Thermodynamics of the ColE1 cruciform. Comparisons between probing and topological experiments using single topoisomers

The sensitivity of the ColE1 cruciform to four enzyme and chemical probes of secondary structure has been studied as a function of plasmid topology. Purified topoisomers of pColIR515 have been probed with S1 nuclease, Bal31 nuclease, phage T4 endonuclease VII or osmium tetroxide, and site-specific reaction quantified. Closely similar profiles of reactivity as a function of linking difference were obtained for each probe. Electrophoresis of the pure topoisomers on polyacrylamide/agarose gels revealed a discontinuity in migration as a function of linking difference. Above a threshold linking difference, topoisomers exhibit pronounced reduction in mobility. The linking difference at which this band shift is found correlates precisely with that required for site-specific reaction with the four probes. We conclude that both probing and topological methods are valuable in the study of cruciform structure in supercoiled DNA. The band shift has been measured with accuracy to allow the calculation of the twist change that accompanies the transition, corresponding to delta Tw = -3.2 +/- 0.1. Using this value together with the critical linking difference we calculate a free energy of formation for this structure delta G = 18.4 +/- 0.5 kcal mol-1 (1 kcal = 4.184 kJ).     

Winding of the DNA helix by divalent metal ions.

When supercoiled pBR322 DNA was relaxed at 0 or 22 degrees C by topoisomerase I in the presence of the divalent cations Ca2+, Mn2+ or Co2+, the resulting distributions of topoisomers observed at 22 degrees C had positive supercoils, up to an average delta Lk value of +8.6 (for Ca2+at 0 degrees C), corresponding to an overwinding of the helix by 0.7 degrees/bp. An increase of the divalent cation concentration in the reaction mixture above 50 mM completely reversed the effect. When such ions were present in agarose electrophoresis gels, they caused a relaxation of positively supercoiled DNA molecules, and thus allowed a separation of strongly positively supercoiled topoisomers. The effect of divalent cations on DNA adds a useful tool for the study of DNA topoisomers, for the generation as well as separation of positively supercoiled DNA molecules.     

Conformational isomerization of the Holliday junction associated with a cruciform during branch migration in supercoiled plasmid DNA

The variable positions of a branch-migrating cruciform junction in supercoiled plasmid DNA were mapped following cleavage of the DNA with bacteriophage T7 endonuclease I. T7 endonuclease I specifically cleaved, and thereby resolved, the Holliday junction existing at the base of the cruciform in the circular bacterial plasmid pSA1B.56A. Cruciform extrusion of cloned sequences in pSA1B.56A (containing a 322 base-pair inverted repeat insert composed of poxvirus telomeric sequences) topologically relaxed the plasmid substrate in vitro. Thus, numerous crossover positions were identified within the region of cloned sequences, reflecting the range of superhelical densities in the native plasmid preparation. Endonuclease I-sensitive crossover positions, mapped to both strands of the viral insert following the T7 endonuclease I digestion of either plasmid preparations or individual topoisomers, were regularly separated by approximately ten nucleotides. The appearance of sensitive crossovers every ten nucleotides corresponds to a change in linking difference (delta Lk) of +/- 2 in the circular core domain of the plasmid during branch point migration. In contrast, individual topoisomers of a plasmid preparation differ in linking number in increments of +/- 1. Thus, the observed linearization of each individual topoisomer following enzyme treatment, as a result of resolution of the crossovers associated with each topoisomer, showed that branch point migration to sensitive crossover positions must have occurred facilely. T7 endonuclease I randomly resolved across either axis of the cruciform, though some discrimination (related to the sequence specificity of the enzyme) was observed. The ten-nucleotide spacing between sensitive crossover positions is accounted for by an isomerization of the cruciform junction on branch point migration. An hypothesis is that this isomerization was imposed upon the cruciform junction by the change in helix twist (delta Tw) in the two branches that compose the topologically closed, circular domain of the plasmid. T7 endonuclease I may discriminate between the various isomeric forms and cleave a sensitive conformation that appears with every turn of branch migration which leads to the extrusion, or absorption, of two turns of helix from the circular core.     

Torsional tension in metaphase chromosome DNA]

The torsional tension in DNA of isolated metaphase chromosomes from murine fibroblasts was measured by the microfluorescent method. The method is based on the ability of a fluorescent dye ethidium bromide to compensate for the negative torsional tension in topologically closed DNA by intercalation between DNA base pairs. The value of the relative twist difference delta Tw/Tw = -0.1 was found in a bulk (about 3/4) of unconstrained chromosomal DNA. In interphase nuclei, the torsionally stressed DNA comprises about 15%, with value of delta Tw/Tw = -0.075. We suppose that the tension in chromosomal DNA was created in the prophase stage of mitosis by condensines, the drivers of chromosomal condensation.     

Interactions of Drosophila DNA topoisomerase II with left-handed Z-DNA in supercoiled minicircles.

The native form of Drosophila melanogaster DNA topoisomerase II was purified from Schneider's S3 tissue culture cells and studied with two supercoiled minicircle preparations, mini and mini-CG, 354 bp and 370 bp in length, respectively. Mini-CG contains a d(CG)7 insert which assumes a left-handed Z-DNA conformation in negative supercoiled topoisomers with a negative linking number difference - delta Lk greater than or equal to 2. The interactions of topoisomerase II with topoisomer families of mini and mini-CG were studied by band-shift gel electrophoresis in which the individual topoisomers and their discrete or aggregated protein complexes were resolved. A monoclonal anti-Z-DNA IgG antibody (23B6) bound and aggregated only mini-CG, thereby confirming the presence of Z-DNA. Topoisomerase II bound and relaxed mini-CG more readily than mini. In both cases, there was a preference for more highly negatively supercoiled topoisomers. The topoisomerase II inhibitor VM-26 induced the formation of stable covalent DNA-protein intermediates. In addition, the non-hydrolyzable GTP analogue GTP gamma S inhibited the binding and relaxation activities. Experiments to detect topoisomerase cleavage sites failed to elicit specific loci on either minicircle preparation. We conclude that Drosophila topoisomerase II is able to bind and process small minicircles with lengths as short as 360 bp and negative superhelix densities, - sigma, which can exceed 0.1. Furthermore, the enzyme has a preferential affinity for topoisomers containing Z-DNA segments and relaxes these molecules, presumably by cleavage external to the inserts. Thus, a potentially functional relationship between topoisomerase II, an enzyme regulating the topological state of DNA-chromatin in vivo, and left-handed Z-DNA, a conformation stabilized by negative supercoiling, has been established.     

Bulge loops used to measure the helical twist of RNA in solution.

Bulge loops are commonly found in helical segments of cellular RNAs. When incorporated into long double-stranded RNAs, they may introduce points of flexibility or permanent bend that can be detected by the altered electrophoretic gel mobility of the RNA. We find that a single An or Un bulge loop near the middle of a long RNA helix significantly retards the RNA during polyacrylamide gel electrophoresis if n greater than or equal to 2. The mobility of an RNA containing two A2 bulges various periodically with the number of base pairs between the bulges. We interpret this to mean that A2 bulges varies periodically with the number of base pairs between the bulges. We interpret this to mean that Z2 bulges form torsionally stiff bends in the helix; the gel mobility reaches a minimum when the total helical twist between the bulges rotates the arms of the molecule into a cis conformation. The gel mobilities are proportional to the predicted end-to-end distance of the RNA if the average RNA helical repeat is 11.8 +/- 0.2 bp/turn and there is no helical twist (3 +/- 9 degrees) associated with the bulge (data obtained in 0.15 M Na+). Other sizes and sequences of bulges have very different effects on RNA helix conformation and flexibility. U2 bulges bend the helix to a much smaller degree than A2 bulges, while longer A or U bulge sequences probably allow bends of 90 degrees or more; all of these may be fairly flexible joints.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Bacillus subtilis primosomal protein DnaD untwists supercoiled DNA

The essential Bacillus subtilis DnaD and DnaB proteins have been implicated in the initiation of DNA replication. Recently, DNA remodeling activities associated with both proteins were discovered that could provide a link between global or local nucleoid remodeling and initiation of replication. DnaD forms scaffolds and opens up supercoiled plasmids without nicking to form open circular complexes, while DnaB acts as a lateral compaction protein. Here we show that DnaD-mediated opening of supercoiled plasmids is accompanied by significant untwisting of DNA. The net result is the conversion of writhe (Wr) into negative twist (Tw), thus maintaining the linking number (Lk) constant. These changes in supercoiling will reduce the considerable energy required to open up closed circular plectonemic DNA and may be significant in the priming of DNA replication. By comparison, DnaB does not affect significantly the supercoiling of plasmids. Binding of the DnaD C-terminal domain (Cd) to DNA is not sufficient to convert Wr into negative Tw, implying that the formation of scaffolds is essential for duplex untwisting. Overall, our data suggest that the topological effects of the two proteins on supercoiled DNA are different; DnaD opens up, untwists and converts plectonemic DNA to a more paranemic form, whereas DnaB does not affect supercoiling significantly and condenses DNA only via its lateral compaction activity. The significance of these findings in the initiation of DNA replication is discussed.     

Chromatin reconstitution on small DNA rings. V. DNA thermal flexibility of single nucleosomes.

The thermal flexibility of DNA minicircles reconstituted with single nucleosomes was measured relative to the naked minicircles. The measurement used a new method based on the electrophoretic properties of these molecules, whose mobility strongly depended on the DNA writhe, either of the whole minicircle, when naked, or of the extranucleosomal loop, when reconstituted. The experiment was as follows. The DNA length was first increased by one base-pair (bp), and the correlative shift in mobility resulting from the altered DNA writhe was recorded. Second, the gel temperature was increased so that the former mobility was restored. Under these conditions, the untwisting of the thermally flexible DNA due to the temperature shift exactly compensates for the increase in the DNA mean twist number resulting from the one bp addition. The relative thermal flexibility was then calculated as the ratio between the increases in temperature measured for the naked and the reconstituted DNAs, respectively. The figure, 0.69 (+/- 0.07), was used to derive the length of DNA in interaction with the histones, 109 (+/- 25) bp. Such length was in good agreement with the mean value of 115 bp we have previously obtained from the distribution of the angles between DNAs at the entrance and exit of similar nucleosomes measured from high resolution electron microscopy. This consistency further reinforces our previous conclusion that minicircle-reconstituted nucleosomes, with 1.3(109/83) to 1.4(115/83) turns of superhelical DNA, show no crossing of entering and exiting DNAs when the loop is in its most probable configuration, and therefore, that these nucleosomes behave topologically as "single-turn" particles. The present data are also within the range of values, 50 to 100 bp of thermally rigid DNA per nucleosome, obtained by others for yeast plasmid chromatin, suggesting that the "single-turn" particle notion may be extended to this particular case of naturally-occurring H1-free chromatin. However, these data are quite different from the 230 bp figure derived from thermal measurements of reconstituted H1-free minichromosomes. It is proposed that nucleosome interactions occurring in this chromatin, but not in yeast chromatin, may be partly responsible for the discrepancy.     

Effect of trypsinization and histone H5 addition on DNA twist and topology in reconstituted minichromosomes.

Free DNA in solution exhibits an untwisting of the double helix with increasing temperature. We have shown previously that when DNA is reconstituted with histones to form nucleosome core particles, both the core DNA and the adjacent linker DNA are constrained from thermal untwisting. The origin of this constraint is unknown. Here we examine the effect of two modifications of nucleosome structure on the constraint against thermal untwisting, and also on DNA topology. In one experiment, we removed the highly positively charged histone amino and carboxy termini by trypsinization. Alternatively, we added histone H5, a histone H1 variant from chick erythrocytes. Neither of these modifications had any major effect on DNA topology or twist in the nucleosome.     

The elastic rod model for DNA and its application to the tertiary structure of DNA minicircles in mononucleosomes.

Explicit solutions to the equations of equilibrium in the theory of the elastic rod model for DNA are employed to develop a procedure for finding the configuration that minimizes the elastic energy of a minicircle in a mononucleosome with specified values of the minicircle size N in base pairs, the extent w of wrapping of DNA about the histone core particle, the helical repeat h(0)b of the bound DNA, and the linking number Lk of the minicircle. The procedure permits a determination of the set Y(N, w, h(0)b) of integral values of Lk for which the minimum energy configuration does not involve self-contact, and graphs of writhe versus w are presented for such values of Lk. For the range of N of interest here, 330 < N < 370, the set Y(N, w, h(0)b) is of primary importance: when Lk is not in Y(N, w, h(0)b), the configurations compatible with Lk have elastic energies high enough to preclude the occurrence of an observable concentration of topoisomer Lk in an equilibrium distribution of topoisomers. Equilibrium distributions of Lk, calculated by setting differences in the free energy of the extranucleosomal loop equal to differences in equilibrium elastic energy, are found to be very close to Gaussian when computed under the assumption that w is fixed, but far from Gaussian when it is assumed that w fluctuates between two values. The theoretical results given suggest a method by which one may calculate DNA-histone binding energies from measured equilibrium distributions of Lk.     

Torsional rigidity of DNA and length dependence of the free energy of DNA supercoiling

By analyzing the Boltzmann populations of DNA topoisomers that differ only in their linking numbers, the dependence of the free energy delta G tau of DNA supercoiling on the linking number alpha has been determined for DNA rings as small as 200 base-pairs (bp) in length. All experimental data can be fitted by the relation delta G tau = K (alpha-alpha)2, where alpha is a constant for a given DNA at a given set of conditions and K is a DNA length-dependent proportionality constant. For DNA rings with length N larger than 2000 bp, K is inversely proportional to N and the product NK is nearly a constant around 1150 RT X bp. For rings smaller than 2000 bp NK increases steadily with decreasing N; for a 200 bp ring NK is 3900 RT X bp. The increase in NK when N decreases can be interpreted as a result of the decrease in the contribution of the fluctuation in the writhing number to the equilibrium distribution in alpha. Assuming that the writhing contribution approaches zero for DNA rings 200 bp in size, the torsional rigidity of the DNA double helix is calculated to be 2.9 X 10(-19) erg cm. In addition, the large value of K for the small circles allows precise calculation of the helical repeat of DNA. For the 210 bp rings, the repeat is measured to be 10.54 bp.     

Thermodynamics of Long Supercoiled Molecules: Insights from Highly Efficient Monte Carlo Simulations

Supercoiled DNA polymer models for which the torsional energy depends on the total twist of molecules (Tw) are a priori well suited for thermodynamic analysis of long molecules. So far, nevertheless, the exact determination of Tw in these models has been based on a computation of the writhe of the molecules (Wr) by exploiting the conservation of the linking number, Lk = Tw + Wr, which reflects topological constraints coming from the helical nature of DNA. Because Wr is equal to the number of times the main axis of a DNA molecule winds around itself, current Monte Carlo algorithms have a quadratic time complexity, O(L²), with respect to the contour length (L) of the molecules. Here, we present an efficient method to compute Tw exactly, leading in principle to algorithms with a linear complexity, which in practice is O(L^1.2). Specifically, we use a discrete wormlike chain that includes the explicit double-helix structure of DNA and where the linking number is conserved by continuously preventing the generation of twist between any two consecutive cylinders of the discretized chain. As an application, we show that long (up to 21 kbp) linear molecules stretched by mechanical forces akin to magnetic tweezers contain, in the buckling regime, multiple and branched plectonemes that often coexist with curls and helices, and whose length and number are in good agreement with experiments. By attaching the ends of the molecules to a reservoir of twists with which these can exchange helix turns, we also show how to compute the torques in these models. As an example, we report values that are in good agreement with experiments and that concern the longest molecules that have been studied so far (16 kbp).     

The effect of changes in the bulk dielectric constant on the DNA torsional properties

The effect of changes in the bulk dielectric constant on the DNA torsional properties was evaluated from plasmid circularization reactions. In these reactions, pUC18 previously linearized by EcoRI digestion was recircularized with T4 DNA ligase. The bulk dielectric constant of the reaction medium was decreased by the addition of different concentrations of neutral solutes: ethylene glycol, glycerol, sorbitol, and sucrose, or increased by the addition of glycine. The topoisomers generated by the ligase reaction were resolved by agarose-gel electrophoresis. The DNA twist energy parameter (kappa), which is an apparent torsional constant, was determined by linearization of the Gaussian topoisomers' distribution. It was observed that the twist energy parameter for the given solutes is almost linearly dependent on the bulk dielectric constant. In the reaction buffer, the twist energy parameter was determined to be 1100 +/- 100. By decreasing the dielectric constant to 74 with the addition of sorbitol, the value of the parameter reaches kappa = 900 +/- 100, whereas the addition of ethylene glycol leads to kappa = 400 +/- 50. Upon addition of glycine, which resulted in a dielectric constant equal to 91, the value of the twist energy parameter increased to kappa = 1750 +/- 100.     

Effect of Bending Strain on the Torsion Elastic Constant of DNA

The torsion constants of both circular and linear forms of the same 181 bp DNA were investigated by time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium. The ratio of intrinsic ethidium binding constants of the circular and linear species was determined from the relative fluorescence intensities of intercalated and non-intercalated dye in each case. Possible changes in secondary structure were also probed by circular dichroism (CD) spectroscopy. Upon circularization, the torsion constant increased by a factor of 1.42, the intrinsic binding constant for ethidium increased by about fourfold, and the CD spectrum underwent a significant change. These effects are attributed to an altered secondary structure induced by the bending strain. Quantitative agreement between torsion constants obtained from the present FPA studies and previous topoisomer distribution measurements on circular DNAs containing 205 to 217 bp removes a long-standing apparent discrepancy between those two methods. After storage at 4°C for eight months, the torsion constant of the circular DNA increased by about 1.25-fold, whereas that of the linear DNA remained unchanged. For these aged circles, both the torsion constant and intrinsic binding constant ratio lie close to the corresponding values obtained previously for a 247 bp DNA by analyzing topoisomer distributions created in the presence of various amounts of ethidium. The available evidence strongly implies that torsion constants measured for small circular DNAs with less than 250 bp are specific to the altered secondary structure(s) therein, and are not applicable to linear and much larger circular DNAs with lower mean bending strains.     

Torsional and bending rigidity of the double helix from data on small DNA rings

We have calculated the variance of equilibrium distribution of a circular wormlike polymer chain over the writhing number, [Wr)2), as a function of the number of Kuhn statistical segments, n. For large n these data splice well with our earlier results obtained for a circular freely jointed polymer chain. Assuming that [delta Lk)2) = [delta Tw)2) we have compared our results with experimental data on the chain length dependence of the [delta Lk)2) value recently obtained by Horowitz and Wang for small DNA rings. This comparison has shown an excellent agreement between theory and experiment and yielded a reliable estimate of the torsional and bending rigidity parameters. Namely, the torsional rigidity constant is C = 3.0.10(-19) erg cm, and the bending rigidity as expressed in terms of the DNA persistence length is a = 500 A. The obtained value of C agrees well with earlier estimates by Shore and Baldwin as well as by Horowitz and Wang whereas the a value is in accord with the data of Hagerman. We have found the data of Shore and Baldwin on the chain length dependence of the [delta Lk)2) value to be entirely inconsistent with our theorectical results.     

Sequence-dependent nucleosome structural and dynamic polymorphism. Potential involvement of histone H2B N-terminal tail proximal domain

Relaxation of nucleosomes on an homologous series (pBR) of ca 350-370 bp DNA minicircles originating from plasmid pBR322 was recently used as a tool to study their structure and dynamics. These nucleosomes thermally fluctuated between three distinct DNA conformations within a histone N-terminal tail-modulated equilibrium: one conformation was canonical, with 1.75 turn wrapping and negatively crossed entering and exiting DNAs; another was also "closed", but with these DNAs positively crossed; and the third was "open", with a lower than 1.5 turn wrapping and uncrossed DNAs. In this work, a new minicircle series (5S) of similar size was used, which contained the 5S nucleosome positioning sequence. Results showed that DNA in pBR nucleosomes was untwisted by approximately 0.2 turn relative to 5S nucleosomes, which DNase I footprinting confirmed in revealing a approximately 1 bp untwisting at each of the two dyad-distal sites where H2B N-terminal tails pass between the two gyres. In contrast, both nucleosomes showed untwistings at the dyad-proximal sites, i.e. on the other gyre, which were also observed in the high-resolution crystal structure. 5S nucleosomes also differ with respect to their dynamics: they hardly accessed the positively crossed conformation, but had an easier access to the negatively crossed conformation. Simulation showed that such reverse effects on the conformational free energies could be simply achieved by slightly altering the trajectories of entering and exiting DNAs. We propose that this is accomplished by H2B tail untwisting at the distal sites through action at a distance ( approximately 20 bp) on H3-tail interactions with the small groove at the nucleosome entry-exit. These results may help to gain a first glimpse into the two perhaps most intriguing features of the high-resolution structure: the alignment of the grooves on the two gyres and the passage of H2B and H3 N-terminal tails between them.