The nuclear scaffold exhibits DNA-binding sites selective for supercoiled DNA

Binding of exogenous DNA to the nuclear scaffold was investigated using a plasmid DNA (pBR322, EcoRI site deleted) of various topological forms and nuclear subfractions with different levels of nuclear DNA depletion. When supercoiled DNA was incubated with histone-depleted nuclei (nuclear halo), a dose-dependent binding of the DNA occurred, whereas no binding was observed with relaxed and linear forms of DNA. The bound DNA was released upon linearization with BamHI or digestion of the scaffolding structure with proteinase K. Extensive digestion of the halo with micrococcal nuclease generated additional sites which bind both relaxed and linear DNA. In the presence of a large excess of calf thymus DNA, these sites were effectively blocked and the specificity to supercoiled DNA was restored. The binding of all forms of DNA was abolished by heat-denatured DNA. There was no detectable change in linking number of the scaffold-associated supercoils. Competitive binding was observed between supercoiled DNAs with unrelated sequences, indicating that no specific nucleotide sequence is required for the binding. RNA was found to be a weak competitor. A DNA binding assay performed on electrophoretic blots of solubilized nuclear scaffold revealed a protein component with apparent molecular weight of 120,000 which retained selective binding to supercoils. These results suggest that the nuclear scaffold possesses DNA-binding sites for torsionally strained domains of chromatin and that an integral protein factor is involved in the binding. Implications of the findings are discussed in connection with proposed functions of the nuclear scaffold and topoisomerase II.     

Dynamics in the isomerization of intramolecular DNA triplexes in supercoiled plasmids.

We report here kinetic and thermodynamic studies on differential isomerization of intramolecular Pyr*Pur.Pyr triplexes in supercoiled plasmids. Two structural isomers of the triplex exist: one with the 3'-half of the Pyr strand as the third strand (H-y3 form) and the other with the 5'-half as the third strand (H-y5 form). The relative populations of the two triplex isomers was determined using the chemical probe with diethyl pryrocarbonate as a function of incubation time. The results demonstrated that triplexes were formed rapidly after a pH change from pH 8.0 to 5.0 and that the initial population of the two isomers exponentially changed with incubation time to reach true thermodynamic equilibrium with a time constant of 0.6-10 h, depending on temperature and the presence of Mg2+. The results clearly demonstrated that interconversion occurs between the two isomers and that the presence of Mg2+ generally retarded the interconversion rates. Kinetic and thermodynamic analyses of the relative populations of the two isomers revealed that the apparent energy barrier for transition from duplex to the H-y3 form is higher than that to the H-y5 form, but H-y3 is more stable in enthalpy terms than H-y5. Therefore, H-y3 is kinetically inferior but thermodynamically favored at higher supercoil levels in plasmids. The presence of Mg2+ resulted in both a kinetic and a thermodynamic preference for H-y5 formation, relative to the H-y3 form.     

Intrachain reactions of supercoiled DNA simulated by Brownian dynamics

We considered an irreversible biochemical intrachain reaction of supercoiled DNA as a random event that occurs, with certain probability, at the instant of collision between two reactive groups bound to distant DNA sites. Using the Brownian dynamics technique, we modeled this process for a supercoiled DNA molecule of 2.5 kb length in dilute aqueous solution at an NaCl concentration of 0.1 M. We calculated the mean reaction time tau(Sigma) as a function of the intrinsic second-order rate constant k(I), the reaction radius R, and the contour separation S of the reactive groups. At the diffusion-controlled limit (k(I) --> infinity), the kinetics of reaction are determined by the mean time tau(F) of the first collision. The dependence of tau(F) on R is close to inversely proportional, implying that the main contribution to the productive collisions is made by bending of the superhelix axis. At sufficiently small k(I), the mean reaction time can be satisfactory approximated by tau(Sigma) = tau(F)(app) + 1/(k(I)c(L)), where c(L) is the local concentration of one reactive group around the other, and tau is an adjustable parameter, which we called the apparent time of the first collision. The value of tau depends on R very weakly and is approximately equal to the mean time of the first collision caused by mutual reptation of two DNA strands forming the superhelix. The quasi-one-dimensional reptation process provides the majority of productive collisions at small k(I) values.     

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.     

Rate of B to Z structural transition of supercoiled DNA

The forward rate of the B to Z transition induced by negative supercoiling of plasmid DNA containing an alternating C-G sequence has been examined using the binding of Z-DNA-specific antibodies to follow the transition. DNA samples of a plasmid containing a d(pCpG)16 X d(pCpG)16 insert were supercoiled to different extents and appropriate amounts of ethidium were bound to the DNAs to relax them and to keep the alternating C-G sequence in the right-hand helical form. Following the rapid removal of ethidium by passage through a column of cation exchange resin, the DNA becomes negatively supercoiled, which induces the flipping of the helical hand of the C-G insert. The rate of the transition is strongly dependent on the degree of supercoiling. The transition is complete in less than 50 seconds for a DNA with a specific linking difference (superhelical density) sigma of -0.09. For the same DNA, the half-time of the transition is about two minutes at sigma = -0.07 and about a factor of 10 slower at sigma = -0.05.     

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.     

Osmium tetroxide probing of local DNA structure in linear and supercoiled plasmids containing curvature-inducing sequences

Recombinant plasmids pK1A108, pK3A108, pK4A108 and pK5/6T217 containing 80 +/- 1 base pair inserts with different curvature-inducing sequences were studied using the DNA structure probe osmium tetroxide in the presence of pyridine (Os, py). The insertion sequences of the plasmids pK1A108, pK3A108, and pK4A108 are strongly related while the degree of curvature increases from pK1A108 (no curvature) less than pK3A108 less than pK4A108 less than pK5/6T217. The Os, py probe reacts selectively with single-stranded and distorted double-stranded regions in the DNA double helix. Nuclease S1 was used to recognize and cleave regions made permanently single-stranded due to osmium recognize and cleave regions made permanently single-stranded due to osmium modification. In linearized plasmids treatment with Os, py produced no S1-detectable site-specific modification. This result is in agreement with models suggested for DNA curvature; in general, continuous base pairing and base stacking is considered through different sequence blocks as well as through structural junctions. Os, py-probing of the plasmids in the supercoiled state also resulted in no S1-detectable site-specific modification within the inserts of pK1A108, pK3A108, and pK4A108 plasmids (while the regions containing inverted repeat nucleotide sequences in these plasmids were site-specifically modified). In contrast, supercoiled pK5/6T217 DNA was site-specifically modified within the curvature-inducing insert sequence. The nucleotide sequence of the insert of this plasmid strongly differs from the insertion sequences of the other three plasmids; it is extremely AT-rich and contains regularly arranged dAGAGA and dATATA sequences. The structural distortion observed in supercoiled pK5/6T217 is most probably due to the presence of these sequences in a particular arrangement in the insertion sequence.     

Intramolecular DNA triplexes in supercoiled plasmids. I. Effect of loop size on formation and stability

The capacity of four oligopurine.oligopyrimidine (pur.pyr) sequences with different lengths of interruptions in the center [GAA)4(N)n(GAA)4G) (n = 3, 5, 7, and 9) to adopt intramolecular DNA triplexes was evaluated in recombinant plasmids. The hyperreactive patterns of the pur.pyr inserts to specific chemical probes (OsO4, diethyl pyrocarbonate, and dimethyl sulfate) at the base pair level demonstrate that intramolecular triplexes with identical 12-base triads in the stem but with different loop sizes (4, 6, 8, and 10 bases) can form in supercoiled plasmids. Furthermore, the extent of OsO4 modification was measured as a function of temperature and of average negative supercoil density. In addition, the transition free energy of B-DNA to triplexes at pH 4.5 was determined by two-dimensional electrophoresis. These comparative studies show that longer loops require more supercoil energy for triplex formation and are less thermostable than triplexes with shorter loops. Also, it may be that not only the loop size but the base composition of the loop region affects the structural transition and triplex stability. Thus, these results significantly broaden the range of natural pur.pyr sequences that may adopt triplexes.     

Structure of plectonemically supercoiled DNA

Using electron microscopy and topological methods, we have deduced an average structure for negatively supercoiled circular DNA in solution. Our data suggest that DNA has a branched plectonemic (interwound) form over the range of supercoiling tested. The length of the superhelix axis is constant at 41% of the DNA length, whereas the superhelix radius decreases essentially hyperbolically as supercoiling increases. The number of supercoils is 89% of the linking deficit. Both writhe and twist change with supercoiling, but the ratio of the change in writhe to the change in twist is fixed at 2.6:1. The extent of branching of the superhelix axis is proportional to the length of the plasmid, but is insensitive to superhelix density. The relationship between DNA flexibility constants for twisting and bending calculated using our structural data is similar to that deduced from previous studies. The extended thin form of plectonemically supercoiled DNA offers little compaction for cellular packaging, but promotes interaction between cis-acting sequence elements that may be distant in primary structure. We discuss additional biological implications of our structural data.     

Site-specific labeling of supercoiled DNA

Visualization of site-specific labels in long linear or circular DNA allows unambiguous identification of various local DNA structures. Here we describe a novel and efficient approach to site-specific DNA labeling. The restriction enzyme SfiI binds to DNA but leaves it intact in the presence of calcium and therefore may serve as a protein label of 13 bp recognition sites. Since SfiI requires simultaneous interaction with two DNA recognition sites for stable binding, this requirement is satisfied by providing an isolated recognition site in the DNA target and an additional short DNA duplex also containing the recognition site. The SfiI/DNA complexes were visualized with AFM and the specificity of the labeling was confirmed by the length measurements. Using this approach, two sites in plasmid DNA were labeled in the presence of a large excess of the helper duplex to compete with the formation of looped structures of the intramolecular synaptic complex. We show that the labeling procedure does not interfere with the superhelical tension-driven formation of alternative DNA structures such as cruciforms. The complex is relatively stable at low and high pH (pH 5 and 9) making the developed approach attractive for use at conditions requiring the pH change.     

Dimensions of plectonemically supercoiled DNA

With a view to determine the configuration and regularity of plectonemically supercoiled DNA, we have measured the small angle neutron scattering from pUC18 plasmid in saline solutions. Furthermore, we have derived the mathematical expression for the single chain scattering function (form factor) of a superhelical structure, including the longitudinal and transverse interference over the plectonemic pitch and radius, respectively. It was found that an interwound configuration describes the data well, provided interactions among supercoils are accounted for in the second virial approximation. The opening angle was observed to be relatively constant and close to 58 degrees, but it was necessary to include a significant distribution in radius and pitch. For diluted supercoils with vanishing mutual interaction, the derived structural results agree with independent measurements, including the distribution in linking number deficit as determined by gel electrophoresis. With increasing plasmid concentration, prior and covering the transition to the liquid-crystalline phase, the radius and pitch are seen to decrease significantly. The latter observation shows that compaction of negatively supercoiled DNA by confinement results in a decrease in writhing number at the cost of a positive twist exerted on the DNA duplex. It is our conjecture that the free energy associated with this excess twist is of paramount importance in controlling the critical boundaries pertaining to the transition to the anisotropic, liquid-crystalline phase.     

Behavior of supercoiled DNA.

We study DNA supercoiling in a quantitative fashion by micromanipulating single linear DNA molecules with a magnetic field gradient. By anchoring one end of the DNA to multiple sites on a magnetic bead and the other end to multiple sites on a glass surface, we were able to exert torsional control on the DNA. A rotating magnetic field was used to induce rotation of the magnetic bead, and reversibly over- and underwind the molecule. The magnetic field was also used to increase or decrease the stretching force exerted by the magnetic bead on the DNA. The molecule's degree of supercoiling could therefore be quantitatively controlled and monitored, and tethered-particle motion analysis allowed us to measure the stretching force acting on the DNA. Experimental results indicate that this is a very powerful technique for measuring forces at the picoscale. We studied the effect of stretching forces ranging from 0.01 pN to 100 pN on supercoiled DNA (-0.1 < sigma < 0.2) in a variety of ionic conditions. Other effects, such as stretching-relaxing hysteresis and the braiding of two DNA molecules, are discussed.     

Drug-induced relaxation of supercoiled plasmid DNA in Bacillus subtilis and induction of the SOS response.

Whereas treatment with many different drugs led to induction of the SOS response in Bacillus subtilis, only inhibitors of DNA gyrase subunit B and, unexpectedly, polyether antibiotics (membrane ionophores) led to relaxation of supercoiled plasmid DNA. However, treatment with DNA gyrase subunit B inhibitors but not with polyethers led to SOS induction. Thus, the presence of underwound supercoiled DNA was not sufficient to induce the SOS response. Possible mechanisms by which polyethers induce relaxation of supercoiled DNA in vivo are discussed.     

Elastic model of highly supercoiled DNA

The equilibrium shapes of supercoiled DNA are investigated by employing an elastic model. First, a set of Euler equations is derived to determine the equilibrium shapes under ring-closure conditions. Two exact solutions that describe circular and figure-8 shapes are obtained. Using these and their topological properties, the configuration change from the circular to the figure-8 form is discussed. Second, more intricate structures of supercoiling DNA are studied by a numerical analysis. Among a class of configurations, the shape that has the minimum elastic energy is explicitly determined. Poisson's ratio, the ratio of the self-avoiding radius to the total length, and the deficit (or excess) of the linking number ΔLk are found to be the important parameters. We conclude that the topology and the elastic theory of looped DNA explain the essential features of the supercoiling phenomena.     

Molecular mechanics model of supercoiled DNA

We describe a pseudo-atomic model of supercoiled DNA. Each base-pair of the DNA is represented in the model by three particles placed in a plane. The particle triplets are stacked to model stacked base-pairs in double-helical DNA, and closed circular conformations are generated to investigate supercoiling. This model is less detailed than all-atom models, which are too computationally demanding to be used to study supercoiling. On the other hand, this model contains details at the base-pair level and is therefore more elaborate than elastomechanical models. A potential energy function is written in terms of a set of internal co-ordinates defined to resemble a limited number of helical parameters. The modeled helical parameters, helical twist, base-roll, tilt and rise, are the most important parameters of the global shape of DNA. Experimentally measured mechanical properties of DNA are used to define the forces holding the particles together. We then use a procedure incorporating energy minimization and molecular dynamics to locate low energy conformations of the model DNA. The model was found to behave very much like rubber-tubing and elastomechanical models. The conformations and the effects of supercoiling pressure (a number proportional to the degree to which the total twist of the DNA has been altered from its natural value) on these conformations are all very similar to those observed in the latter two models. We also used this model to examine the effects of supercoiling pressure, base-sequence and mechanical properties on the conformations and energies of five sequences. The sequences studied include models of naturally straight DNA and DNA with static or natural bends.     

Nanomechanics of negatively supercoiled diaminopurine-substituted DNA

Single molecule experiments have demonstrated a progressive transition from a B- to an L-form helix as DNA is gently stretched and progressively unwound. The particular sequence of a DNA segment defines both base stacking and hydrogen bonding that affect the partitioning and conformations of the two phases. Naturally or artificially modified bases alter H-bonds and base stacking and DNA with diaminopurine (DAP) replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded DAP:T base pairs. Both unmodified and DAP-substituted DNA transitioned from a B- to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing and the ease of this transition may prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length than B-DNA. However, left-handed DAP-substituted DNA was twice as stiff as unmodified L-DNA. Unmodified DNA and DAP-substituted DNA have very distinct mechanical characteristics at physiological levels of negative supercoiling and tension.     

Bidirectional sequencing of supercoiled plasmid DNA

In this paper we show that restriction DNA fragments can prime DNA synthesis of a homologous supercoiled plasmid DNA. Using the dideoxyribonucleotide chain terminator method, newly synthesized truncated chains can be detached from the primers by restriction enzyme digestion. Therefore, by choosing DNA fragments flanked by two different restriction enzymes sites, nucleotide sequence information can be simultaneously obtained on both regions of the DNA surrounding the restriction fragment. The advantage of this sequencing approach over current methods is that no prior knowledge of the primary sequence is needed to find the nucleotide sequence of a given DNA fragment. Thus, synthetic primers are not required and internal sequences of a given clone can be easily accessed without the need of fragmenting the original construct. The method has been used with rapid plasmid preparations, thus considerable time and effort can be saved in the gathering of nucleotide sequence information.