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Short arginine-rich proteins called protamines mediate the near crystalline DNA packaging in most vertebrate sperm cells. Protamines are synthesized during spermiogenesis and condense the paternal genome into a transcriptionally inactive state in late-stage spermatids. Protamines from eutherian mammals, including bulls and humans, also contain multiple cysteine residues that form intra- and interprotamine sulfur-sulfur bonds during the final stages of sperm maturation. Although the cross-linked protamine network is known to stabilize the resulting nucleoprotamine structure, little is known about the role of disulfide bonds on DNA condensation in the mammalian sperm. Using small angle x-ray scattering, we show that isolated bull nuclei achieve slightly lower DNA packing densities compared to salmon nuclei despite salmon protamine lacking cysteine residues. Surprisingly, reduction of the intermolecular sulfur-sulfur bonds of bull protamine results in tighter DNA packing. Complete reduction of the intraprotamine disulfide bonds ultimately leads to decondensation, suggesting that disulfide-mediated secondary structure is also critical for proper protamine function. Lastly, comparison of multiple bull collections showed some to have aberrant x-ray scattering profiles consistent with incorrect disulfide bond formation. Together, these observations shed light on the biological functions of disulfide linkages for in vivo DNA packaging in sperm chromatin.

Releasing the packaged viral DNA into the host cell is an essential process to initiate viral infection. In many double-stranded DNA bacterial viruses and herpesviruses, the tightly packaged genome is hexagonally ordered and stressed in the protein shell, called the capsid. DNA condensed in this state inside viral capsids has been shown to be trapped in a glassy state, with restricted molecular motion in vitro. This limited intracapsid DNA mobility is caused by the sliding friction between closely packaged DNA strands, as a result of the repulsive interactions between the negative charges on the DNA helices. It had been unclear how this rigid crystalline structure of the viral genome rapidly ejects from the capsid, reaching rates of 60,000 bp/s. Through a combination of single-molecule and bulk techniques, we determined how the structure and energy of the encapsidated DNA in phage λ regulates the mobility required for its ejection. Our data show that packaged λ-DNA undergoes a solid-to-fluid–like disordering transition as a function of temperature, resulting locally in less densely packed DNA, reducing DNA–DNA repulsions. This process leads to a significant increase in genome mobility or fluidity, which facilitates genome release at temperatures close to that of viral infection (37 °C), suggesting a remarkable physical adaptation of bacterial viruses to the environment of Escherichia coli cells in a human host.

DNA in the human Herpes simplex virus type 1 (HSV-1) capsid is packaged to a tight density. This leads to tens of atmospheres of internal pressure responsible for the delivery of the herpes genome into the cell nucleus. In this study we show that despite its liquid crystalline state inside the capsid, the DNA is fluid-like which facilitates its ejection into the cell nucleus during infection. We found that the sliding friction between closely packaged DNA strands, caused by interstrand repulsive interactions, is reduced by the ionic environment, mimicking that of epithelial cells and neurons susceptible to herpes infection. However, variations in the ionic conditions corresponding to neuronal activity can restrict DNA mobility in the capsid, making it more solid-like. This can inhibit intranuclear DNA release and interfere with viral replication. In addition, the temperature of the human host (37°C) induces a disordering transition of the encapsidated herpes genome which reduces interstrand interactions and provides genome mobility required for infection.

Protamines are small, highly positively charged peptides used to package DNA at very high densities in sperm nuclei. Tight DNA packing is considered essential for the minimization of DNA damage by mutagens and reactive oxidizing species. A striking and general feature of protamines is the almost exclusive use of arginine over lysine for the positive charge to neutralize DNA. We have investigated whether this preference for arginine might arise from a difference in DNA condensation by arginine and lysine peptides. The forces underlying DNA compaction by arginine, lysine, and ornithine peptides are measured using the osmotic stress technique coupled with X-ray scattering. The equilibrium spacings between DNA helices condensed by lysine and ornithine peptides are significantly larger than the interhelical distances with comparable arginine peptides. The DNA surface-to-surface separation, for example, is some 50% larger with polylysine than with polyarginine. DNA packing by lysine rich peptides in sperm nuclei would allow much greater accessibility to small molecules that could damage DNA. The larger spacing with lysine peptides is caused by both a weaker attraction and a stronger short-range repulsion relative to that of the arginine peptides. A previously proposed model for binding of polyarginine and protamine to DNA provides a convenient framework for understanding the differences between the ability of lysine and arginine peptides to assemble DNA.

The DNA structure in phage capsids is determined by DNA–DNA interactions and bending energy. The effects of repulsive interactions on DNA interaxial distance were previously investigated, but not the effect of DNA bending on its structure in viral capsids. By varying packaged DNA length and through addition of spermine ions, we transform the interaction energy from net repulsive to net attractive. This allowed us to isolate the effect of bending on the resulting DNA structure. We used single particle cryo-electron microscopy reconstruction analysis to determine the interstrand spacing of double-stranded DNA encapsidated in phage λ capsids. The data reveal that stress and packing defects, both resulting from DNA bending in the capsid, are able to induce a long-range phase transition in the encapsidated DNA genome from a hexagonal to a cholesteric packing structure. This structural observation suggests significant changes in genome fluidity as a result of a phase transition affecting the rates of viral DNA ejection and packaging.

Using osmotic stress coupled with X-ray scattering, we have directly examined the salt sensitivity of the intermolecular forces between helices in condensed protamine-DNA arrays. Thermodynamic forces are measured from the dependence of DNA helical interaxial spacings on external salt concentration or the osmotic pressure applied by neutral polymer solutions in equilibrium with the condensed phase. Force curves of salmon protamine-DNA condensates are highly dependent on salt species and concentration, indicating salt binding to protamine-DNA complexes. This dependence of the forces on salt species follows the Hofmeister series for anions. Chaotropic anions bind more tightly to protamine-DNA arrays than kosmotropic anions, thus more greatly disrupting the attractive thermodynamic forces. Variations with cation type are small compared with those observed for anions. Further, osmotic stress is used to estimate the number of ions bound in the condensed phase through a Gibbs-Duhem relationship. We estimate that at equilibrium, ∼1 Br(-) is bound per protamine molecule at 200 mM NaBr concentration. Remarkably, this one bound anion results in a change of ∼12% in the surface-to-surface distance between DNA helices. Potential biological implications of this attractive force salt sensitivity are discussed.

The DNA binding stringency of restriction endonucleases is crucial for their proper function. The x-ray structures of the specific and non-cognate complexes of the restriction nuclease EcoRV are considerably different suggesting significant differences in the hydration and binding free energies. Nonetheless, the majority of studies performed at pH 7.5, optimal for enzymatic activity, have found less than a 10-fold difference between EcoRV binding constants to the specific and nonspecific sequences in the absence of divalent ions. We used a recently developed self-cleavage assay to measure EcoRV-DNA competitive binding and to evaluate the influence of water activity, pH and salt concentration on the binding stringency of the enzyme in the absence of divalent ions. We find the enzyme can readily distinguish specific and nonspecific sequences. The relative specific-nonspecific binding constant increases strongly with increasing neutral solute concentration and with decreasing pH. The difference in number of associated waters between specific and nonspecific DNA-EcoRV complexes is consistent with the differences in the crystal structures. In spite of the large pH dependence of the sequence specificity, the osmotic pressure dependence indicates little change in structure with pH. The large osmotic pressure dependence means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowded environment of the cell. In addition to divalent ions, water activity and pH are key parameters that strongly modulate binding specificity of EcoRV.

Understanding and manipulation of the forces assembling DNA/RNA helices have broad implications for biology, medicine, and physics. One subject of significance is the attractive force between dsDNA mediated by polycations of valence ≥3. Despite extensive studies, the physical origin of the “like-charge attraction” remains unsettled among competing theories. Here we show that triple-strand DNA (tsDNA), a more highly charged helix than dsDNA, is precipitated by alkaline-earth divalent cations that are unable to condense dsDNA. We further show that our observation is general by examining several cations (Mg 2+ , Ba 2+ , and Ca 2+ ) and two distinct tsDNA constructs. Cation-condensed tsDNA forms ordered hexagonal arrays that redissolve upon adding monovalent salts. Forces between tsDNA helices, measured by osmotic stress, follow the form of hydration forces observed with condensed dsDNA. Probing a well-defined system of point-like cations and tsDNAs with more evenly spaced helical charges, the counterintuitive observation that the more highly charged tsDNA (vs. dsDNA) is condensed by cations of lower valence provides new insights into theories of polyelectrolytes and the biological and pathological roles of tsDNA. Cations and tsDNAs also hold promise as a model system for future studies of DNA–DNA interactions and electrostatic interactions in general.

Understanding the strength and specificity of interactions among biologically important macromolecules that control cellular functions requires quantitative knowledge of intermolecular forces. Controlled DNA condensation and assembly are particularly critical for biology, with separate repulsive and attractive intermolecular forces determining the extent of DNA compaction. How these forces depend on the charge of the condensing ion has not been determined, but such knowledge is fundamental for understanding the basis of DNA-DNA interactions. Here, we measure DNA force-distance curves for a homologous set of arginine peptides. All forces are well fit as the sum of two exponentials with 2.4- and 4.8-Å decay lengths. The shorter-decay-length force is always repulsive, with an amplitude that varies slightly with length or charge. The longer-decay-length force varies strongly with cation charge, changing from repulsion with Arg1 to attraction with Arg2. Force curves for a series of homologous polyamines and the heterogeneous protein protamine are quite similar, demonstrating the universality of these forces for DNA assembly. Repulsive amplitudes of the shorter-decay-length force are species-dependent but nearly independent of charge within each species. A striking observation was that the attractive force amplitudes for all samples collapse to a single curve, varying linearly with the inverse of the cation charge.

Many specific sequence DNA binding proteins locate their target sequence by first binding to DNA nonspecifically, then by linearly diffusing or hopping along DNA until either the protein dissociates from the DNA or it finds the recognition sequence. We have devised a method for measuring one-dimensional diffusion along DNA based on the ratio of the dissociation rate of protein from DNA fragments containing one specific binding site to the dissociation rate from DNA fragments containing two specific binding sites. Our extensive measurements of dissociation rates and specific-nonspecific relative binding constants of the restriction nuclease EcoRI enable us to determine the diffusion rate of nonspecifically bound protein along the DNA. By varying the distance between the two binding sites, we confirm a linear diffusion mechanism. The sliding rate is relatively insensitive to salt concentration and osmotic pressure, indicating that the protein moves smoothly along the DNA probably following the helical phosphate-sugar backbone of DNA. We calculate a diffusion coefficient for EcoRI of 3 x 10(4) bp(2) s(-)(1) EcoRI is able to diffuse approximately 150 bp, on average, along the DNA in 1 s. This diffusion rate is about 2000-fold slower than the diffusion of free protein in solution. A factor of 40-50 can be accounted for by rotational friction resulting from following the helical path of the DNA backbone. Two possibilities could account for the remaining activation energy: salt bridges between the DNA and the protein are transiently broken, or the water structure at the protein-DNA interface is disrupted as the two surfaces move past each other.

By combining single-molecule magnetic tweezers and osmotic stress on DNA assemblies, we separate attractive and repulsive components of the total intermolecular interaction between multivalent cation condensed DNA. Based on measurements of several different cations, we identify two invariant properties of multivalent cation-mediated DNA interactions: repulsive forces decay exponentially with a 2.3 +/- 0.1 A characteristic decay length and the attractive component of the free energy is always 2.3 +/- 0.2 times larger than the repulsive component of the free energy at force-balance equilibrium. These empirical constraints are not consistent with current theories that attribute DNA-DNA attractions to a correlated lattice of counterions. The empirical constraints are consistent with theories for Debye-Hückel interactions between helical line charges and with the order-parameter formalism for hydration forces. Each of these theories posits exponentially decaying attractions and, if we assume this form, our measurements indicate a cation-independent, 4.8 +/- 0.5 A characteristic decay length for intermolecular attractions between condensed DNA molecules.

We have measured forces generated by multivalent cation-induced DNA condensation using singlemolecule magnetic tweezers. In the presence of cobalt hexammine, spermidine, or spermine, stretched DNA exhibits an abrupt configurational change from extended to condensed. This occurs at a well-defined condensation force that is nearly equal to the condensation free energy per unit length. The multivalent cation concentration dependence for this condensation force gives the apparent number of multivalent cations that bind DNA upon condensation. The measurements show that the lower critical concentration for cobalt hexammine as compared to spermidine is due to a difference in ion binding, not a difference in the electrostatic energy of the condensed state as previously thought. We also show that the resolubilization of condensed DNA can be described using a traditional Manning– Oosawa cation adsorption model, provided that cation–anion pairing at high electrolyte concentrations is taken into account. Neither overcharging nor significant alterations in the condensed state are required to describe the resolubilization of condensed DNA. The same model also describes the spermidine 3+ /Na + phase diagram measured previously.

Releasing the packaged viral DNA into the host cell is an essential process to initiate viral infection. In many double-stranded DNA bacterial viruses and herpesviruses, the tightly packaged genome is hexagonally ordered and stressed in the protein shell, called the capsid. DNA condensed in this state inside viral capsids has been shown to be trapped in a glassy state, with restricted molecular motion in vitro. This limited intracapsid DNA mobility is caused by the sliding friction between closely packaged DNA strands, as a result of the repulsive interactions between the negative charges on the DNA helices. It had been unclear how this rigid crystalline structure of the viral genome rapidly ejects from the capsid, reaching rates of 60,000 bp/s. Through a combination of single-molecule and bulk techniques, we determined how the structure and energy of the encapsidated DNA in phage λ regulates the mobility required for its ejection. Our data show that packaged λ-DNA undergoes a solid-to-fluid-like disordering transition as a function of temperature, resulting locally in less densely packed DNA, reducing DNA-DNA repulsions. This process leads to a significant increase in genome mobility or fluidity, which facilitates genome release at temperatures close to that of viral infection (37 °C), suggesting a remarkable physical adaptation of bacterial viruses to the environment of Escherichia coli cells in a human host.

Using the osmotic stress technique together with a self-cleavage assay we measure directly differences in sequestered water between specific and nonspecific DNA-BamHI complexes as well as the numbers of water molecules released coupled to specific complex formation. The difference between specific and nonspecific binding free energy of the BamHI scales linearly with solute osmolal concentration for seven neutral solutes used to set water activity. The observed osmotic dependence indicates that the nonspecific DNA-BamHI complex sequesters some 120-150 more water molecules than the specific complex. The weak sensitivity of the difference in number of waters to the solute identity suggests that these waters are sterically inaccessible to solutes. This result is in close agreement with differences in the structures determined by x-ray crystallography. We demonstrate additionally that when the same solutes that were used in competition experiments are used to probe changes accompanying the binding of free BamHI to its specific DNA sequence, the measured number of water molecules released in the binding process is strikingly solute-dependent (with up to 10-fold difference between solutes). This result is expected for reactions resulting in a large change in a surface exposed area.

The physical forces that underlie the exclusion of solutes from macromolecular surfaces can be probed in a similar way as the measurement of forces between macromolecules in condensed arrays using the osmotic stress technique and x-ray scattering. We report here the dependence of alcohol exclusion or, equivalently, the preferential hydration of DNA on the spacing between helices in condensed arrays. The actual forces describing exclusion are quite different from the commonly assumed steric crowding coupled with weak binding. For a set of 12 nonpolar alcohols, exclusion is due to repulsive hydration interactions with the charged DNA surface. Exclusion amplitudes do not depend simply on size, but rather on the balance between alkyl carbons and hydroxyl oxygens. Polyols are included at very close spacings. The distance dependence of polyol inclusion, however, is quite different from nonpolar alcohol exclusion, suggesting the underlying mechanism of interaction is different.

We take advantage of our previous observation that neutral osmolytes can strongly slow down the rate of DNA-protein complex dissociation to develop a method that uses osmotic stress to 'freeze' mixtures of DNA-protein complexes and prevent further reaction enabling analysis of the products. We apply this approach to the gel mobility shift assay and use it to modify a self-cleavage assay that uses the nuclease activity of the restriction endonucleases to measure sensitively their specific binding to DNA. At sufficiently high concentrations of neutral osmolytes the cleavage reaction can be triggered at only those DNA fragments with initially bound enzyme. The self-cleavage assay allows measurement of binding equilibrium and kinetics directly in solution avoiding the intrinsic problems of gel mobility shift and filter binding assays while providing the same sensitivity level. Here we compare the self-cleavage and gel mobility shift assays applied to the DNA binding of EcoRI and BamHI restriction endonucleases. Initial results indicate that BamHI dissociation from its specific DNA sequence is strongly linked to water activity with the half-life time of the specific complex increasing approximately 20-fold from 0 to 1 osmolal betaine.

We have measured the dependencies of both the dissociation rate of specifically bound EcoRI endonuclease and the ratio of non-specific and specific association constants on water activity, salt concentration, and pH in order to distinguish the contributions of these solution components to specific and non-specific binding. For proteins such as EcoRI that locate their specific recognition site efficiently by diffusing along non-specific DNA, the specific site dissociation rate can be separated into two steps: an equilibrium between non-specific and specific binding of the enzyme to DNA, and the dissociation of non-specifically bound protein. We demonstrated previously that the osmotic dependence of the dissociation rate is dominated by the equilibrium between specific and non-specific binding that is independent of the osmolyte nature. The remaining osmotic sensitivity linked to the dissociation of non-specifically bound protein depends significantly on the particular osmolyte used, indicating a change in solute-accessible surface area. In contrast, the dissociation of non-specifically bound enzyme accounts for almost all the pH and salt-dependencies. We observed virtually no pH-dependence of the equilibrium between specific and non-specific binding measured by the competition assay. The observed weak salt-sensitivity of the ratio of specific and non-specific association constants is consistent with an osmotic, rather than electrostatic, action. The seeming lack of a dependence on viscosity suggests the rate-limiting step in dissociation of non-specifically bound protein is a discrete conformational change rather than a general diffusion of the protein away from the DNA.

Water often acts as a critical reactant in cellular reactions. Its role can be detected by modulating water activity with osmotic agents. We describe the principles behind this 'osmotic stress' strategy, and survey the ubiquity of water effects on molecular structures that have aqueous, solute-excluding regions. These effects are seen with single-functioning molecules such as membrane channels and solution enzymes, as well as in the molecular assembly of actin, the organization of DNA and the specificity of protein/DNA interactions.

The forces that govern DNA double helix organization are being finally systematically measured. The non-specific longer-range interactions--such as electrostatic interactions, hydration, and fluctuation forces--that treat DNA as a featureless rod are reasonably well recognized. Recently, specific interactions--such as those controlled by condensing agents or those consequent to helical structure-are beginning to be recognized, quantified and tested.

The free energy difference between complexes of the restriction nuclease EcoRI with nonspecific DNA and with the enzyme's recognition sequence is linearly dependent on the water chemical potential of the solution, set using several very different solutes, ranging from glycine and glycerol to triethylene glycol and sucrose. This osmotic dependence indicates that the nonspecific complex sequesters some 110 waters more than the specific complex with the recognition sequence. The insensitivity of the difference in number of waters released to the solute identity further indicates that this water is sequestered in a space that is sterically inaccessible to solutes, most likely at the protein-DNA interface of the nonspecific complex. Calculations based on the structure of the specific complex suggest that the apposing DNA and protein surfaces in the nonspecific complex retain approximately a full hydration layer of water.

Simultaneous measurements on the packing and energetics of high-density liquid crystalline DNA phases show that the crowding of long DNA polyelectrolytes at ever increasing concentrations is accomplished through straightening of the random coils that the double helix assumes in dilute solution. X-ray scattering by ordered phases reveals that the local straightening of the molecules is also accompanied by their progressive immobilization and confinement within the molecular 'cages' created by neighboring molecules. These effects can be clearly observed through the measured energies of DNA packing under osmotic stress and through the changes in structural and dynamic characteristics of X-ray scattering from DNA in ordered arrays at different concentrations. The character of the confinement of large DNA motions for a wide range of DNA concentrations is dominated by the soft potentials of direct interaction. We do not see the power-law variation of energy vs. volume expected from space-filling fluctuations of molecules that enjoy no interaction except the hard clash of steric repulsion. Rather, in highly concentrated DNA mesophases we see a crowding of molecules through electrostatic or hydration repulsion that confines their movements and positions. This view is based on directly measured packing energies as well as on concurrently measured structural parameters while the DNA double helices are condensed under an externally applied osmotic pressure.

We find that netropsin and netropsin analogue protect DNA from EcorI restriction nuclease cleavage by inhibiting the binding of EcoRI to its recognition site. The drug -- EcoRI competitive binding constants measured by a electrophoretic gel mobility shift assay are in excellent agreement with the nuclease protection results for the netropsin analogue and in reasonable agreement for netropsin itself. Crystal structures of complexes show that netropsin and EcoRI recognize different regions of the DNA helix and would not be expected to compete for binding to the restriction nuclease site. The large distortions in DNA structure caused by EcoRI binding are most likely responsible for an indirect structural competition with netropsin binding. The structural change in the netropsin binding region induced by EcoRI binding to its region essentially prevents drug association. Given the reciprocal nature of competition, binding of netropsin to a minimally perturbed structure then also makes the association of EcoRI energetically more costly. Since many sequence specific DNA binding proteins significantly bend or distort the DNA helix, drugs that compete indirectly can be as effective as drugs that act through a direct steric inhibition.

Direct transfer of proteins between DNA helices is a recognized important feature of the recognition site search process. Direct transfer is characterized by a dissociation rate that depends on total DNA concentration. This is taken as evidence for the formation of an intermediate DNA-protein-DNA ternary complex. We find that the dissociation rate of EcoRI-DNA-specific complexes at 80 mM NaCl depends on the concentration of competitor oligonucleotide suggesting that direct transfer contributes to EcoRI dissociation. This dependence on competitor DNA concentration is not seen at 180 mM salt. A careful examination of the salt concentration dependence of the dissociation rate, however, shows that the predictions for the formation of a ternary complex are not observed experimentally. The findings can be rationalized by considering that just after dissociating from a DNA fragment the protein remains in close proximity to that fragment, can reassociate with it, and diffuse back to the recognition site rather than bind to an oligonucleotide in solution, a hopping excursion. The probability that a protein will bind to an oligonucleotide during a hop can be approximately calculated and shown to explain the data. A dependence of the dissociation rate of a DNA-protein complex on competitor DNA concentration does not necessarily mean direct transfer.

The electrophoretic mobility shift assay (EMSA) is one of the most popular tools in molecular biology for measuring DNA-protein interactions. The technique uses polyacrylamide gel electrophoresis to separate DNA-protein or RNA-protein complexes from free DNA or RNA. Polyacrylamide gels stabilize DNA-protein, RNA-protein, or protein-protein complexes by a crowding or caging mechanism. Still every technique has its limitations. EMSA, as standardly practiced today, works well for complexes with association binding constants Ka>109 M−1 under normal conditions of salt and pH. Many DNA-protein complexes are not stable enough so that they dissociate while moving through the gel matrix giving smeared bands that are difficult to quantitate reliably. We take advantage of our previous observation that neutral osmolytes can strongly slow down the rate of DNA-protein complex dissociation to develop a method that uses osmotic stress to stabilize complexes in the gel matrix as well as in the solution. In this work we demonstrate that the addition of the osmolyte triethylene glycol to polyacrylamide gels dramatically stabilizes labile restriction endonuclease EcoRI complexes with nonspecific DNA sequences enabling quantitation of binding using the electrophoretic mobility shift assay. The significant improvement of the technique resulting from the addition of osmolytes to the gel matrix greatly extends the range of binding constants of protein-DNA complexes that can be investigated using this widely used assay. Extension of this approach to other techniques used for separating bound and free components such as gel chromatography and capillary electrophoresis is straightforward.

In spermatogenesis, chromatin histones are replaced by arginine-rich protamines to densely compact DNA in sperm heads. Tight packaging is considered necessary to protect the DNA from damage. We have previously observed that the net attraction between salmon protamine condensed DNA helices was much smaller for DNA condensed by the equivalent homo-arginine peptide. We hypothesized that this is caused by the neutral amino acids present in protamines. To better understand the nature of the forces condensing protamine-DNA assemblies and their dependence on amino acid content, the effect of neutral and negatively charged amino acids on DNA-DNA intermolecular forces was studied using model peptides containing six arginines. The component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance have been determined by the osmotic stress technique coupled with x-ray scattering as a function of the chemistry, position, and number of the amino acid inserted. Neutral amino acids inserted into hexa-arginine increase the short-range repulsion; while only slightly decreasing the longer-ranged attraction. The decrease in net attraction between salmon protamine condensed helices compared with arginine homopeptides can be well explained by amino acid content alone. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both these observation have biological implications for protamine-DNA packaging in sperm heads.

Directly measured forces between DNA helices in ordered arrays have been reduced to simple force coefficients and mathematical expressions for the interactions between pairs of molecules. The tabulated force parameters and mathematical expressions can be applied to parallel molecules or, by transformation, to skewed molecules of variable separation and mutual angle. This "toolbox" of intermolecular forces is intended for use in modelling molecular interactions, assembly, and conformation. The coefficients characterizing both the exponential hydration and the electrostatic interactions depend strongly on the univalent counterion species in solution, but are only weakly sensitive to anion type and temperature (from 5 to 50 degrees C). Interaction coefficients for the exponentially varying hydration force seen at spacings less than 10 to 15 A between surfaces are extracted directly from pressure versus interaxial distance curves. Electrostatic interactions are only observed at larger spacings and are always coupled with configurational fluctuation forces that result in observed exponential decay lengths that are twice the expected Debye-Huckel length. The extraction of electrostatic force parameters relies on a theoretical expression describing steric forces of molecules "colliding" through soft exponentially varying direct interactions.

In spermatogenesis, chromatin histones are replaced by arginine-rich protamines to densely compact DNA in sperm heads. Tight packaging is considered necessary to protect the DNA from damage. To better understand the nature of the forces condensing protamine-DNA assemblies and their dependence on amino acid content, the effect of neutral and negatively charged amino acids on DNA-DNA intermolecular forces was studied using model peptides containing six arginines. We have previously observed that the neutral amino acids in salmon protamine decrease the net attraction between protamine-DNA helices compared with the equivalent homo-arginine peptide. Using osmotic stress coupled with x-ray scattering, we have investigated the component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance as a function of the chemistry, position, and number of the amino acid inserted. Neutral amino acids inserted into hexa-arginine increase the short range repulsion while only slightly affecting longer range attraction. The amino acid content alone of salmon protamine is enough to rationalize the forces that package DNA in sperm heads. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both of these observations have biological implications for protamine-DNA packaging in sperm heads.

The DNA binding stringency of restriction endonucleases is crucial for their proper function. The x-ray structures of the specific and non-cognate complexes of the restriction nuclease EcoRV are considerably different suggesting significant differences in the hydration and binding free energies. Nonetheless, the majority of studies performed at pH 7.5, optimal for enzymatic activity, have found less than a 10-fold difference between EcoRV binding constants to the specific and nonspecific sequences in the absence of divalent ions. We used a recently developed self-cleavage assay to measure EcoRV-DNA competitive binding and to evaluate the influence of water activity, pH and salt concentration on the binding stringency of the enzyme in the absence of divalent ions. We find the enzyme can readily distinguish specific and nonspecific sequences. The relative specific-nonspecific binding constant increases strongly with increasing neutral solute concentration and with decreasing pH. The difference in number of associated waters between specific and nonspecific DNA-EcoRV complexes is consistent with the differences in the crystal structures. In spite of the large pH dependence of the sequence specificity, the osmotic pressure dependence indicates little change in structure with pH. The large osmotic pressure dependence means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowded environment of the cell. In addition to divalent ions, water activity and pH are key parameters that strongly modulate binding specificity of EcoRV.

Using solution synchrotron x-ray scattering, we measure the variation of DNA-DNA $d$ spacings in bacteriophage $\ensuremath{\lambda}$ with mono-, di-, and polyvalent salt concentrations, for wild-type [$48.5\ifmmode\times\else\texttimes\fi{}{10}^{3}$ base pairs (bp)] and short-genome-mutant (37.8 kbp) strains. From the decrease in $d$ spacings with increasing salt, we deduce the relative contributions of DNA self-repulsion and bending to the energetics of packaged phage genomes. We quantify the DNA-DNA interaction energies within the intact phage by combining the measured $d$ spacings in the capsid with measurements of osmotic pressure in DNA assemblies under the same salt conditions in bulk solution. In the commonly used Tris-Mg buffer, the DNA-DNA interaction energies inside the phage capsids are shown to be about $1kT/\mathrm{bp}$, an order of magnitude larger than the bending energies.

Binding of Zn 2+ to the 5 S RNA gene sequence of Xenopus borealis results in strong bending of the DNA, as inferred from transient electric birefringence data. The effect is specific for Zn 2+ ; several other divalent ions are not able to induce a bend of a similar magnitude. Using five different fragments that span the binding sequence, we are able to estimate a bend magnitude of at least 55 ° centered at base-pair +65 within the gene. This places the bend within the binding domain of the gene-regulatory protein transcription factor (TF) IIIA. Recent evidence has shown that the protein-DNA complex is also bent. Although our data do not allow us directly to link the two bends, our results suggest that TFIIIA could form a folded structure by stabilizing the same bent conformation that is induced by binding of Zn 2+ . The chemistry of Zn 2+ binding to DNA, and the sequence around the bend center, suggest that the bend is most probably caused by joint co-ordination of Zn 2+ to the N-7 groups of stacked purine residues.

Recent work has shown that pressures inside dsDNA phage capsids can be as high as many tens of atmospheres; it is this pressure that is responsible for initiation of the delivery of phage genomes to host cells. The forces driving ejection of the genome have been shown to decrease monotonically as ejection proceeds, and hence to be strongly dependent on the genome length. Here we investigate the effects of ambient salts on the pressures inside phage-lambda, for the cases of mono-, di-, and tetravalent cations, and measure how the extent of ejection against a fixed osmotic pressure (mimicking the bacterial cytoplasm) varies with cation concentration. We find, for example, that the ejection fraction is halved in 30 mM Mg(2+) and is decreased by a factor of 10 upon addition of 1 mM spermine. These effects are calculated from a simple model of genome packaging, using DNA-DNA repulsion energies as determined independently from x-ray diffraction measurements on bulk DNA solutions. By comparing the measured ejection fractions with values implied from the bulk DNA solution data, we predict that the bending energy makes the d-spacings inside the capsid larger than those for bulk DNA at the same osmotic pressure.

Corrigendum: Solid-to-fluid DNA transition inside HSV-1 capsid close to the temperature of infection

DNA in vertebrate sperm nuclei is organized differently from any other cell. DNA is compacted in tightly packed (∼ 400 mg/ml) hexagonal arrays. Since DNA repair is absent in sperm, dense packaging of DNA is considered necessary to protect against damage by reactive oxidizing species. DNA damage correlates with infertility and likely contributes to miscarriages and birth defects. Our goal is to characterize protamine defects that result in DNA mispackaging. Small (∼30-50 amino acids), arginine rich (50-70%) peptides called protamines assemble DNA in sperm nuclei. Protamines will spontaneously condense DNA from dilute solution into close-packed, liquid crystalline arrays. Both sperm nuclei and reconstituted protamine-DNA arrays are sufficiently ordered that the interhelical spacing or packing density can be determined by x-ray scattering.Insufficient protamine to neutralize all DNA has been linked to excess damage. Salmon sperm nuclei can be fractionated on sucrose density gradients. The interhelical scattering maxima of the heavy and light fractions overlap indicating that all nuclei have a tightly packed fraction of DNA. The lighter fraction, however, shows a scattering profile that is skewed toward larger spacings. Part of the DNA is not as well packed as the majority. Adding excess protamine to the light fraction recovers the scattering profile of the heavy fraction.Protamines are initially serine phosphorylated when replacing histones on DNA. Incomplete dephosphorylation has also been linked to DNA damage. Herring protamines can be partially phosphorylated with protein kinase A. We find that even modest phosphorylation that reduces the protamine charge by only 5% will increase the water volume accessible to oxidizing species in reconstituted DNA-protamine assemblies by 50%.

We have investigated the salt sensitivity of the hexagonal-to-cholesteric phase transition of spermidine-condensed DNA. This transition precedes the resolubilization of precipitated DNA that occurs at high spermidine concentration. The sensitivity of the critical spermidine concentration at the transition point to the anion species and the NaCl concentration indicates that ion pairing of this trivalent ion underlies this unusual transition. Osmotic pressure measurements of spermidine salt solutions are consistent with this interpretation. Spermidine salts are not fully dissociated at higher concentrations. The competition for DNA binding among the fully charged trivalent ion and the lesser charged complex species at higher concentrations significantly weakens attraction between DNA helices in the condensed state. This is contrary to the suggestion that the binding of spermidine at higher concentrations causes DNA overcharging and consequent electrostatic repulsion.

The interaction of the alcohols 2-methyl-2,4-pentanediol (MPD) and 2-propanol and of glycerol with condensed spermidine(3+)-DNA arrays are investigated with direct force measurements using osmotic stress coupled with X-ray scattering. Thermodynamic forces between DNA helices are measured from the dependence of helical interaxial spacings on the osmotic pressure applied by poly(ethylene glycol) solutions in equilibrium with the DNA phase. The sensitivity of these forces to solute concentration can be transformed into a change in the number of excess or deficit solutes or waters in the DNA phase by applying the Gibbs-Duhem equation. The alcohols examined are excluded from the condensed DNA array and strongly affect the osmotic stress force curves. DNA is preferentially hydrated. MPD is significantly more excluded than 2-propanol. The exclusion of these alcohols, however, is not due to a steric repulsion since glycerol that is intermediate in size between MPD and 2-propanol does not observably affect DNA force curves. As the distance between DNA helices varies, the change in the number of excess waters is independent of alcohol concentration for each alcohol. These solutes are acting osmotically on the condensed array. The distance dependence of exclusion indicates that repulsive water structuring forces dominate the interaction of alcohols with the DNA surface. The exclusion measured for these condensed arrays can quantitatively account for the effect of these alcohols on the precipitation of DNA from dilute solution by spermidine(3+).

We recently showed that a nonspecific complex of the restriction nuclease EcoRI with poly (dI-dC) sequesters significantly more water at the protein-DNA interface than the complex with the specific recognition sequence. The nonspecific complex seems to retain almost a full hydration layer at the interface. We now find that at low osmotic pressures a complex of the restriction nuclease EcoRI with a DNA sequence that differs by only one base pair from the recognition site (a 'star' sequence) sequesters about 70 waters more than the specific one, a value virtually indistinguishable from nonspecific DNA. Unlike complexes with oligo (dI-dC) or with a sequence that differs by two base pairs from the recognition sequence, however, much of the water in the 'star' sequence complex is removed at high osmotic pressures. The energy of removing this water can be calculated simply from the osmotic pressure work done on the complex. The ability to measure not only the changes in water sequestered by DNA-protein complexes for different sequences, but also the work necessary to remove this water is a potentially powerful new tool for coupling inferred structural changes and thermodynamics.

The binding of a netropsin analogue to random sequence DNA, monitored by CD, is seen dependent on the concentration of neutral solutes. The binding free energy decreases linearly with solute osmolal concentration and the magnitude of the effect is insensitive to the chemical identity of the solute fur betaine, sorbitol, and triethylene glycol. These solutes appear to modulate binding through their effect on water activity and changes in the hydration of the drug and DNA in the complex reaction, not through a direct interaction with the reactants or the product. The dependence of binding constant on solute concentration can be interpreted as an additional binding of some 50–60 extra solute excluding water molecules by the complex. A water sensitivity of drug binding is further seen from the dependence of binding constants on the type of anion in solution. Anions in the Hofmeister series strongly affect bulk water free energies and entropies. The differences in netropsin analogue binding to DNA with Cl−, F−, and CIO are consistent with the effect observed with neutral solutes. The ability to measure changes in water binding associated with a specific DNA interaction is a first step toward correlating changes in hydration with the strength and specificity of binding. © 1995 John Wiley & Sons, Inc.

Pressurized DNA storage and pressure-driven DNA ejection in bacteriophage lambda provide an attractive model system for investigating DNA packaging in tight spaces. Moreover, lambda phage holds therapeutic promise as an alternative to antibiotics and vehicle for gene delivery. A wide variety of experimental and theoretical tools have been employed to interrogate DNA mechanics in lambda phage. Normally triggered ejection leads to loss of all DNA from the phage head; partial DNA ejection can be achieved by either counteracting the DNA pressure with external osmotic pressure or reducing the DNA-DNA repulsion with multivalent cations. Our objective in this study was to systematically map the lengths of DNA left inside partially ejected lambda phage. We determined the expected length of DNA inside the phage by monitoring the amount of ejected DNA with UV spectroscopy and assayed the actual length left directly with field-inversion gel electrophoresis. Previously used experimental methods, such as UV spectroscopy, were examined and used to assess the results from our modified protocols. Surprisingly, we found that lengths of retained DNA were concentrated around multiple specific lengths rather than the single equilibrium length that would fit thermodynamic models of DNA packaging in lambda phage. These DNA lengths occurred at regular intervals and highlighted unexpected conformational variants, sequence specific interactions, or discontinuities in DNA packaging. Structurally relevant patterns were observed when cobalt hexamine, magnesium chloride, spermine, and bis(ethyl) spermine were used as counterions. Taken together, our results show that thermodynamic models of DNA packaging in lambda phage need to be reevaluated to account for the size specific ejection that has been observed in our study.

Biological systems under extreme conditions often resort to unusual structures to achieve special functions for survival. The “bioengineering” principles of such “extreme” structures may inspire biomimic designs of functional materials. Here we give several examples of such naturally occurring unusual structures probed with x-ray scattering. i) In bacterial spores, the spore coat appears to comprise of laminar layers of quasi-2D crystals with periodicity of ∼1 nm. Such ordered assembly may be responsible for the spore resistance to heat, toxic chemicals, and mechanical disruption. ii) In starved bacterial cells, the DNA packaging protein (DPS) is over-produced to compact the chromosomal DNA close to crystalline density to protect the genomic integrity and facilitate homologous recombination. iii) In bacterial cells with over-produced DNA, mild treatment with antibiotics can lead to liquid-crystalline DNA that responds to external osmotic stress. Future work includes 1) uncovering the molecular basis of characterized structures and 2) extending into systems such as cells under radiation or heat and cancer cells.

We have determined the rise per base pair and persistence length of A-form DNA in trifluoroethanol solutions for fragments 350-900 base pairs in length that best describe rotational diffusion coefficients determined by transient electric birefringence. The 2.6 A spacing between base pairs found in crystal and fiber A-form structures is preserved in solution. The persistence length is about 1500 A, or about three times longer than for B-form DNA. There is no apparent electrostatic contribution to the persistence length in the salt concentration range 0.2-2.0 mM Na cacodylate. This suggests an even closer association between DNA and its neutralizing counterions than predicted by condensation theory, perhaps due to a sheath of trifluoroethanol excluded water surrounding the A-form helix.

Transient electric dichroism measurements have been used to observe the rotational relaxation times of 145 base pair fragments of poly (dGm5dC) and random sequence DNA to solution. From these the lengths of the fragments are calculated and the interbase pair separation or rise per base pair (RPB) calculated. The observations show that even in low salt, the addition of very low concentrations of trivalent Co(NH3)63+ results in a transition of the dGm5dC polymer from B-form to Z-form with a change in the RPB from 3.4 +/- .06A to 3.7 +/- .06A, the latter form defined by the criterion of an inverted circular dichroism spectra similar to that observed at high salt in the absence of Co(NH3)63+. The 145 base pair DNA and poly (dGm5dC) are found to be essentially fully extended rods in low salt (0.2 - 2 mM Na+) solutions.

We have analyzed the structure of complexes between DNA gyrase and four defined DNA fragments by electric dichroism. Both the extrapolated dichroism and relaxation time of these complexes suggest that a single turn of DNA is wrapped around the enzyme with the entry and exit points located close together. The average angle between the DNA tails emerging from the particle is about 120 degrees. This structure is consistent with that seen by electron microscopy. Addition of ATP or the non-hydrolyzable ATP analog 5'-adenylyl-beta, gamma-imidodiphosphate results in a structural change of the complex, consistent with the DNA tails now being wrapped around the protein. The significance of these observations with respect to the mechanism of DNA supercoiling by DNA gyrase is discussed.

Values of the apparent intrinsic dichroism of the 260-nm transition of duplex poly(dG-dC) and poly(dG-m5dC) in both B- and Z-conformations are determined from electric dichroism (ED) measurements by extrapolation of the data at high field strengths. These values are compared to each other and to values of the intrinsic dichroism calculated for the Z- and B-forms generated respectively from the crystal structure coordinates [(1981) A. H. J. Wang, G. J. Quigley, F. J. Kolpak, G. Van der Marel, T. H. Van Boom & A. Rich, Science211, 171–176.] of the d(CpGpCpGpCpGp) hexamer and from the coordinates [(1982) A. V. Fratini, M. L. Kopka, H. R. Drew & R. E. Dickerson, J. Biol. Chem.257, 14686–14707.]. of the terminal CpGpCpGp base pairs of the crystal of straight B-form dodecamer d(CpGpCpGpApApTpTpCpGpCpGp). Direct association of the dichroism with the orientation of the optical transition moments with respect to the helix axis of poly(dG-dC) implies that the average tilt of the bases is much different than the values calculated from the structural coordinates. The experimental values for both the B- and Z-forms are, however, almost identical to those observed with fragments of “random” sequence B-form DNA of the same molecular length. It is argued that this descrepancy and other anomalies reported in the literature are due to the theoretically predicted unusual high field behavior associated with diffuse ion atmosphere polarization. Unlike either permanent dipoles or induced moments based on bond polarizabilities, the orienting dipole energy from the polarization of the counterions surrounding DNA approaches a finite limiting value, dependent on DNA length, at very high field strengths. This can result in extrapolated dichroisms that do not reflect structure at perfect orientation, but rather structure at some limiting orientation. Consequently, we believe, the conclusion drawn from ED measurements on short fragments of DNA that the bases are highly tilted from the perpendicular to the helix axis by propeller twisting or otherwise is suspect.

The high mobility group proteins, HMG 14 and 17, have been associated with the chromatin of active genes (refs 1-8), although how they function is not known. We use sedimentation and electric dichroism to investigate the effect of HMG 14 and 17 on the condensation of chicken erythrocyte chromatin into higher order structure. We find no evidence that excess HMG 14 and 17 induce an extended configuration, either in bulk chromatin or in the chromatin of the chicken beta-globulin gene.

Various assays and physicochemical methods were used to observe changes taking place in membrane proteins from erythrocytes that had been stored up to 42 days at 4°C. Red cell membranes were found to decrease in hydrophobicity, flexibility, and sulfhydryl content on storage. These changes are not due to proteolysis, but related to an unresolved change in the structural characteristics of the lipoproteins comprising the ‘membrane superstructure’.

Rather than acting by modifying van der Waals or electrostatic double layer interactions or by directly bridging neighboring molecules, polyvalent ligands bound to DNA double helices appear to act by reconfiguring the water between macromolecular surfaces to create attractive long range hydration forces. We have reached this conclusion by directly measuring the repulsive forces between parallel B-form DNA double helices pushed together from the separations at which they have self organized into hexagonal arrays of parallel rods. For all of the wide variety of "condensing agents" from divalent Mn to polymeric protamines, the resulting intermolecular force varies exponentially with a decay rate of 1.4–1.5 A, exactly one-half that seen previously for hydration repulsion. Such behavior qualitatively contradicts the predictions of all electrostatic double layer and van der Waals force potentials previously suggested. It fits remarkably well with the idea, developed and tested here, that multivalent counterion adsorption reorganizes the water at discrete sites complementary to unadsorbed sites on the apposing surface. The measured strength and range of these attractive forces together with their apparent specificity suggest the presence of a previously unexpected force in molecular organization.

The assembly of double stranded DNA helices with divalent manganese ion is favored by increasing temperature. Direct force measurements, obtained from the osmotic stress technique coupled with x-ray diffraction, show that the force characteristics of spontaneously precipitated Mn(2+)-DNA closely resemble those observed previously by us for other counterion condensed DNA assemblies. At temperatures below the critical one for spontaneous assembly, we have quantitated the changes in entropy and manganese ion binding associated with the transition from repulsive to attractive interactions between helices mediated by osmotic stress. The release of structured water surrounding the DNA helix to the bulk solution is the most probable source of increased entropy after assembly. Increasing the water entropy of the bulk solution by changing the manganese salt anion from CI- to ClO4- predictably and quantitatively increases the transition entropy. This is further evidence for the dominating role of water in the close interaction of polar surfaces.

We describe recent studies of the 30 nm fiber, in which we have used electric dichroism to determine the orientation of chromatosomes within the chromatin fiber. We show that data obtained with chromatin from a variety of sources, and with varying spacer length, suggest a similar packing of the chromatosomes in all 30 nm fibers. We also discuss factors that might affect the stability of the fiber, particularly in such a way as to render the DNA it contains more accessible for transcription.

DNA renaturations under nucleation-rate-limiting conditions on simple DNA such as bacterial and bacteriophage DNA show significant deviation from ideal second-order kinetics when followed by optical density measurements at 260 nm. Ideal second-order kinetics yield linear plots when the data is plotted in the standard reciprocal second-order (RSO) manner. The observed deviations from ideal second-order behavior take the form of steadily downward-curving RSO plots. In this paper, experiments are presented for E. coli and T2 DNA documenting this non-ideal behavior. Since experiments usine, T4, T5 ind B. subtilis DNA yield identical non-ideal behavior, this behavior appears to be a property of DNA renaturation followed by optical density, not a peculiarity of a particular DNA. Identical non-ideal behavior is also seen in kinetics followed by S1 nuclease assay. A theory is developed to explain this deviation from ideal second-order kinetics. The theory also explains why kinetics followed by hydroxyapatite chromalography show nearly ideal second-order kinetics. In contrast to the approach taken by others in developing equations that describe S1 nuclease monitored reactions, our view is that non-ideal second-order kinetics are fundamentally due to the reaction of free single strands to yield partially helical duplex species. Later reactions of these species tend to reduce the deviations from non-ideal second-order kinetics.

The physical properties of chromatin have been explored at the level of individual nucleosomes, extended polynucleosome filaments, and folded polynucleosome fibers. To compare the physical properties of bulk chromatin with the properties of chromatin in transcriptionally active genes, we have examined the stability of the 30 nm chromatin fiber in the neighborhood of the adult s-globin gene in chicken red cells. We find that the DNA in the neighborhood of this gene is indistinguishable from bulk chromatin in its degree of compaction and in its reaction with the HMG proteins HMG 14/17.