Circular dichroism of superhelical DNA

The circular dichroism (CD) spectra of a number of superhelical DNA's have been measured. The introduction of negative superhelical turns causes an increase in magnitude of the positive band around 280 mμ, while the trough around 250mμ is little affected. For two samples of λb2b5c DNA (20 Mdalton) containing different number of negative superhelical turns, the magnitude of the positive band relative to that of the nicked control increases with increasing number of superhelical turns. In 2M NaCl, the small (1.45 Mdalton) superhelical DNA from E. coli 15 shows an unusually large difference in CD compared with that of the same DNA with a few single-chain scissions per molecule. This large difference is not observed in a medium containing p. 0.11M NaCl. These results indicate that the double helix in a superhelical DNA is perturbed somewhat due to the bending and torsional forces in such a molecule. The magnitude of such structural alteration seems to depend on the number of superhelical turns per unit length, the size of the DNA molecule, as well as the ionic medium.     

Further analysis of the altered secondary structure of superhelical DNA. Sensitivity to methylmercuric hydroxide a chemical probe for unpaired bases

A previous study in our laboratory of the reaction of formaldehyde with super-helical DNAs (φX replicative form and PM2) has led to a model for superhelical DNA in which there is a region or regions of altered secondary structure containing unpaired bases. Similar experiments using the nicked circular DNA gave no evidence of interruptions of base pairing. In this study we present additional data, which support the above model as well as extending our analysis of the secondary structure of superhelical DNA and the dynamics of the early denaturation process. In a series of experiments involving the binding of methyl-mercury as a chemical probe of unpaired bases, we obtained the following results. (1) Initially, both s⁰_20w and the buoyant density of the superhelical form of phage PM2 DNA increased as a function of methylmercuric hydroxide concentration, whereas the nicked form did not. (2) This initial binding is accompanied by an increase in superhelical content τ from ?41 to ?46 turns. (3) The binding analysis allows us to estimate that 3.7% of the bases contain methylmercury in this phase of the transition. This is in excellent agreement with the extent of formylation. (4) Such a preformylated molecule shows a shift in the transition to lower mercurial concentrations. These results are interpreted as follows. The initial increase in ?τ excludes the possibility that binding occurs to normal base-paired structures, since this would produce a coupled unwinding of duplex and superhelical turns. The additive effects of formylation and methylmercury binding support the concept that both chemical probes attack the same sites and induce similar structural changes. Thus the evidence clearly supports the view that superhelical DNA contains localized region(s) of interrupted base pairing. Recent studies from other laboratories using single strand-specific endonucleases are in complete agreement with this model.     

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.     

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.     

Cross-linking and relaxation of supercoiled DNA by psoralen and light.

Photoreaction of 4,5',8-trimethylpsoralen with superhelical ColE1 and ColE1amp DNA was studied. Changes in mobilities in agarose gels, formation of interstrand cross-links, and DNA strand breaks were determined. Psoralen and light treatment removed negative superhelical turns, and extensive treatments failed to produce positive superhelical turns in covalently closed plasmid DNA. The rate of relaxation of superhelical turns by psoralen Photobinding appeared to be directly proportional to the number of superhelical turns remaining. A unique reaction mechanism is presented to explain these results. By this interpretation the initial rate of psoralen photobinding to superhelical DNA was estimated to be 3 times that for linear DNA, and the ratio of cross-linking to monofuctional adducts appears to be dependent on the superhelical conformation of the DNA. The estimated ratio of psoralen molecules bound to DNA strand breaks was 1.7 . 10(4):1, and 70% of this breakage is caused by the light alone.     

Supercoils in human DNA.

The three-dimensional structure of a double-stranded DNA molecule may be described by distinguishing the helical turns of the DNA duplex from any superhelical turns that might be superimposed upon the duplex turns. There are characteristic changes in the hydrodynamic properties of superhelical DNA molecules when they interact with intercalating agents. The hydrodynamic properties of nuclear structures released by gently lysing human cells are changed by intercalating agents in this characteristic manner. The characteristic changes are abolished by irradiating the cells with gamma-rays but may be restored by incubating the cells at 37 degrees C after irradiation. These results are interpreted as showing that human DNA is supercoiled. A model for the structure of the chromosome is suggested.     

The number of superhelical turns in native virion SV40 DNA and minicol DNA determined by the band counting method.

By a method of overlapping the results obtained after agarose gel electrophoresis under two different sets of conditions, it has become possible to determine the number of superhelical turns in a given DNA by counting the bands present after partially relaxing the DNA (Keller and Wendel, 1974) with highly purified nicking-closing (N-C) enzyme from LA9 mouse cell nuclei. Because native supercoiled DNA is heterogeneous with respect to superhelix density, an average number of superhelical turns was determined. Virion SV40 DNA contains 26 +/- 0.5 superhelical turns, and native Minicol DNA contains 19 +/- 0.5 superhelical turns. The above are values at 0.2 M NaCl and at 37 degrees C, the condition under which the enzymatic relaxations were performed. The superhelix densities determined by the band counting method have been compared with superhelix densities determined by buoyant equilibrium in PDl-CsCl gradients. The Gray, Upholt, and Vinograd (1971) calculation procedure has been used for evaluating the superhelix densities by the latter method with the new statement, however, that relaxed DNA has zero superhelical turns. Comparison of the superhelix densities obtained by both methods permits a calculation of an unwinding angle for ethidium. The mean value from experiments with SV40 DNA is 23 +/- 3 degree. The average number of superhelical turns in SV40, 26, combined with the value, 21, obtained by both Griffith (1975) and Germond et al. (1975) for the average number of nucleosomes per SV40 genome, yields an average of 1.25 superhelical turns per 1/21 of the SV40 genome. If the regions of internucleosomal DNA are fully relaxed, 1.25 correesponds to the average number of superhelical turns with a nucleosome. When analyzed under identical conditions, the limit product generated by ligating a nicked circular substrate in the presence of 0.001 M Mg2+ at 37 degrees C (ligation conditions) is slightly more positively supercoiled than the limit product obtained when the N-C reaction is performed in 0.2 M NaCl at 37 degrees C. The difference in superhelix density as measured in gels between the two sets of limit products for both Minicol and SV40 DNAs is 0.0059 +/- 0.0005. This result indicates that the DNA duplex is overwound in the ligation solvent relative to its state in 0.2 M NaCl.     

Conformational variation in superhelical deoxyribonucleic acid.

Sedimentation experiments have shown that superhelical DNA undergoes a sharp structural transition at low ionic strength. Light-scattering experiments show that this is due to a change in conformation of the DNA rather than to a change in interactions among DNA molecules. The results show that two possible conformations can occur for superhelical DNA under routine experimental conditions and may explain the discrepancies in the number of early unwinding sites exposed by different techniques.     

Purification and demonstration of the enzymatic character of the nicking-closing protein from mouse L cells

A rapid procedure for the purification of the nicking-closing enzyme from mouse L cells is described. The procedure reproducibly provides high yields of enzyme. A purity of greater than 90% is obtained in five steps. The enzymatic character of the nicking-closing activity has been demonstrated. On the average 20 PM2 DNA I molecules are completely relaxed by one enzyme molecule. The enzyme releases superhelical turns from closed circular DNA by providing a swivel through a sequence of successive nicking and closing events. It is not known yet whether the release of superhelical turns proceeds via a one hit or a multiple hit mechanism.     

Light-scattering studies on supercoil unwinding

It has been shown previously that supercoiled [unk]X174 bacteriophage intracellular DNA (mol.wt. 3.2x10(6)) with superhelix density, sigma=-0.025 (-12 superhelical turns) at 25 degrees C is best represented as a Y shape. In this work two techniques have been used to unwind the supercoil and study the changes in tertiary structure which result from changes in the secondary structure. The molecular weights from all experiments were in the range 3.2x10(6)+/-0.12x10(6). In experiments involving temperature change little change in the Y shape was observed between sigma=-0.027 (-13 superhelical turns, 14.9 degrees C) and sigma=-0.021 (-10 superhelical turns, 53.4 degrees C) as evidenced by the root-mean-square radius and the particle-scattering factor P(theta). However, at sigma=-0.0176 (-8 superhelical turns, 74.5 degrees C) the root-mean-square radius fell to between 60 and 70nm from 90nm indicating a large structural change, as did alterations in the P(theta) function. In experiments with the intercalating dye proflavine from values of bound proflavine of 0-0.06mol of dye/mol equiv. of nucleotide which correspond to values of sigma from -0.025 to -0.0004 (-12 to 0 superhelical turns) a similar transition was found when the superhelix density was changed by the same amount, and the molecule was shown to go through a further structural change as the unwinding of the duplex proceeded. At sigma=-0.018 (-9 superhelical turns) the structure was compatible with a toroid, and at sigma=-0.0004 it was compatible with a circle but at no point in the sequence of structure transitions was the structure compatible with the conventional straight interwound model normally visualized as the shape of supercoiled DNA.     

Supercoiled DNA is interwound in liquid crystalline solutions.

Two structures have been proposed for supercoiled DNA: it is idealized either as a toroidal ring or as a rod of two interwound duplex chains. The latter model is the most widely depicted but the evidence remains controversial. We have worked with monomers and dimers of two plasmids, pUC8 and pKS414, of similar size and natural superhelical density. pKS414 contains a bend promoting sequence whereas pUC8 does not. In concentrated solutions these plasmids form a partially ordered liquid crystalline phase which is found, using neutron diffraction, to consist of a hexagonally packed assembly of parallel rod-like particles. This shape strongly suggests an interwound conformation for which some structural parameters are deduced. The mass/unit length obtained by combining the area of the hexagonal lattice and the concentration is approximately 3.6 times that of linear DNA. This implies a shallow superhelical pitch angle approximately 36 degrees which, when combined with the known number of supercoil turns, yields the pitch approximately 360 A and radius approximately 80 A for the supercoil. Oriented X-ray fibre diffraction patterns at 92% relative humidity indicate a B type duplex structure. Nicked circular plasmids also form liquid crystals but their behaviour, as a function of concentration, differs from that of the superhelical plasmids.     

Interaction between DNA and an Escherichia coli protein omega

An E. coli protein, designated ω, has been purified at least 1000-fold. Treatment of a eovalently closed DNA duplex containing negative superhelical turns with ω results in the loss of most of the superhelical turns. The loss of superhelical turns follows a gradual course rather than a one-hit mechanism. This reaction does not require a cofactor. No other change in the physical properties of the DNA could be detected. The DNA remains covalently closed. Its ultraviolet absorption spectrum, circular dichroism, buoyant density in CsCl, sedimentation properties in neutral media containing varying amounts of ethidium and in an alkaline medium, and its susceptibility toward Neurospora endonuclease, are not significantly different from an untreated DNA containing the same number of superhelical turns. Thus it appears that ω is capable of introducing a “swivel” reversibly into a DNA. A plausible mechanism is postulated.     

Enzymatic cleavage of superhelical DNA in a liquid crystal state]

Superhelical pBR322 DNA molecules form liquid-crystalline dispersions in water-salt solutions containing poly(ethyleneglycol). The formation of the liquid-crystalline dispersions from superhelical DNA molecules results in the appearance of two sites inside the DNA molecules that are split by Micrococcal nuclease. The first site of digestion does not differ from the standard site split by this enzyme in water-salt solutions, whereas the second one represents a new site specific only for the DNA molecules forming liquid-crystalline dispersions. Splitting of the DNA molecule through the first site is accompanied by formation of its linear form; splitting of a new site results in the formation of two linear DNA fragments with molecular masses equal to half of the initial DNA molecules. Enzyme digestion of superhelical DNA molecules forming liquid-crystalline dispersions induces a reformation of the "nonspecific" space organization of dispersions to the cholesteric one. A hypothetic model for packing of the superhelical DNA molecules inside liquid-crystalline dispersions and its transformation under enzyme action is suggested.     

Simian virus 40 DNA extracted from infected cells with sodium deoxycholate no longer reflects its in vivo superhelix density.

When simian virus 40 DNA is extracted from infected cells with low concentrations of sodium deoxycholate, which selectively extract non-encapsidated simian virus 40 DNA, the DNA has a lower average number of superhelical turns than the DNA extracted from purified viral particles. During extraction, a partial deproteinization of the DNA by a concentration of detergent that did not inactivate a nicking-closing activity led to the removal of some superhelical turns. The DNA extracted in this way no longer reflected its in vivo number of superhelical turns.     

Linker histone interaction shows divalent character with both supercoiled and linear DNA

The interaction of linker histone H1 with both linear and superhelical double-stranded DNA has been investigated at low ionic strengths. Gel mobility retardation experiments demonstrate strikingly different behavior for the two forms of DNA. First, the experiments strongly suggest that linker histone binds to superhelical DNA in a negatively cooperative mode. In contrast, binding of linker histone to linear DNA under the conditions employed here shows no cooperativity. Second, binding of linker histone to linear DNA results in aggregation of histone-DNA complexes, even at very low levels of input histone H1. Because H1 has been shown to interact as a monomer, this aggregation is evidence of the divalent character of the linker histone, for without H1's ability to bind to two duplex strands of DNA, aggregation could not occur. Although aggregation can be made to occur with superhelical DNA, it can do so only at near-saturation levels of input histone H1. Finally, in direct competition, linker histone binds to superhelical DNA to the complete exclusion of linear DNA, indicating that the linker histone's function is related to the crossover structures that differentiate superhelical DNA from linear DNA. We develop a model that explains the observed behavior of binding of linker histone to superhelical DNA that is consistent with both the divalent character of the linker histone and the negative cooperativity by which linker histone and superhelical DNA interact.     

Sedimentation coefficient of polyoma virus DNA

The sedimentation coefficient of the twisted circular form of polyoma virus DNA is calculated from the Kirkwood sedimentation–diffusion equation, the structure being assumed to be a rigid double superhelix. Agreement with the experimental sedimentation coefficient can be obtained, with the use of an experimental value for the number of superhelical turns, when the pitch of the superhelix is intermediate between its minimal and maximal possible values. Another model, which has been proposed for polyoma DNA at low ionic strengths, may be visualized as a superhelical structure wound about a torus. Calculations of sedimentation coefficients for this model agree qualitatively with experimental data at ionic strengths Below 10^?2M.     

The use of Haemophilus gallinarum DNA-relaxing enzyme to investigate the relationship between the number of superhelical turns and the molecular weight in a negatively twisted DNA.

Covalently closed-circular, superhelical DNAs, including viral DNAs, bacterial plasmid DNAs, and bacteriophage replicative-form DNA, were treated with a small amount of Haemophilus gallinarum DNA-relaxing enzyme to generate incompletely relaxed DNA molecules. Each sample consisted of a set of closed-circular DNA molecules differing by one turn in their number of superhelical turns. The DNA samples were analyzed by agarose gel electrophoresis under conditions such that the electrophoretic mobility was a function of the number of turns. The numbers of superhelical turns (at 37 degrees C in 20 mM Tris-HCl (pH 7.5)-5 mM MgCl2) in the DNAs of pSC101 (5.8 megadaltons), Colicin E1 (4.2 megadaltons), pMR4 (4.0 megadaltons; recombinant between pBR322 and lambda DNA fragment), phi X174 replicative-form (RF) I, Simian virus 40 (SV40), and polyoma virus (3.4--3.6 megadaltons each), and lambda dv021 (2.05 megadaltons) were estimated to be 36, 27, 23--24, 20--21, 20--21, 20--21, and 11--13, respectively. It appears that the number of superhelical turns is mainly a function of the molecular weight of the DNA, at least in the substrates tested here.     

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