Trivalent counterion condensation on DNA measured by pulse gel electrophoresis

Pulse gel electrophoresis was used to measure the reduction of mobilities of λ-DNA-Hind III fragments ranging from 23.130 to 2.027 kilobase pairs in Tris borate buffer solutions mixed with either hexammine cobalt(III), or spermidine³⁺ trivalent counterions that competed with Tris⁺ and Na⁺ for binding onto polyion DNA. The normalized titration curves of mobility were well fit by the two-variable counterion condensation theory. The agreement between measured charge fraction neutralized and counterion condensation prediction was good over a relatively wide range of trivalent cation concentrations at several solution conditions (pH, ionic strength). The effect of ionic strength, trivalent cation concentration, counterion structure, and DNA length on the binding were discussed based on the experimental measurements and the counterion condensation theory. © 1996 John Wiley & Sons, Inc.     

Calculations on the effect of methylation on the electrostatic stability of the B- and Z-conformers of DNA

The three-dimensional Poisson–Boltzmann equation for the distribution of counterion charge density around double-helical DNA has been solved for solutions of .01M, .10M, and .20M monovalent salt. The polymers, poly[d(CpGp)] and poly[d(m⁵CpGp)], were studied in the B- and the Z-conformations. The effect of methylation on the relative stabilities of these conformers in solutions of different ionic strengths is known to favor the Z-form. Accumulation of charge density around the B- and the Z-conformers is compared in detail. The relative electrostatic stabilities of the B- and Z-conformers in .01M, .10M, and .20M solutions are compared and discussed in terms of the ion–DNA interactions and the self-energy of the structured ionic environment. The ion–DNA interaction energies, termed “phosphate screening,” monotonically decrease with ionic strength and are consistent with a B-to-Z conformation change induced in either polymer by increased electrolyte concentration. However, these calculated energies alone do not account for the fact that the ionic strength at the midpoint of the transition of the methylated polymer is substantially lower than that of its unmethylated analogues. The phosphate screening effect is counterbalanced by changes in the self-energy required for the creation of the structured counterion environment. This self-energy of the electrolyte environment monotonically increases with ionic strength. Methylation-induced shifts in the overall conformational equilibria depend on the relative changes of these competing effects. Increasing salt concentration is calcualted to favor the Z-conformer. The effect of methylation, lowering the ionic strength of the transition midpoint, is proposed to originate in minor structural changes in the Z-form of the polymer, making the groove more accessible to counterions in the G(3′ – 5′)C region. This allows a redistribution of counterion density and a lowering of the self-energy of the ionic environment, conferring added stability to the Z-conformation, as indicated by calculations of relative entropies. The experimentally observed temperature dependence of the B-to-Z transition, however, cannot be explained without assuming the release of bound water. Maps of the calculated three-dimensional structure at the counterion distribution near the surface of these molecules in both the B- and the Z-forms are also presented.     

Electrophoretic Mobility of DNA in Solutions of High Ionic Strength

The free-solution mobilities of small single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) have been measured by capillary electrophoresis in solutions containing 0.01–1.0 M sodium acetate. The mobility of dsDNA is greater than that of ssDNA at all ionic strengths because of the greater charge density of dsDNA. The mobilities of both ssDNA and dsDNA decrease with increasing ionic strength until approaching plateau values at ionic strengths greater than ∼0.6 M. Hence, ssDNA and dsDNA appear to interact in a similar manner with the ions in the background electrolyte. For dsDNA, the mobilities predicted by the Manning electrophoresis equation are reasonably close to the observed mobilities, using no adjustable parameters, if the average distance between phosphate residues (the b parameter) is taken to be 1.7 Å. For ssDNA, the predicted mobilities are close to the observed mobilities at ionic strengths ≤0.01 M if the b-value is taken to be 4.1 Å. The predicted and observed mobilities diverge strongly at higher ionic strengths unless the b-value is reduced significantly. The results suggest that ssDNA strands exist as an ensemble of relatively compact conformations at high ionic strengths, with b-values corresponding to the relatively short phosphate-phosphate distances through space.     

Growth of linear charged micelles

The electrostatics of micellar growth is reviewed and extended for solutions containing excess salt. In dilute solution the expansion of a linear micelle with increasing salt concentration is explained for a wide range of ionic strength. When the micellar charge density is very high, counterions condense nonuniformly onto the micellar rod. In that case the micelle may contract upon the addition of salt. In semidilute solutions the excluded-volume effect is an additional factor complicating the ionic strength dependence of micellar growth.     

Electrostatic effects on the stability of condensed DNA in the presence of divalent cations.

Cylindrical cell model Poisson-Boltzmann (P-B) calculations are used to evaluate the electrostatic contributions to the relative stability of various DNA conformations (A, B, C, Z, and single-stranded (ss) with charge spacings of 3.38 and 4.2 A) as a function of interhelix distance in a concentrated solution of divalent cations. The divalent ion concentration was set at 100 mM, to compare with our earlier reports of spectroscopic and calorimetric experiments, which demonstrate substantial disruption of B-DNA geometry. Monovalent cations neutralize the DNA phosphates in two ways, corresponding to different experimental situations: 1) There is no significant contribution to the ionic strength from the neutralizing cations, corresponding to DNA condensation from dilute solution and to osmotic stress experiments in which DNA segments are brought into close proximity to each other in the presence of a large excess of buffer. 2) The solution is uniformly concentrated in DNA, so that the neutralizing cations add significantly to those in the buffer at close DNA packing. In case 1), conformations with lower charge density (Z and ssDNA) have markedly lower electrostatic free energies than B-DNA as the DNA molecules approach closely, due largely to ionic entropy. If the divalent cations bind preferentially to single-stranded DNA or a distorted form of B-DNA, as is the case with transition metals, the base pairing and stacking free energies that stabilize the double helix against electrostatic denaturation may be overcome. Strong binding to the bases is favored by the high concentration of divalent cations at the DNA surface arising from the large negative surface potential; the surface concentration increases sharply as the interhelical distance decreases. In case 2), the concentration of neutralizing monovalent cations becomes very large and the electrostatic free energy difference between secondary structures becomes small as the interhelical spacing decreases. Such high ionic concentrations will be expected to modify the stability of DNA by changing water activity as well as by screening electrostatic interactions. This may be the root of the decreased thermal stability of DNA in the presence of high concentrations of magnesium ions.     

Evaluating the Effect of Ionic Strength on Duplex Stability for PNA Having Negatively or Positively Charged Side Chains

The enhanced thermodynamic stability of PNA:DNA and PNA:RNA duplexes compared with DNA:DNA and DNA:RNA duplexes has been attributed in part to the lack of electrostatic repulsion between the uncharged PNA backbone and negatively charged DNA or RNA backbone. However, there are no previously reported studies that systematically evaluate the effect of ionic strength on duplex stability for PNA having a charged backbone. Here we investigate the role of charge repulsion in PNA binding by synthesizing PNA strands having negatively or positively charged side chains, then measuring their duplex stability with DNA or RNA at varying salt concentrations. At low salt concentrations, positively charged PNA binds more strongly to DNA and RNA than does negatively charged PNA. However, at medium to high salt concentrations, this trend is reversed, and negatively charged PNA shows higher affinity for DNA and RNA than does positively charged PNA. These results show that charge screening by counterions in solution enables negatively charged side chains to be incorporated into the PNA backbone without reducing duplex stability with DNA and RNA. This research provides new insight into the role of electrostatics in PNA binding, and demonstrates that introduction of negatively charged side chains is not significantly detrimental to PNA binding affinity at physiological ionic strength. The ability to incorporate negative charge without sacrificing binding affinity is anticipated to enable the development of PNA therapeutics that take advantage of both the inherent benefits of PNA and the multitude of charge-based delivery technologies currently being developed for DNA and RNA.     

Brownian dynamics simulations of probe and self-diffusion in concentrated protein and DNA solutions.

We have developed a Brownian dynamics algorithm for simulating probe and self-diffusion in concentrated solutions of DNA and protein. In these simulations, proteins are represented as spheres with radii given by their hydrodynamic radii, while DNA is modeled as a wormlike chain of hydrodynamically equivalent spherical frictional elements. The molecular interaction potentials employed by the program allow for intramolecular stretching and bending motions of the DNA chains, short-range Lennard-Jones interactions, and long-range electrostatic interactions. To test the program, we have carried out simulations of bovine serum albumin (BSA) probe diffusion and DNA self-diffusion in solutions of short-chain DNA as a function of both DNA concentration and solution ionic strength. In addition, we report on simulations of BSA self-diffusion as a function of BSA concentration and ionic strength. Based on a comparison to available experimental data, we find that our simulations accurately predict these transport properties under conditions of physiological salt concentration and predict the stronger concentration dependence observed at lower salt concentrations. These results are discussed in light of the nature of the intermolecular interactions in such systems and the approximations and limitations of the simulation algorithm.     

Interaction of tobacco mosaic virus and tobacco mosaic virus protein with bovine serum albumin.

Bovine serum albumin (BSA) causes tobacco mosaic virus (TMV) to crystallize at pH values where both have negative charges. The amount of albumin required to precipitate the virus varies inversely with ionic strength of added electrolyte. At pH values above 5, the precipitating power is greatest when BSA has the maximum total, positive plus negative, charge. Unlike early stages of the crystallization of TMV in ammonium sulfate-phosphate solutions, which can be reversed by lowering the temperature, the precipitation of TMV by BSA is not readily reversed by changes in temperature. The logarithm of the apparent solubility of TMV in BSA solutions, at constant ionic strength of added electrolyte, decreases linearly with increasing BSA concentration. This result and the correlation of precipitating power with total BSA charge suggest that BSA acts in the manner of a salting-out agent. The effect of BSA on the reversible entropy-driven polymerization of TMV protein (TMVP) depends on BSA concentration, pH, and ionic strength. In general, BSA promotes TMVP polymerization, and this effect increases with increasing BSA concentrations. The effect is larger at pH 6.5 than at pH 6. Even though increasing ionic strength promotes polymerization of TMVP in absence of BSA, the effect of increasing ionic strength from 0.08 to 0.18 at pH 6.5 decreases the polymerization-promoting effect of BSA. Likewise, the presence of BSA decreases the polymerization-promoting effect of ionic strength. The polymerization-promoting effect of BSA can be interpreted in terms of a process akin to salting-out. The mutual suppression of the polymerization-promoting effects of BSA and of electrolytes by each other can be partially explained in terms of salting-in of BSA.     

Effect of ion binding on protein transport through ultrafiltration membranes

Electrostatic interactions can have a significant impact on protein transmission through semipermeable membranes. Experimental data for the transport of bovine serum albumin (BSA) through a polyethersulfone ultrafiltration membrane were obtained in different salt solutions over a range of pH and salt concentrations. Net BSA charge under the same conditions was evaluated from mobility data measured by capillary electrophoresis. The results show that specific ionic composition, in addition to solution pH and ionic strength, can strongly affect the rate of protein transport through semipermeable ultrafiltration membranes. The effects of different ions on BSA sieving are due primarily to differences in ion binding to the protein, which leads to significant differences in the net protein charge at a given pH and ionic strength. This effect could be described in terms of an effective protein radius, which accounts for the electrostatic exclusion of the charged protein from the membrane pores. These results provide important insights into the nature of the electrostatic interactions in membrane systems.     

DNA Strands Attached Inside Single Conical Nanopores: Ionic Pore Characteristics and Insight into DNA Biophysics

Single nanopores attract a great deal of scientific interest as a basis for biosensors and as a system to study the interactions and behavior of molecules in a confined volume. Tuning the geometry and surface chemistry of nanopores helps create devices that control transport of ions and molecules in solution. Here, we present single conically shaped nanopores whose narrow opening of 8 or 12 nm is modified with single-stranded DNA molecules. We find that the DNA occludes the narrow opening of nanopores and that the blockade extent decreases with the ionic strength of the background electrolyte. The results are explained by the ionic strength dependence of the persistence length of DNA. At low KCl concentrations (10 mM) the molecules assume an extended and rigid conformation, thereby blocking the pore lumen and reducing the flow of ionic current to a greater extent than compacted DNA at high salt concentrations. Attaching flexible polymers to the pore walls hence creates a system with tunable opening diameters in order to regulate transport of both neutral and charged species.     

Transmission of poly(dT60) single-stranded DNA through polycarbonate track-etched ultrafiltration membranes

The objective of this study was to examine membrane filtration of a single stranded DNA (ssDNA) with 60 thymine nucleotides, and to elucidate the variables controlling its transmission across track-etched porous membranes. Dead end filtration measurements were performed using different pore size membranes (10, 15, and 30 nm) at different transmembrane pressures in solutions with ionic strength ranging from 0 to 1000 mM NaCl. The diffusivity of the ssDNA was determined using fluorescence recovery after photobleaching, yielding hydrodynamic radii ranging from 1.6 to 2.8 nm, with values decreasing with increasing solution ionic strength. Despite the small ssDNA/membrane pore size, nearly 100% rejection was observed for measurements performed with the 10 and 15 nm pore size membranes under low-ionic strength conditions. These high rejections can be attributed to strong repulsive electrostatic ssDNA-membrane interactions. With increasing ionic strength, electrostatic interactions as well as the effective size of the ssDNA decreases and the flexibility of the ssDNA increases, leading to a reduction in ssDNA rejection. A design of experiments approach was used to plan filtration experiments that adequately covered the variable space with a manageable number of experiments. The results yielded an empirical expression relating ssDNA rejection to pore size, solution ionic strength and transmembrane pressure. There was evidence of flow induced elongation at high-transmembrane pressures in the 30 nm pore size membranes, but not in the smaller pore size membranes. These results are consistent with critical flux estimates developed using a free draining model for the ssDNA.     

Do zwitterions contribute to the ionic strength of a solution?

Capillary electrophoresis has been used to determine whether zwitterions contribute to the ionic strength of a solution, by measuring the mobility of a double-stranded DNA oligomer in cacodylate-buffered solutions containing various concentrations of the ionic salt tetraethylammonium chloride (TEA(+)Cl(-)) or the zwitterion tricine(+/-). The mobility of the DNA decreased as the square root of ionic strength, as expected from the Debye-Hückel-Onsager theory of electrophoresis, when TEA(+)Cl(-) was added to the buffer. However, the mobility was independent of the concentration of added tricine(+/-). Hence, zwitterions do not contribute to the ionic strength of a solution.     

Conformational changes modulate the activity of human RAD51 protein

Homologous recombination provides a major pathway for the repair of DNA double-strand breaks in mammalian cells. Defects in homologous recombination can lead to high levels of chromosomal translocations or deletions, which may promote cell transformation and cancer development. A key component of this process is RAD51. In comparison to RecA, the bacterial homologue, human RAD51 protein exhibits low-level strand-exchange activity in vitro. This activity can, however, be stimulated by the presence of high salt. Here, we have investigated the mechanistic basis for this stimulation. We show that high ionic strength favours the co-aggregation of RAD51-single-stranded DNA (ssDNA) nucleoprotein filaments with naked duplex DNA, to form a complex in which the search for homologous sequences takes place. High ionic strength allows differential binding of RAD51 to ssDNA and double-stranded DNA (dsDNA), such that ssDNA-RAD51 interactions are unaffected, whereas those between RAD51 and dsDNA are destabilized. Most importantly, high salt induces a conformational change in RAD51, leading to the formation of extended nucleoprotein filaments on ssDNA. These extended filaments mimic the active form of the Escherichia coli RecA-ssDNA filament that exhibits efficient strand-exchange activity.     

Specificity of binding of single-stranded DNA-binding protein to its target

Single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA (ssDNA) and participate in all genetic processes involving ssDNA, such as replication, recombination, and repair. Here we applied atomic force microscopy to directly image SSB-DNA complexes under various conditions. We used the hybrid DNA construct methodology in which the ssDNA segment is conjugated to the DNA duplex. The duplex part of the construct plays the role of a marker, allowing unambiguous identification of specific and nonspecific SSB-DNA complexes. We designed hybrid DNA substrates with 5'- and 3'-ssDNA termini to clarify the role of ssDNA polarity on SSB loading. The hybrid substrates, in which two duplexes are connected with ssDNA, were the models for gapped DNA substrates. We demonstrated that Escherichia coli SSB binds to ssDNA ends and internal ssDNA regions with the same efficiency. However, the specific recognition by ssDNA requires the presence of Mg(2+) cations or a high ionic strength. In the absence of Mg(2+) cations and under low-salt conditions, the protein is capable of binding DNA duplexes. In addition, the number of interprotein interactions increases, resulting in the formation of clusters on double-stranded DNA. This finding suggests that the protein adopts different conformations depending on ionic strength, and specific recognition of ssDNA by SSB requires a high ionic strength or the presence of Mg(2+) cations.     

Histone H1--DNA interaction. On the mechanism of DNA strands crosslinking by histone H1.

Crosslinking of DNA fibers by histone H1 or phosphorylated on Ser-37 histone H1, and by the individual fragments of the H1 polypeptide chain was studied by the method of turbidimetry. The dependence of the turbidity of DNA-protein complexes on the ionic strength in solution suggests that the condensation of H1.DNA complexes in vitro is apparently due to both specific histone-DNA interactions with the contribution of hydrogen and/or hydrophobic bonds and the formation of polycationic "bridges" fastening the DNA fibers. The effectiveness of the condensation is postulated to be a function of a proportion between the two mechanisms which in turn can be controlled by slight changes in ionic surroundings. The sharp dependence of shrinkage of H1.DNA complexes on ionic strength at "physiological" salt concentrations could provide a mechanism to regulate density and consequently the total activity of chromatin in the cell nuclei. The phosphorylation of histone H1 on Ser-37 by a specific histone kinase does not noticeably affect the pattern of DNA crosslinking by the H1.     

Effect of ionic strength of a solution on the protonation of DNA molecule

The influence of the ionic strength of solution on the DNA molecule protonation was studied by means of circular dichroism (CD), spectrophotometric and potentiometric titration methods over a wide range of the supporting electrolyte concentrations [( NaCl] = 0.0005 divided by 4 M). Consideration of the obtained CD spectra shown that the acidation of the solution induces two cooperative structural transitions in the double stranded DNA molecule in the pre-denaturation pH region. Further decrease in the solution pH results in acidic melting of the DNA molecule. Analysis of the potentiometric data shows that diluted DNA solutions exhibit marked buffer capacity at pH greater than 4.2. A concept of local pH dependent on the electrostatic potential in the vicinity of the polyion was used for interpreting the obtained results. A phase diagram, which describes the polymorphic transformations of the protonated macromolecule, was constructed in terms of pHloc and -log[Na+]. Consideration of this phase diagram allows to hypothesize that: 1) in the neutral diluted DNA solution with a very low supporting electrolyte content the macromolecule exists in a polymorphic state; 2) at [NaCl] greater than or equal to 0.001 M the acid-base equilibrium in the DNA molecule is invariant in respect to the ionic strength of the solution.     

Mechanism of RecO recruitment to DNA by single-stranded DNA binding protein

RecO is a recombination mediator protein (RMP) important for homologous recombination, replication repair and DNA annealing in bacteria. In all pathways, the single-stranded (ss) DNA binding protein, SSB, plays an inhibitory role by protecting ssDNA from annealing and recombinase binding. Conversely, SSB may stimulate each reaction through direct interaction with RecO. We present a crystal structure of Escherichia coli RecO bound to the conserved SSB C-terminus (SSB-Ct). SSB-Ct binds the hydrophobic pocket of RecO in a conformation similar to that observed in the ExoI/SSB-Ct complex. Hydrophobic interactions facilitate binding of SSB-Ct to RecO and RecO/RecR complex in both low and moderate ionic strength solutions. In contrast, RecO interaction with DNA is inhibited by an elevated salt concentration. The SSB mutant lacking SSB-Ct also inhibits RecO-mediated DNA annealing activity in a salt-dependent manner. Neither RecO nor RecOR dissociates SSB from ssDNA. Therefore, in E. coli, SSB recruits RMPs to ssDNA through SSB-Ct, and RMPs are likely to alter the conformation of SSB-bound ssDNA without SSB dissociation to initiate annealing or recombination. Intriguingly, Deinococcus radiodurans RecO does not bind SSB-Ct and weakly interacts with the peptide in the presence of RecR, suggesting the diverse mechanisms of DNA repair pathways mediated by RecO in different organisms.     

Flexible polymer-induced condensation and bundle formation of DNA and F-actin filaments

A simple semi-empirical theory is developed for the ionic strength dependence of the flexible polymer-induced condensation of semiflexible polyelectrolytes such as DNA and F-actin filaments. Critical concentrations of flexible polymer needed for condensation are calculated by comparing the free energies of inserting the semiflexible polyelectrolytes in a solution of flexible polymers, respectively, in their free state, and in their condensed state. Predictions of the theory are compared to experimental data on the condensation of DNA and F-actin filaments induced by the flexible polymer poly(ethylene oxide). The theory also predicts that reentrant decollapse is possible at low ionic strength and high concentrations of flexible polymer, as observed for DNA.     

Intrinsic electric fields and proton diffusion in immobilized protein membranes. Effects of electrolytes and buffers.

We present a theory for proton diffusion through an immobilized protein membrane perfused with an electrolyte and a buffer. Using a Nernst-Planck equation for each species and assuming local charge neutrality, we obtain two coupled nonlinear diffusion equations with new diffusion coefficients dependent on the concentration of all species, the diffusion constants or mobilities of the buffers and salts, the pH-derivative of the titration curves of the mobile buffer and the immobilized protein, and the derivative with respect to ionic strength of the protein titration curve. Transient time scales are locally pH-dependent because of protonation-deprotonation reactions with the fixed protein and are ionic strength-dependent because salts provide charge carriers to shield internal electric fields. Intrinsic electric fields arise proportional to the gradient of an "effective" charge concentration. The field may reverse locally if buffer concentrations are large (greater to or equal to 0.1 M) and if the diffusivity of the electrolyte species is sufficiently small. The "ideal" electrolyte case (where each species has the same diffusivity) reduces to a simple form. We apply these theoretical considerations to membranes composed of papain and bovine serum albumin (BSA) and show that intrinsic electric fields greatly enhance the mobility of protons when the ionic strength of the salts is smaller than 0.1 M. These results are consistent with experiments where pH changes are observed to depend strongly on buffer, salt, and proton concentrations in baths adjacent to the membranes.     

Probing the electrostatic shielding of DNA with capillary electrophoresis

The free solution mobility of a 20-bp double-stranded DNA oligomer has been measured in diethylmalonate (DM) and Tris-acetate buffers, with and without added NaCl or TrisCl. DM buffers have the advantage that the buffering ion is anionic, so the cation composition in the solution can be varied at will. The results indicate that the free solution mobility of DNA decreases linearly with the logarithm of ionic strength when the ionic strength is increased by increasing the buffer concentration. The mobility also decreases linearly with the logarithm of ionic strength when NaCl is added to NaDM buffer or TrisCl is added to TrisDM buffer. Nonlinear effects are observed if the counterion in the added salt differs from the counterion in the buffer. The dependence of the mobility on ionic strength cannot be predicted using the Henry, Debye-Hückel-Onsager, or Pitts equations for electrophoresis. However, the mobilities observed in all buffer and buffer/salt solutions can be predicted within approximately 20% by the Manning equation for electrophoresis, using no adjustable parameters. The results suggest that the electrostatic shielding of DNA is determined not only by the relative concentrations of the various ions in the solution, but also by their equivalent conductivities.