The three-dimensional structure of supercoiled deoxyribonucleic acid in solution. Evidence obtained from the angular distribution of scattered light

1. The size and shape of superhelical double-stranded circular DNA from bacteriophage ØX174 were investigated by light-scattering. The molecular weight of the DNA is 3.17×10⁶ and the root-mean-square radius is 103.5nm. 2. The light-scattering envelopes of various theoretical three-dimensional models for such DNA molecules were calculated by repetitive computational techniques, and the results were compared with the experimental findings. 3. It is concluded that the structure of supercoiled DNA containing −12 superhelical turns in buffer of I0.2 corresponds best to one of the more compact models for superhelix structure such as the branched model, and the commonly employed straight interwound superhelix model is incompatible with the experimental results, at the superhelix density found.     

Dynamic light scattering as a probe of superhelical DNA-intercalating agent interaction

Polarized dynamic light-scattering measurements on superhelical pBR322-plasmid DNA solutions in 0.2M NaCl, 2 mM NaPi, pH 7.0, 2 mM EDTA result in a translational diffusion coefficient D = (3.77 ± 0.10) × 10^?8 cm²/s for the native molecule. Modeling the DNA, in the simplest approximation, as a 10 × 440-nm effective hydrodynamic rigid rod yields a good fit to the apparent diffusion coefficient angular-dependence data up to 70°; the model fails at higher angles, probably due to the effects of flexibility or branching of the rod. Diffusion coefficient titration experiments with a platinum complex intercalating agent (PtTS) result in a titratable superhelix density of σ = ?0.079 ± 0.008 under our experimental conditions, corresponding to about 34 superhelical turns in the native DNA. The DNA contour length predicted by our two independent results, the rod dimensions and the number of superhelical turns, is in excellent agreement with the contour length calculated from the number of base pairs, supporting the hydrodynamic approximation of an effective rodlike structure for this small DNA molecule in solution.     

Biosynthetic studies of the Y base in yeast phenylalanine tRNA. Incorporation of guanine

Replicating polyoma virus DNA, pulse-labeled with ³H-thymidine, was isolated from infected mouse embryo cells by velocity sedimentation in neutral sucrose and purified by benzoylated-naphthoylated DEAE-cellulose chromatography. Nascent strands, prepared by heat denaturation of purified replicative intermediate, banded at a slightly higher buoyant density in neutral cesium sulfate gradients than single strands derived from superhelical viral DNA. Treatment of nascent strands with a mixture of ribonucleases 1A and T1 shifted their buoyant density to that of single strands derived from superhelical viral DNA. These results indicate that an oligoribonucleotide component is covalently associated with replicating polyoma DNA strands.     

Studies on deoxyribonucleoprotein structure. Small-angle X-ray scattering in solutions of various ionic strengths.

The cross-sectional radius of gyration of the deoxyribonucleoprotein (DNP) threads was measured by small-angle X-ray scattering in a wide range of ionic strengths (from 0.0005 to 2 M NaCl). For DNP in a solution of low ionic strength, this value is 30 Å. The increase of ionic strength results in partial deproteinization of DNP, while the cross-sectional radius of gyration varies from 25 Å for DNP in 0.7 M NaCl to 10 Å for DNP in 2 M NaCl. It is suggested that gradual deproteinization by the increase of NaCl concentration causes conformational changes, which are associated with the alteration of the DNP superstructure. The data are interpreted on the basis of the superhelical model of DNA packing in DNP; however, the coexistence of superhelical and unfolded regions in the DNP structure is also a possibility.     

Dynamic model for sedimentation of supercoiled molecules.

An experimental curve for the sedimentation of SV40 DNA has been fitted by using a three parameter theory. The chain stiffness parameter is evaluated at each point, and the general behavior as a function of the number of loops (superhelix density) has been described. The chain stiffness, like the sedimentation ratio, also depends on the superhelical density in a complicated way.     

Theoretical model for the equilibrium behavior of DNA superhelices

A statistical-mechanical model for superhelical DNA is presented. The partition function for a DNA superhelix is written by using a combinatorial approach in order to allow for the known relation between the number of superhelical twists and the states of the base pairs in the double helix. While the theory allows any factors which might contribute to the free energy of superhelical twisting to be included in the statistical weights of the superhelical twists, only the reduction in configurational entropy is considered in this paper. Similarities between an imperfectly matched DNA double helix and a DNA superhelix are used in the derivation of expressions for the entropy of superhelical DNA. Although the partition function is presented in a general form, permitting many equilibrium properties of DNA superhelices to be treated, the application considered in this paper is the calculation of helix–coil transition curves. Several experimentally observed features of such transitions are predicted. For example, the curves are bimodal, with an early and a late transition relative to that of a nicked molecule. The results are very sensitive to the volume within which two parts of the double helix must meet when forming a superhelical twist. The free energy of superhelix formation is calculated, and the results are compared with those obtained from the data of Bauer and Vinograd for ethidium bromide intercalation. In the present model, the free energy increases less sharply with an increase in the number of superhelical twists than observed experimentally, indicating that factors other than configurational entropy probably make important contributions to the free energy of superhelix formation.     

Isolation and Characterization of Kinetoplast DNA Networks and Minicircles from Crithidia fasciculata*

SYNOPSIS. Covalently closed kinetoplast DNA networks have been isolated from stationary phase Crithidia fasciculata cells by a technic involving selective pelleting of the networks at a low centrifugal field. Approximately 62% of the kinetoplast DNA of the cell was recovered free of nuclear DNA by simple differential centrifugation. Purified kinetoplast DNA networks were visualized both in the electron microscope and in the light microscope. Closed networks sedimented as a homogeneous band both in neutral and alkaline sucrose, with an s_20w in neutral sucrose of approximately 5 × 10³. Closed monomeric minicircles were isolated from purified networks by mild sonication and band sedimentation in alkaline sucrose. Several physical properties of closed monomeric minicircles were measured. These included molecular weight, buoyant density in CsCl, superhelix density and sedimentation coefficient.     

Superhelix density heterogeneity in closed circular intracellular PM2 DNA

Covalently closed intracellular DNA obtained from Pseudomonas BAL 31 20 min after infection with PM2 phage has been shown to be heterogeneous in superhelix density. Analytical band sedimentation, in the presence of low concentrations of ethidium bromide, has been carried out on fractions centripetal and centrifugal to the mode of a single band of closed circular DNA in a preparative propidium iodide–CsCl buoyant density gradient. Different average sedimentation rates, as well as different band shapes, have been observed for upper and lower fractions centrifuged at a dye concentration near the minimum in s° versus ethidium bromide concentration titrations performed on DNA from proximate intermediate fractions. Similar differences, although not as pronounced, have been obtained at a dye concentration corresponding to a point in the steep region of the titrations. Differential band sedimentation experiments performed on the same fractions have confirmed these results. Differential band sedimentation experiments on similarly fractionated mature PM2 I DNA (closed circular form) have shown slight differences in the relative sedimentation rates of upper and lower fractions at dye concentrations corresponding to the steep regions in the titrations. The same experiments, when performed on nicked circular DNA obtained from heating both the mature and intracellular fractions, showed no evidence of differences in sedimentation coefficients. Such results may indicate slight heterogeneity in the superhelix density of viral PM2 I DNA; however, the degree of this heterogeneity would be somewhat less than that of the intracellular DNA. The possibility that superhelix density heterogeneity may arise from displacement loops, which have been found at low levels in intracellular PM2 DNA, has been subjected to experimental tests. Unless such structures are originally present and removed by the isolation procedure, this possibility may be rejected.     

Brownian dynamics simulations of supercoiled DNA with bent sequences.

The recently presented Brownian dynamics model for superhelical DNA is extended to include local curvature of the DNA helix axis. Here we analyze the effect of a permanent bend on the structure and dynamics of an 1870-bp superhelix with delta Lk = -10. Furthermore, we define quantitative expressions for computing structural parameters such as loop positions, superhelix diameter, and plectonemic content for trajectories of superhelical DNA, and assess the convergence toward global equilibrium. The structural fluctuations in an interwound superhelix, as reflected in the change in end loop positions, seem to occur by destruction/creation of loops rather than by a sliding motion of the DNA around its contour. Their time scale is on the order of 30-100 microseconds. A permanent bend changes the structure and the internal motions of the DNA drastically. The position of the end loop is fixed at the permanent bend, and the local motions of the chain are enhanced near the loops. A displacement of the bend from the end loop to a position inside the plectonemic part of the superhelix results in the formation of a new loop and the disappearance of the old one; we estimate the time involved in this process to be about 0.5 ms.     

Evidence for allosteric transitions in secondary structure induced by superhelical stress

Previous studies suggest that the global secondary structures of native supercoiled and equilibrium linear DNAs may differ somewhat. Recent evidence also indicates that metastable secondary structure commonly persists following complete relaxation of the superhelical stress by intercalating dyes or by the action of topoisomerase I. In this work, the torsion constants (alpha) of pBR322, pUC8 and M13mp7 (replicative form) DNAs are determined by time-resolved fluorescence polarization anisotropy at various times subsequent to linearization. In all three cases, the torsion constants are relatively low immediately after linearization, and evolve for eight to ten weeks before reaching their apparent equilibrium values. It is shown in detail how the persistence of metastable secondary structure, subsequent to relaxation of superhelical stress, necessarily implies that one or more transitions in equilibrium secondary structure are induced as the superhelix density is varied from zero to native, or vice versa. Samples of pUC8 dimer (5434 base-pairs) with different superhelix densities are prepared by the action of topoisomerase I in the presence of various amounts of ethidium. Their median linking number differences are determined by standard band counting methods. The translational diffusion coefficient (Do) and the plateau diffusion coefficient (Dplat) characterizing internal motions over short distances (225 A) are determined by dynamic light-scattering. The torsion constant (alpha) between base-pairs and the circular dichroism spectrum are also measured for each sample. Curves of Dplat, Do, alpha and molar ellipticity ([theta]) (at the minimum near 250 nm) versus superhelix density (sigma) are constructed. The curve of Do versus sigma is very similar to that for sedimentation coefficient versus sigma for simian virus 40 (SV40) and polyoma DNAs. The curves of Dplat, Do, alpha and [theta] versus sigma show that, with increasing negative superhelix density, a structural transition occurs near sigma = -0.020 to an intermediate state with low torsion constant, and a second structural transition occurs near sigma = -0.035 to a state that exhibits more normal properties by sigma = -0.048. These data are consistent with the hypothesis that supercoiling induces two successive allosteric transitions to alternative global secondary structures. The data are much less consistent with the hypothesis that supercoiling induces some radical secondary structure at one or a few sites of small extent at sigma = -0.020, and at other sites at sigma = -0.035, or with hypotheses based on changes in tertiary structure alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intrinsic viscosity of native and single-stranded T7 DNA and its relationship to sedimentation coefficient

The intrinsic viscosity and sedimentation coefficient, of native and single-stranded T7 DNA have been determined at 25°C as a function of ionic strength in neutral and alkaline NaCl. The relationship between [η] and S_,w is well represented by the Mandelkern-Flory equation over the entire range of conditions between 0.0013 and 1M Na⁺. An apparent discrepancy between the two methods at moderate to high ionic strengths is probably due to a change in V with ionic strength. It appears that [η] is a more sensitive and reliable measure of molecular expansion for native DNA, S_,w but is a better index of conformational change in single strands, since [η] becomes too small to measure conveniently at high ionic strengths. At moderate to high ionic strengths, denaturation leads to a decrease in [η], although unfolded single strands retain considerable viscosity. At sufficiently low ionic strength, the intrinsic viscosity of the single strands becomes higher than that of native DNA, and the effective volume of a single strand approaches that of the native molecule.     

Application of Polyelectrolyte Theory to the Study of the B-Z Transition in DNA (1)

Abstract

We have used the polyelectrolyte theory to study the ionic strength dependence of the B-Z equilibrium in DNA. A DNA molecule is molded as an infinitely long continuously charged cylinder of radius a with reduced linear charge density q. The parameters a and q for the B and Z forms were taken from X-ray data: a _B = 1nm, q _B = 4.2, a _z = 0.9 nm and q _z = 3.9. A simple theory shows that at low ionic strengths (when Debye screening length r _D>>a) the electrostatic free energy difference F ^el _Bz = F ^el _Z - F ^el _B increases with increasing ionic strength since q _B>q_z. At high ionic strengths (when r _D<<a) the F ^el _BZ would go on growing with increasing ionic strength if the inequality q _B/^a _B<q_z/a _z were valid. In the converse case when q _z/q _B<a_z/a _B the F ^el _BZ value decreases with increasing salt concentration at high ionic strength. Since X-ray data correspond to the latter case, theory predicts that the F ^el _BZ value reaches a maximum at an intermediate ionic strength of about 0.1 M (where r _D～a). We also performed rigorous calculations based on the Poisson-Boltzmann equation. These calculations have confirmed the above criterion of nonmonotonous behaviour of the F ^el _BZ value as a function of ionic strength. Different theoretical predictions for the B-Z transition in linear and superhelical molecules are discussed. Theory predicts specifically that at a very low ionic strength the Z form may prove to be more stable than the B form. Thus, one can observe the Z-B-Z transition with increasing ionic strength. In the light of our theoretical findings we discuss numerous experimental data on the B-Z transition in linear and superhelical DNA.     

Changes in chromatin folding in solution

The formation of higher order structures by nucleosome oligomers of graded sizes with increasing ionic strength has been studied in solution, by measuring sedimentation coefficients. Nucleosome monomers and dimers show no effect of ionic strength at the concentrations used, while trimers to pentamers show a linear dependence of the logarithm of sedimentation coefficient upon the logarithm of ionic strength between 5 and 25 mm, but no dependence above 25 mm. Between pentamer and hexamer a change occurs and the linear relationship is observed up to ionic strength 125 mm with hexamer and above.The simple power-law dependence of the sedimentation coefficient upon the ionic strength (s ∝ Iⁿ) is observed up to nucleosome 30mers, but by 60mer a jump in the sedimentation coefficient occurs between ionic strengths 45 and 55 mm, with the power-law applying both above and below the jump. Removal of histone H1 and non-histone proteins lowers the overall sedimentation rate and abolishes the jump.Cross-linking large oligomers at ionic strength 65 mm stabilizes the structure in the conformation found above the jump, leading to a simple power-law dependence throughout the range of ionic strength for cross-linked material. Cleavage of the cross-links restores the jump, presumably by allowing the conformational transition that causes it. Large oligomers are indistinguishable in sedimentation behaviour whether extracted from nuclei at low ionic strength or at 65 mm and maintained in the presence of salt.We interpret these results, together with the detailed electron microscopic studies reported by Thoma et al. (1979) under similar salt conditions, as showing the histone H1-dependent formation of superstructures of nucleosomes in solution induced by increasing ionic strength. The unit of higher order structure probably contains five or six nucleosomes, leading to the change in stability with hexamer. Although this size corresponds to the lower limit of size suggested for “superbeads” (Renz et al., 1977), we see no evidence that multiples of six nucleosomes have any special significance as might be predicted if superbeads had any structural importance. Rather, our results are compatible with a continuous pattern of condensation, such as a helix of nucleosomes (see e.g. Finch & Klug, 1976). The jump in sedimentation observed between ionic strengths 45 and 55 mm, together with the effect of cross-linking, suggests the co-operative stabilization of this structure at higher ionic strengths. A plausible hypothesis is that the turns of the solenoid are not tightly bonded in the axial direction below 45 mm, but come apart due to the hydrodynamic shearing forces in the larger particles leading to less compact structures with slower sedimentation rates. Above 55 mm the axial bonding is strong enough to give a stable structure of dimensions compatible with the 30 nm structures observed in the cell nucleus.     

Energetics at the DNA Supercoiling Transition

Twisting a DNA molecule held under constant tension is accompanied by a transition from a linear to a plectonemic DNA configuration, in which part of the applied twist is absorbed in a superhelical structure. Recent experiments revealed the occurrence of an abrupt extension change at the onset of this transition. To elucidate its origin we study this abrupt DNA shortening using magnetic tweezers. We find that it strongly depends on the length of the DNA molecule and the ionic strength of the solution. This behavior can be well understood in the framework of a model in which the energy per writhe for the initial plectonemic loop is larger than for subsequent turns of the superhelix. By quantitative data analysis, relevant plectoneme energies and other parameters were extracted, providing good agreement with a simple theory. As a direct confirmation of the initial-loop model, we find that for a kinked DNA molecule the abrupt extension change occurs at significantly lower twist than the subsequent superhelix formation. This should allow pinning of the plectoneme position within supercoiled DNA if a kinked substrate is used, and enable the detection of enzymes and proteins which, themselves, bend or kink DNA.     

Variance of Writhe for Wormlike DNA Rings with Excluded Volume

Abstract

We have calculated the variance of the equilibrium distribution of a circular wormlike polymer chain over the writhing number, <((Wr)² )>, with allowance for the excluded volume effects. Within this model the <((Wr)² )> value is a function of the number of Kuhn statistical segments, n, and the chain diameter, d measured in Kuhn statistical lengths, b. Simulated DNA chains varied from 200 to 10,000 base pairs and the d value varied from 0.02 to 0.2. Theory predicts a considerable ionic strength dependence of the DNA superhelix energy as a consequence of the change in the DNA diameter. A comparison with the available experimental data has yielded an estimate of the DNA torsional rigidity, the Kuhn statistical length, and the effective diameter of the double helix under conditions of the complete screening of the DNA electrostatic potential.     

The problems of eukaryotic and prokaryotic DNA packaging and in vivo conformation posed by superhelix density heterogeneity

Systems for gel electrophoresis in the presence of one of the intercalative unwinding ligands, ethidium or chloroquine, have been developed which permit the resolution of highly supercoiled closed circular DNA molecules differing by unit values of the topological winding number, alpha. All native closed circular DNAs examined, including the viral and intracellular forms of SV40 and polyoma DNA, bacterial plasmid DNAs, and the double stranded closed circular DNA genome of the marine bacteriophage, PM2, are more heterogeneous with respect to the number of superhelical turns present than are the thermal distributions observed in the limit products of the action of nicking-closing (N-C) enzyme on the respective DNAs. In the cases of SV40 and polyoma, where it has been shown that the supercoiling is a combined consequence of the binding of the four nucleosomal histones, H2a, H2b, H3 and H4, and the action of N-C enzyme, the breadth of the distributions within the form I DNAs poses specific problems since the work of other laboratories indicates that the number of nucleosomes on the respective minichromosomes falls within a narrow distribution of 21. If it is assumed that all nucleosomes have identical structures, and that the DNA within a nucleosome is not free to rotate, the native DNA would be anticipated to be less heterogeneous than the thermal equilibrium mixtures present in N-C enzyme relaxed SV40 and polyoma DNAs.The absolute number of superhelical turns (at 37 degrees C in 0.2 M NaCl) in virion polyoma DNA has been determined to be 26 +/- 1, which is the same value obtained for virion SV40 DNA. This is consistent with the observations that polyoma DNA has a higher molecular weight, a lower superhelix density, but the same number of nucleosomes as SV40 DNA. In addition, the distributions within the virion and intracellular form I DNAs of both SV40 and polyoma were found to be indistinguishable.Images     

X-Ray Structure of the Nucleosome Core Particle

Abstract

Two monoclinic crystal forms (P2₁,C2) of chicken erythrocyte nucleosomes have been under study in this laboratory. The x-ray structure of the P2₁ crystal form has been solved to 15 Å resolution. The B-DNA superhelix has a relatively uniform curvature, with only several local distortions observed in the superhelix. The individual histone domains have been localized and specific contacts between each histone and the DNA can be observed. Histone contacts to the inner surface of the DNA superhelix occur predominantly at the minor groove sites. Most of the histone core is contained within the inner surface of the superhelical DNA, except for part of H2A which extends between the DNA gyres near the terminus of the DNA. No part of H2A blocks the DNA terminus or would prevent a smooth exit of the DNA into the linker region. A similar extension of a portion of histone H4 between the DNA gyres occurs close to the dyad axis. Both unique nucleosomes in the P2₁ asymmetric unit demonstrate good dyad symmetry and are similar to each other throughout the histone core and DNA regions.     

Analysis of cooperativity and ion effects in the interaction of quinacrine with DNA

The interaction of quinacrine with calf thymus DNA was monitored at several different ionic strengths using spectrophotometric and equilibrium dialysis techniques. The binding results can be explained, assuming each base pair is a potential binding site, using a model containing two negative cooperative effects: (1) ligand exclusion at binding sites adjacent to a filled binding site and (2) ligand–ligand negative cooperativity at adjacent filled binding sites. The logarithm of the observed equilibrium constant (K_obs) determined by this model varies linearily with log[Na⁺], as predicted by the ion condensation theory for polyelectrolytes. When the log K_obs plot is correlated for sodium release by DNA in the intercalation conformational change, the predicted number of ion pairs between the ligand and DNA is approximately two, as expected for the quinacrine dication. Even though K_obs depends strongly on ionic strength, the ligand negative cooperativity parameter ω was found to be indpendent of ionic strength within experimental error. This finding is also in agreement with the ion condensation theory, which predicts a relatively constant amount of condensed counterion on the DNA double helix over this ionic strength range. Drugs would, therefore, experience a relatively constant ionic environment when complexed to DNA even though the ionic conditions of the solvent could change considerably.