Plasmid migration using orthogonal-field-alternation gel electrophoresis.

The migration properties of a series of supercoiled plasmids ranging in size from 4 to 16 kilobases (kb) have been analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE). These circular DNAs enter the gel and are well resolved. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a wide range of pulse times, from 10 to 120 seconds, as well as over a broad range of total running times, from 6 to 24 hours. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of pBR322 shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.     

Effect of ethidium on the torsion constants of linear and supercoiled DNAs.

The torsion elastic constants (alpha) of linear pBR322 (4363 bp) and pUC8 (2717 bp) DNAs and supercoiled pBR322 and pJMSII (4375 bp) DNAs are measured in 0.1 M NaCl as a function of added ethidium/base-pair (EB/BP) ratio by studying the fluorescence polarization anisotropy (FPA) of the intercalated ethidium. The time-resolved FPA is measured by using a picosecond dye laser for excitation and time-correlated single photon counting detection. Previously developed theory for the emission anisotropy is generalized to incorporate rotations of the transition dipole due to excitation transfer. The excitation transfers are simulated by a Monte Carlo procedure (Genest et al., Biophys. Chem. 1 (1974) 266-278) and the consequent rotations of the transition dipole are superposed on the Brownian rotations. After accounting for excitation transfer, the torsion constants of the linear DNAs are found to be essentially independent of intercalated ethidium up to a binding ratio r = 0.10 dye/bp. Dynamic light scattering measurements on linear pUC8 DNA confirm that the torsion constant is independent of binding ratio up to r = 0.20 dye/bp. If alpha d denotes the torsion constant between ethidium and a base-pair, and alpha 0 that between two base-pairs, then our data imply that alpha d/alpha 0 lies in the range 0.65 to 1.64 with a most probable value of 1.0. The torsion constants of supercoiled DNAs decrease substantially with increasing binding ratio even after accounting for excitation transfer. At the binding ratio r* = 0.064, where the superhelix density vanishes and superhelical strain is completely relaxed, the torsion constant of the supercoiled pBR322 DNA/dye complex lies below that of the corresponding linear DNA/dye complex by about 30%. This contradicts the conventional view according to which linear, nicked circular, and supercoiled DNA/dye complexes with r = r* should coexist with the same concentration of free dye, display the same distribution of bound dye, and exhibit identical secondary structures, twisting and bending rigidities, and FPA dynamics. These and other observations imply the existence of metastable secondary structure in freshly relaxed supercoiled DNAs. A tentative explanation is presented for these and other unexpected observations on supercoiled DNAs.     

Effects of chloroquine on the torsional dynamics and rigidities of linear and supercoiled DNAs at low ionic strength

The magnitude and uniformity of the torsion elastic constant (alpha) of linear and supercoiled pBR322 DNAs are measured in 3 mM Tris as a function of added chloroquine/basepair ratio (chl/bp) by studying the fluorescence polarization anisotropy of intercalated ethidium dye. The time-resolved FPA is measured using a picosecond dye-laser for excitation and time-correlated single-photon counting detection. For both linear and supercoiled DNAs, alpha remains uniform except at the very highest chl/bp ratio examined. For the linear DNA, alpha decreases from 5.0 x 10(-12) dyne-cm at chl/bp = 0 to about 3.5 x 10(-12) dyne-cm at chl/bp = 0.5, and remains at that value up to chl/bp = 5, whereupon it increases back up to its original value. For the supercoiled DNA, alpha remains constant at about 5.2 x 10(-12) dyne-cm from chl/bp = 0 up to chl/bp = 5, whereupon it increases in parallel with the linear DNA. The effect of chloroquine on the secondary structure, torsion constant, and torsional dynamics evidently differs substantially between linear and supercoiled DNAs, even under conditions where the supercoiled DNA is completely relaxed and both DNAs bind the same amount of dye. This strongly contradicts any notion that the local structures of linear and relaxed supercoiled DNA/dye complexes with the same binding ratio are identical. The increase in apparent alpha at chl/bp = 5 for both DNAs may be due to stacking of the chloroquine in the major groove and consequent stiffening of the filament.     

On the origin of the temperature dependence of the supercoiling free energy.

Monte Carlo simulations using temperature-invariant torsional and bending rigidities fail to predict the rather steep decline of the experimental supercoiling free energy with increasing temperature, and consequently fail to predict the correct sign and magnitude of the supercoiling entropy. To illustrate this problem, values of the twist energy parameter (E(T)), which governs the supercoiling free energy, were simulated using temperature-invariant torsion and bending potentials and compared to experimental data on pBR322 over a range of temperatures. The slope, -dE(T)/dT, of the simulated values is also compared to the slope derived from previous calorimetric data. The possibility that the discrepancies arise from some hitherto undetected temperature dependence of the torsional rigidity was investigated. The torsion elastic constant of an 1876-bp restriction fragment of pBR322 was measured by time-resolved fluorescence polarization anisotropy of intercalated ethidium over the range 278-323 K, and found to decline substantially over that interval. Simulations of a 4349-bp model DNA were performed using these measured temperature-dependent torsional rigidities. The slope, -dE(T)/dT, of the simulated data agrees satisfactorily with the slope derived from previous calorimetric measurements, but still lies substantially below that of Duguet's data. Models that involve an equilibrium between different secondary structure states with different intrinsic twists and torsion constants provide the most likely explanation for the variation of the torsion constant with T and other pertinent observations.     

Dynamics of supercoiled and linear pTZ18U plasmids observed with a long-lifetime metal-ligand complex

The metal-ligand complex, [Ru(bpy)2(dppz)]2+ (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) (Ru-BD), was used as a spectroscopic probe for studying nucleic acid dynamics. The Ru-BD complex displays a long lifetime of over 100 ns and a molecular light switch property upon DNA binding due to shielding of its dppz ligand from water. To further show the usefulness of this luminophore (Ru-BD) for probing DNA dynamics, we examined its intensity and anisotropy decays when intercalated into supercoiled and linear pTZ18U plasmids using frequency-domain fluorometry with a light-emitting diode (LED) as the modulated light source. Compared to the supercoiled plasmids with an average intensity decay time of 120.8 ns at 25 degrees C, we obtained somewhat longer lifetimes for the linear plasmids ((tau) = 141.4 ns at 25 degrees C), suggesting a more efficient shielding from water by the linear plasmids. The anisotropy decay data also showed longer rotational correlation times for the linear plasmids (495 and 35 ns at 25 degrees C) as compared to the supercoiled plasmids (412 and 27 ns at 25 degrees C). The slow and fast rotational correlation times appear to be consistent with the bending and torsional motions of the plasmids, respectively. The anisotropy values were quite similar, although the values of the supercoiled plasmids were slightly higher in both the steady-state and anisotropy decay measurements. These results indicate that Ru-BD can be applied in the study of both bending and torsional dynamics of nucleic acids.     

DNA gyrase can supercoil DNA circles as small as 174 base pairs.

DNA gyrase introduces negative supercoils into closed-circular DNA using the free energy of ATP hydrolysis. Consideration of steric and thermodynamic aspects of the supercoiling reaction indicates that there should be a lower limit to the size of DNA circle which can be supercoiled by gyrase. We have investigated the supercoiling reaction of circles from 116-427 base pairs (bp) in size and have determined that gyrase can supercoil certain relaxed isomers of circles as small as 174 bp, dependent on the final superhelix density of the supercoiled product. Furthermore, this limiting superhelical density (-0.11) is the same as that determined for the supercoiling of plasmid pBR322. We also find that although circles in the range 116-152 bp cannot be supercoiled, they can nevertheless be relaxed by gyrase when positively supercoiled. These data suggest that the conformational changes associated with the supercoiling reaction can be carried out by gyrase in a circle as small as 116 bp. We discuss these results with respect to the thermodynamics of DNA supercoiling and steric aspects of the gyrase mechanism.     

Monte Carlo simulations of supercoiling free energies for unknotted and trefoil knotted DNAs.

A new Monte Carlo (MC) algorithm is proposed for simulating inextensible circular chains with finite twisting and bending rigidity. This new algorithm samples the relevant Riemann volume elements in a uniform manner, when the constraining potential vanishes. Simulations are performed for filaments comprising 170 subunits, each containing approximately 28 bp, which corresponds to a DNA length of 4770 bp. The bending rigidity is chosen to yield a persistence length, P = 500 A, and the intersubunit potential is taken to be a hard-cylinder potential with diameter d = 50 A. This value of d yields the same second virial coefficient as the electrostatic potential obtained by numerical solution of the Poisson-Boltzmann equation for 150 mM salt. Simulations are performed for unknotted circles and also for trefoil knotted circles using two different values of the torsional rigidity, C = (2.0 and 3.0) x 10(-19) dyne cm2. In the case of unknotted circles, the simulated supercoiling free energy varies practically quadratically with linking difference delta l. The simulated twist energy parameter ET, its slope dET/dT, and the mean reduced writhe <w>/delta l for C = 3 x 10(-19) dyne cm2 all agree well with recent simulations for unknotted circles using the polygon-folding algorithm with identical P, d, and C. The simulated ET vs. delta l data for C = 2.0 x 10(-19) dyne cm2 agree rather well with recent experimental data for p30 delta DNA (4752 bp), for which the torsional rigidity, C = 2.07 x 10(-19) dyne cm2, was independently measured. The experimental data for p30 delta are enormously more likely to have arisen from C = 2.0 x 10(-19) than from C = 3.0 x 10(-19) dyne cm2. Serious problems with the reported experimental assessments of ET for pBR322 and their comparison with simulated data are noted. In the case of a trefoil knotted DNA, the simulated value, (ET)tre, exceeds that of the unknotted DNA, (ET)unk, by approximately equal to 1.40-fold at magnitude of delta l = 1.0, but declines to a plateau about 1.09-fold larger than (ET)unk when magnitude of delta l > or = 15. Although the predicted ratio, (ET)tre/(ET)unk approximately equal to 1.40, agrees fairly well with recent experimental measurements on a 5600-bp DNA, the individual measured ET values, like some of those reported for pBR322, are so large that they cannot be simulated using P = 500 A, d = 50 A, and any previous experimental estimate of C.     

Left-handed Z-DNA helices in polymers, restriction fragments, and recombinant plasmids

Studies on DNA polymers, restriction fragments, and recombinant plasmids have revealed the following: A) A family of left-handed DNA conformations exists for (dC-dG)n.(dC-dG)n. The observation of a particular conformation is dependent on the salt, the salt concentration and dehydrating agent. B) In sodium acetate solutions, (dC-dG)n.(dC-dG)n forms left-handed, psi(+)-condensed structures as detected by Raman spectroscopy and circular dichroism. C) (dT-dG)n.(dC-dA)n undergoes a right-to-left-handed transition only when reacted with AAF and at high salt concentrations. D) Transitions observed for polymer DNAs also are observed for restriction fragments containing both (dC-dG).(dC-dG) and (dT-dG).(dC-dA) sequences, but the transitions in the fragments generally require higher salt concentrations than observed for the polymers. E) Studies with recombinant plasmids containing (dC-dG) sequences from 10 to 58 bp in length demonstrate that left-handed Z-DNA segments can exist contiguous to B-DNA segments. F) Negative supercoil density (sigma less than or equal to -0.072) is sufficient to convert the (dC-dG) regions in those plasmids into left-handed structures under physiological ionic conditions (200 mM NaCl). G) The favorable free energy contribution of methylation in stabilizing the Z form in fragments and plasmids is approximately offset by the unfavorable free energy contributions of the B/Z junctions. H) Sl and BAL 31 nucleases recognize aberrant structural features at the confluence of the B and Z regions. I) Detailed mapping of Sl nuclease cleavage on supercoiled plasmids shows that the nuclease sensitive regions extend over at least five to ten bp. J) Even though the (dT-dG)n.(dC-dA)n polymer requires base modification and high salt conditions to undergo the R----L transition, supercoiling (sigma less than or equal to -0.07) can supply enough energy to allow a plasmid containing the intervening sequence of a human fetal globin gene with (dT-dG).(dC-dA) sequences to undergo a R----L transition.     

Interaction of chloroquine with linear and supercoiled DNAs. Effect on the torsional dynamics, rigidity, and twist energy parameter

The magnitude and uniformity of the torsion elastic constant (alpha) of linear pBR322 DNA and supercoiled pBR322 DNAs with high-twist (sigma = -0.083) and normal-twist (sigma = -0.48) are measured in 0.1 M NaCl as a function of added chloroquine/base-pair ratio (chl/bp) by studying the fluorescence polarization anisotrophy (FPA) of intercalated ethidium dye. The time-resolved FPA is measured by using a picosecond dye laser for excitation and time-correlated single-photon counting detection. A general theory is developed for the binding of ligands that unwind superhelical DNAs, and the simultaneous binding of two different intercalators is treated in detail. The equilibrium constant (K) for binding chloroquine to linear pBR322 DNA and the number (r) of bound chloroquines per base pair are determined from the relative amplitude ratio of the slow (normally intercalated) and fast (free) components in the decay of the (probe) ethidium fluorescence intensity as a function of chl/bp. For chloroquine binding to supercoiled pBR322 DNAs, the intrinsic binding constant is assumed to be the same as for the linear DNA, but the twist energy parameter ET (N times the free energy to change the linking number from 0 to 1 in units of kBT) is regarded as adjustable. Using the best-fit ET, the binding ratios r are calculated for each chl/bp ratio. Twist energy parameters are also determined for ethidium binding to these supercoiled DNAs by competitive dialysis. For chloroquine binding, we obtain ET = 360 and 460 respectively for the normal-twist and high-twist supercoiled DNAs. For ethidium binding the corresponding values are ET = 280 +/- 70 and 347 +/- 50. Like other dye-binding values, these are substantially lower than those obtained by ligation methods. In the absence of chloroquine, the torsion constants of all three DNAs are virtually identical, alpha = (5.0 +/- 0.4) x 10(-12) dyn.cm. For linear pBR322 DNA, the magnitude and uniformity of alpha remain unaltered by intercalated chloroquine up to r = 0.19. This finding argues that the FPA is not significantly relaxed by diffusion of any kinks or solitons. If alpha d denotes the torsion constant between a dye and a base pair and alpha 0 that between two base pairs, then our data imply that alpha d/alpha 0 lies in the range 0.65-1.64, with a most probable value of 1.0.(ABSTRACT TRUNCATED AT 400 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.     

Analysis of amplified DNAs from drug-resistant Leishmania by orthogonal-field-alternation gel electrophoresis. The effect of size and topology on mobility

Amplified extrachromosomal DNAs from antifolate-resistant Leishmania are 30-75 kilobase (kb) supercoiled molecules that resolve on orthogonal-field-alternation gel electrophoresis (OFAGE) gels. These DNAs comigrate with smaller supercoiled plasmids (7-8 kb), and their mobility is not a simple function of their size. The properties of the amplified DNAs were investigated to determine if an unusual structure accounts for the observed mobility of the amplified DNAs by OFAGE; however, their topological properties were similar to those of standard Escherichia coli plasmids. The migration of a series of supercoiled plasmids ranging in size from 6 to 91 kb was analyzed by OFAGE, and a triphasic pattern was observed. The mobilities of plasmids between 20 and 60 kb increase with size, whereas the migration of plasmids between 6 and 20 and 60 and 91 kb is inversely proportional to size. Like smaller plasmids, the large supercoiled DNAs show a pulse time-independent mobility by OFAGE. The mobility of amplified DNA from Leishmania is in accord with that of the plasmid markers. Therefore, it is primarily the size of the amplified extrachromosomal DNAs from Leishmania, rather than an unusual superhelical density or topological structure, that results in the previously unexplained migration pattern.     

Monte Carlo simulations of supercoiled DNAs confined to a plane.

Recent advances in atomic force microscopy (AFM) have enabled researchers to obtain images of supercoiled DNAs deposited on mica surfaces in buffered aqueous milieux. Confining a supercoiled DNA to a plane greatly restricts its configurational freedom, and could conceivably alter certain structural properties, such as its twist and writhe. A program that was originally written to perform Monte Carlo simulations of supercoiled DNAs in solution was modified to include a surface potential. This potential flattens the DNAs to simulate the effect of deposition on a surface. We have simulated transfers of a 3760-basepair supercoiled DNA from solution to a surface in both 161 and 10 mM ionic strength. In both cases, the geometric and thermodynamic properties of the supercoiled DNAs on the surface differ significantly from the corresponding quantities in solution. At 161 mM ionic strength, the writhe/twist ratio is 1.20-1.33 times larger for DNAs on the surface than for DNAs in solution and significant differences in the radii of gyration are also observed. Simulated surface structures in 161 mM ionic strength closely resemble those observed by AFM. Simulated surface structures in 10 mM ionic strength are similar to a minority of the structures observed by AFM, but differ from the majority of such structures for unknown reasons. In 161 mM ionic strength, the internal energy (excluding the surface potential) decreases substantially as the DNA is confined to the surface. Evidently, supercoiled DNAs in solution are typically deformed farther from the minimum energy configuration than are the corresponding surface-confined DNAs. Nevertheless, the work (Delta A(int)) done on the internal coordinates, which include uniform rotations at constant configuration, during the transfer is positive and 2.6-fold larger than the decrease in internal energy. The corresponding entropy change is negative, and its contribution to Delta A(int) is positive and exceeds the decrease in internal energy by 3.6 fold. The work done on the internal coordinates during the solution-to-surface transfer is directed primarily toward reducing their entropy. Evidently, the number of configurations available to the more deformed solution DNA is vastly greater than for the less deformed surface-confined DNA.     

High mobility group protein 1 preferentially conserves torsion in negatively supercoiled DNA

HMG 1 is known to bind to a variety of DNAs and to unwind nicked and closed circular DNA. We now report evidence that it has a significantly higher unwinding angle on negatively supercoiled DNA than on the other torsional forms. The degree of unwinding observed on nicked circular DNA depends on the purity of the HMG 1 preparation used. HMG 1 from CM-Sephadex has an unwinding angle of 28.8 degrees, compared to 7.2 degrees for the purer preparation obtained from Mono S, suggesting that contaminating strand-separating activity is removed by the additional purification step. The subsequent studies on closed circular forms of DNA were all performed using the purer HMG 1. After preincubation of highly negatively supercoiled DNA (sigma = -0.040) with HMG 1, the DNA-protein mixture was relaxed with Escherichia coli topoisomerase I. At molar ratios of less than 100:1 (HMG 1 to DNA), negatively supercoiled DNA displays a dose-dependent change in the linking number, indicating an unwinding angle of 57.6 degrees. HMG 1 protects 50% of highly negatively supercoiled DNA from E. coli topoisomerase I at a molar ratio of 100:1, and protects all supercoils at a molar ratio of 200:1, indicating saturation of the DNA at this concentration. HMG 1 also protects highly negatively supercoiled DNA from calf thymus topoisomerase I, with an apparent unwinding angle of 57.6 degrees. Moderately negatively supercoiled DNA (sigma = -0.018), but not moderately positively supercoiled DNA (sigma = +0.011), competes for the protective effect of HMG 1 on highly negatively supercoiled DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of plasmid DNA topology among mesophilic and thermophilic eubacteria and archaebacteria.

Several plasmid DNAs have been isolated from mesophilic and thermophilic archaebacteria. Their superhelical densities were estimated at their host strain's optimal growth temperature, and in some representative strains, the presence of reverse gyrase activity (positive DNA supercoiling) was investigated. We show here that these plasmids can be grouped in two clusters with respect to their topological state. The group I plasmids have a highly negatively supercoiled DNA and belong to the mesophilic archaebacteria and all types of eubacteria. The group II plasmids have DNA which is close to the relaxed state and belong exclusively to the thermophilic archaebacteria. All archaebacteria containing a relaxed plasmid, with the exception of the moderately thermophilic methanogen Methanobacterium thermoautotrophicum Marburg, also exhibit reverse gyrase activity. These findings show that extrachromosomal DNAs with very different topological states coexist in the archaebacterial domain.     

Histone stoichiometry and DNA circularization in archaeal nucleosomes.

Recombinant (r)HMfB (archaealhistone B fromMethanothermusfervidus) formed complexes with increasing stability with DNA molecules increasing in length from 52 to 100 bp, but not with a 39 bp molecule. By using125I-labeled rHMfB-YY (an rHMfB variant with I31Y and M35Y replacements) and32P-labeled 100 bp DNA, these complexes, designated archaeal nucleosomes, have been shown to contain an archaeal histone tetramer. Consistent with DNA bending and wrapping, addition of DNA ligase to archaeal nucleosomes assembled with 88 and 128 bp DNAs resulted in covalently-closed monomeric circular DNAs which, following histone removal, were positively supercoiled based on their electrophoretic mobilities in the presence of ethidium bromide before and after relaxation by calf thymus topoisomerase I. Ligase addition to mixtures of rHMfB with 53 or 30 bp DNA molecules also resulted in circular DNAs but these were circular dimers and trimers. These short DNA molecules apparently had to be ligated into longer linear multimers for assembly into archaeal nucleosomes and ligation into circles. rHMfB assembled into archaeal nucleosomes at lower histone to DNA ratios with the supercoiled, circular ligation product than with the original 88 bp linear version of this molecule. Archaeal histones are most similar to the globular histone fold region of eukaryal histone H4, and the results reported are consistent with archaeal nucleosomes resembling the structure formed by eukaryal histone (H3+H4)2tetramers.