on force, salt and HU concentration. Previous experiments at sub-physiological salt levels revealed that low concentrations of HU could compact DNA, whereas larger HU concentrations formed a DNA-stiffening complex. Here we report that this bimodal binding behavior depends sensitively on salt concentration. Only the compaction mode was observed for 150 mM and higher NaCl levels, i.e. for physiological salt concentrations. Similar results were obtained for the more physiological salt K-glutamate. Real-time studies of dissociation kinetics revealed that HU unbound slowly (minutes to hours under the conditions studied) but completely for salt concentrations at or above 100 mM NaCl; the lifetime of HU complexes was observed to increase with the HU concentration at which the complexes were formed, and to decrease with salt concentration. Higher salt levels of 300 mM NaCl completely eliminated observable HU binding to DNA. Finally, we observed that the dissociation kinetics depend on force applied to the DNA: increased applied force in the sub-piconewton range accelerates dissociation, suggesting a mechanism for DNA tension to regulate chromosome structure and gene expression.

Unlabelled

Off-rates of proteins from the DNA double helix are widely considered to be dependent only on the interactions inside the initially bound protein-DNA complex and not on the concentration of nearby molecules. However, a number of recent single-DNA experiments have shown off-rates that depend on solution protein concentration, or "facilitated dissociation." Here, we demonstrate that this effect occurs for the major Escherichia coli nucleoid protein Fis on isolated bacterial chromosomes. We isolated E. coli nucleoids and showed that dissociation of green fluorescent protein (GFP)-Fis is controlled by solution Fis concentration and exhibits an "exchange" rate constant (kexch) of ≈10(4) M(-1) s(-1), comparable to the rate observed in single-DNA experiments. We also show that this effect is strongly salt dependent. Our results establish that facilitated dissociation can be observed in vitro on chromosomes assembled in vivo

Importance

Bacteria are important model systems for the study of gene regulation and chromosome dynamics, both of which fundamentally depend on the kinetics of binding and unbinding of proteins to DNA. In experiments on isolated E. coli chromosomes, this study showed that the prolific transcription factor and chromosome packaging protein Fis displays a strong dependence of its off-rate from the bacterial chromosome on Fis concentration, similar to that observed in in vitro experiments. Therefore, the free cellular DNA-binding protein concentration can strongly affect lifetimes of proteins bound to the chromosome and must be taken into account in quantitative considerations of gene regulation. These results have particularly profound implications for transcription factors where DNA binding lifetimes can be a critical determinant of regulatory function.

Determining numbers of proteins bound to large DNAs is important for understanding their chromosomal functions. Protein numbers may be affected by physical factors such as mechanical forces generated in DNA, e.g. by transcription or replication. We performed single-DNA stretching experiments with bacterial nucleoid proteins HU and Fis, verifying that the force-extension measurements were in thermodynamic equilibrium. We, therefore, could use a thermodynamic Maxwell relation to deduce the change of protein number on a single DNA due to varied force. For the binding of both HU and Fis under conditions studied, numbers of bound proteins decreased as force was increased. Our experiments showed that most of the bound HU proteins were driven off the DNA at 6.3 pN for HU concentrations lower than 150 nM; our HU data were fit well by a statistical-mechanical model of protein-induced bending of DNA. In contrast, a significant amount of Fis proteins could not be forced off the DNA at forces up to 12 pN and Fis concentrations up to 20 nM. This thermodynamic approach may be applied to measure changes in numbers of a wide variety of molecules bound to DNA or other polymers. Force-dependent DNA binding by proteins suggests mechano-chemical mechanisms for gene regulation.

The multistep kinetics through which DNA-binding proteins bind their targets are heavily studied, but relatively little attention has been paid to proteins leaving the double helix. Using single-DNA stretching and fluorescence detection, we find that sequence-neutral DNA-binding proteins Fis, HU and NHP6A readily exchange with themselves and with each other. In experiments focused on the Escherichia coli nucleoid-associated protein Fis, only a small fraction of protein bound to DNA spontaneously dissociates into protein-free solution. However, if Fis is present in solution, we find that a concentration-dependent exchange reaction occurs which turns over the bound protein, with a rate of k(exch) = 6 × 10(4) M(-1)s(-1). The bacterial DNA-binding protein HU and the yeast HMGB protein NHP6A display the same phenomenon of protein in solution accelerating dissociation of previously bound labeled proteins as exchange occurs. Thus, solvated proteins can play a key role in facilitating removal and renewal of proteins bound to the double helix, an effect that likely plays a major role in promoting the turnover of proteins bound to DNA in vivo and, therefore, in controlling the dynamics of gene regulation.

Histone H1 binds to linker DNA between nucleosomes, but the dynamics and biological ramifications of this interaction remain poorly understood. We performed single-molecule experiments using magnetic tweezers to determine the effects of H1 on naked DNA in buffer or during chromatin assembly in Xenopus egg extracts. In buffer, nanomolar concentrations of H1 induce bending and looping of naked DNA at stretching forces below 0.6 pN, effects that can be reversed with 2.7-pN force or in 200 mM monovalent salt concentrations. Consecutive tens-of-nanometer bending events suggest that H1 binds to naked DNA in buffer at high stoichiometries. In egg extracts, single DNA molecules assemble into nucleosomes and undergo rapid compaction. Histone H1 at endogenous physiological concentrations increases the DNA compaction rate during chromatin assembly under 2-pN force and decreases it during disassembly under 5-pN force. In egg cytoplasm, histone H1 protects sperm nuclei undergoing genome-wide decondensation and chromatin assembly from becoming abnormally stretched or fragmented due to astral microtubule pulling forces. These results reveal functional ramifications of H1 binding to DNA at the single-molecule level and suggest an important physiological role for H1 in compacting DNA under force and during chromatin assembly.

DNA strand exchange by serine recombinases has been proposed to occur by a large-scale rotation of halves of the recombinase tetramer. Here we provide the first direct physical evidence for the subunit rotation mechanism for the Hin serine invertase. Single-DNA looping assays using an activated mutant (Hin-H107Y) reveal specific synapses between two hix sites. Two-DNA "braiding" experiments, where separate DNA molecules carrying a single hix are interwound, show that Hin-H107Y cleaves both hix sites and mediates multi-step rotational relaxation of the interwinding. The variable numbers of rotations in the DNA braid experiments are in accord with data from bulk experiments that follow DNA topological changes accompanying recombination by the hyperactive enzyme. The relatively slow Hin rotation rates, combined with pauses, indicate considerable rotary friction between synapsed subunit pairs. A rotational pausing mechanism intrinsic to serine recombinases is likely to be crucial for DNA ligation and for preventing deleterious DNA rearrangements.

The binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits that TF off rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key Escherichia coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate [Formula: see text], establishing that FD of Fis occurs at the single-binding site level, and we find that the off rate saturates at large Fis concentrations in solution. Although spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that FD depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF with structure that differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those that we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap.

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.

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis.