DNA Writhe Research Articles

Innovative Tools for DNA Topology Probing in Human Cells Reveal a Build-Up of Positive Supercoils Following Replication Stress at Telomeres and at the FRA3B Fragile Site.

Linear unconstrained DNA cannot harbor supercoils since these supercoils can diffuse and be eliminated by free rotation of the DNA strands at the end of the molecule. Mammalian telomeres, despite constituting the ends of linear chromosomes, can hold supercoils and be subjected to topological stress. While negative supercoiling was previously observed, thus proving the existence of telomeric topological constraints, positive supercoils were never probed due to the lack of an appropriate tool. Indeed, the few tools available currently could only investigate unwound (Trioxsalen) or overwound (GapR) DNA topology (variations in twist) but not the variations in writhe (supercoils and plectonemes). To address this question, we have designed innovative tools aimed at analyzing both positive and negative DNA writhe in cells. Using them, we could observe the build-up of positive supercoils following replication stress and inhibition of Topoisomerase 2 on telomeres. TRF2 depletion caused both telomere relaxation and an increase in positive supercoils while the inhibition of Histone Deacetylase I and II by TSA only caused telomere relaxation. Moving outside telomeres, we also observed a build-up of positive supercoils on the FRA3B fragile site following replication stress, suggesting a topological model of DNA fragility for this site.

Mechanism for conversion between twist and writhe of DNA based on chiral gauge potential

It is well known that the linking number is the sum of the writhing number and twisting number based on white’s formula. In this work, we reveal the underlying physics of the transformation between the twist and the writhe of deoxyribonucleic acid (DNA). In the framework of the Landau model, we describe the local interaction between the base pairs by using the chiral gauge potential. With the chiral gauge potential, we develop a geometrical model where the free energy density is given by the geometrical parameters, including the normal curvature, the geodesic curvature, and the geodesic torsion. It is shown that the chiral gauge potential plays a crucial role in the conversion between the twist and the writhe of DNA. By using the invariance of the self-linking number, we find that when the total twisting number is changed by −Δ Q, the writhing number is changed by Δ Q. Our findings not only deepen the understanding of the topological nature of DNA, but also provide a physical model for the research of the conformation of DNA.

Topoisomerase VI is a chirally-selective, preferential DNA decatenase.

DNA topoisomerase VI (topo VI) is a type IIB DNA topoisomerase found predominantly in archaea and some bacteria, but also in plants and algae. Since its discovery, topo VI has been proposed to be a DNA decatenase; however, robust evidence and a mechanism for its preferential decatenation activity was lacking. Using single-molecule magnetic tweezers measurements and supporting ensemble biochemistry, we demonstrate that Methanosarcina mazei topo VI preferentially unlinks, or decatenates DNA crossings, in comparison to relaxing supercoils, through a preference for certain DNA crossing geometries. In addition, topo VI demonstrates a significant increase in ATPase activity, DNA binding and rate of strand passage, with increasing DNA writhe, providing further evidence that topo VI is a DNA crossing sensor. Our study strongly suggests that topo VI has evolved an intrinsic preference for the unknotting and decatenation of interlinked chromosomes by sensing and preferentially unlinking DNA crossings with geometries close to 90°.

Direct observation of helicase–topoisomerase coupling within reverse gyrase

Reverse gyrases (RGs) are the only topoisomerases capable of generating positive supercoils in DNA. Members of the type IA family, they do so by generating a single-strand break in substrate DNA and then manipulating the two single strands to generate positive topology. Here, we use single-molecule experimentation to reveal the obligatory succession of steps that make up the catalytic cycle of RG. In the initial state, RG binds to DNA and unwinds ∼2 turns of the double helix in an ATP-independent fashion. Upon nucleotide binding, RG then rewinds ∼1 turn of DNA. Nucleotide hydrolysis and/or product release leads to an increase of 2 units of DNA writhe and resetting of the enzyme, for a net change of topology of +1 turn per cycle. Final dissociation of RG from DNA results in rewinding of the 2 turns of DNA that were initially disrupted. These results show how tight coupling of the helicase and topoisomerase activities allows for induction of positive supercoiling despite opposing torque.

Quantitative Study of the Chiral Organization of the Phage Genome Induced by the Packaging Motor

Molecular motors that translocate DNA are ubiquitous in nature. During morphogenesis of double-stranded DNA bacteriophages, a molecular motor drives the viral genome inside a protein capsid. Several models have been proposed for the three-dimensional geometry of the packaged genome, but very little is known of the signature of the molecular packaging motor. For instance, biophysical experiments show that in some systems, DNA rotates during the packaging reaction, but most current biophysical models fail to incorporate this property. Furthermore, studies including rotation mechanisms have reached contradictory conclusions. In this study, we compare the geometrical signatures imposed by different possible mechanisms for the packaging motors: rotation, revolution, and rotation with revolution. We used a previously proposed kinetic Monte Carlo model of the motor, combined with Brownian dynamics simulations of DNA to simulate deterministic and stochastic motor models. We find that rotation is necessary for the accumulation of DNA writhe and for the chiral organization of the genome. We observe that although in the initial steps of the packaging reaction, the torsional strain of the genome is released by rotation of the molecule, in the later stages, it is released by the accumulation of writhe. We suggest that the molecular motor plays a key role in determining the final structure of the encapsidated genome in bacteriophages.

Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase IIα

Human topoisomerase IIα unlinks catenated chromosomes and preferentially relaxes positive supercoils. Supercoil chirality, twist density, and tension determine topoisomerase IIα relaxation rate and processivity. Strand passage rate is determined by the efficiency of transfer segment capture that is modulated by the topoisomerase C-terminal domains. Single-molecule measurements reveal the mechanism of chiral discrimination and tension dependence of supercoil relaxation by human topoisomerase IIα. Type IIA topoisomerases (Topo IIA) are essential enzymes that relax DNA supercoils and remove links joining replicated chromosomes. Human topoisomerase IIα (htopo IIα), one of two human isoforms, preferentially relaxes positive supercoils, a feature shared with Escherichia coli topoisomerase IV (Topo IV). The mechanistic basis of this chiral discrimination remains unresolved. To address this important issue, we measured the relaxation of individual supercoiled and "braided" DNA molecules by htopo IIα using a magnetic tweezers-based single-molecule assay. Our study confirmed the chiral discrimination activity of htopo IIα and revealed that the strand passage rate depends on DNA twist, tension on the DNA, and the C-terminal domain (CTD). Similar to Topo IV, chiral discrimination by htopo IIα results from chiral interactions of the CTDs with DNA writhe. In contrast to Topo IV, however, these interactions lead to chiral differences in relaxation rate rather than processivity. Increasing tension or twist disrupts the CTD-DNA interactions with a subsequent loss of chiral discrimination. Together, these results suggest that transfer segment (T-segment) capture is the rate-limiting step in the strand passage cycle. We propose a model for T-segment capture that provides a mechanistic basis for chiral discrimination and provides a coherent explanation for the effects of DNA twist and tension on eukaryotic type IIA topoisomerases.

Histone H3-variant Cse4-induced positive DNA supercoiling in the yeast plasmid has implications for a plasmid origin of a chromosome centromere

The Saccharomyces cerevisiae 2-μm plasmid is a multicopy selfish genome that resides in the nucleus. The genetic organization of the plasmid is optimized for stable, high-copy propagation in host-cell populations. The plasmid's partitioning system poaches host factors, including the centromere-specific histone H3-variant Cse4 and the cohesin complex, enabling replicated plasmid copies to segregate equally in a chromosome-coupled fashion. We have characterized the in vivo chromatin topology of the plasmid partitioning locus STB in its Cse4-associated and Cse4-nonassociated states. We find that the occupancy of Cse4 at STB induces positive DNA supercoiling, with a linking difference (ΔLk) contribution estimated between +1 and +2 units. One plausible explanation for this contrary topology is the presence of a specialized Cse4-containing nucleosome with a right-handed DNA writhe at a functional STB, contrasted by a standard histone H3-containing nucleosome with a left-handed DNA writhe at a nonfunctional STB. The similarities between STB and centromere in their nucleosome signature and DNA topology would be consistent with the potential origin of the unusual point centromere of budding yeast chromosomes from the partitioning locus of an ancestral plasmid.

Temperature Dependence of DNA Persistence Length

We have determined the temperature dependence of DNA persistence length, a, using two different methods. The first approach was based on measuring the j-factors of short DNA fragments at various temperatures. Fitting the measured j-factors by the theoretical equation allowed us to obtain the values of a for temperatures between 5 and 42 C. The second approach was based on measuring the equilibrium distribution of the linking number between the strands of circular DNA at different temperatures. The major contribution into the distribution variance comes from the fluctuations of DNA writhe in the nicked circular molecules which are specified by the value of a. The computation-based analysis of the measured variances was used to obtain the values of a for temperatures up to 60°C. We found a good agreement between the results obtained by these two methods. Our data show that DNA persistence length strongly depends on temperature and accounting for this dependence is important in quantitative comparison between experimental results obtained at different temperatures.

Manipulating DNA writhe through varying DNA sequences

It is demonstrated that the shapes and magnitudes of DNA writhe can be precisely manipulated solely through maneuvering the nucleotide sequence of DNA and without the assistance of topoisomerases.

Temperature dependence of DNA persistence length

We have determined the temperature dependence of DNA persistence length, a, using two different methods. The first approach was based on measuring the j-factors of short DNA fragments at various temperatures. Fitting the measured j-factors by the theoretical equation allowed us to obtain the values of a for temperatures between 5°C and 42°C. The second approach was based on measuring the equilibrium distribution of the linking number between the strands of circular DNA at different temperatures. The major contribution into the distribution variance comes from the fluctuations of DNA writhe in the nicked circular molecules which are specified by the value of a. The computation-based analysis of the measured variances was used to obtain the values of a for temperatures up to 60°C. We found a good agreement between the results obtained by these two methods. Our data show that DNA persistence length strongly depends on temperature and accounting for this dependence is important in quantitative comparison between experimental results obtained at different temperatures.

Terminal Twist-Induced Writhe of DNA with Intrinsic Curvature

Supercoiling of a closed circular DNA rod may result from an application of terminal twist to the DNA rod by cutting the rod, rotating one of the cut faces as the other being fixed and then sealing the cut. According to White's formula, DNA supercoiling is probably accompanied by a writhe of the DNA axis. Deduced from the elastic rod model for DNA structure, an intrinsically straight closed circular DNA rod does not writhe as subject to a terminal twist, until the number of rotation exceeds a rod-dependent threshold. By contrast, a closed circular DNA rod with intrinsic curvature writhes instantly as subject to a terminal twist. This noteworthy character in fact belongs to many intrinsically curved DNA rods. By solving the dynamic equations, the linearization of the Euler-Lagrange equations governing intrinsically curved DNA rods, this paper shows that almost every clamped-end intrinsically curved DNA rod writhes instantly when subject to a terminal twist (clamped-end DNA rods include closed circular DNA rods and topological domains of open DNA rods). In terms of physical quantities, the exceptions are identified with points in R(6) whose projections onto R(5) (through ignoring the total energy density of a rod) form a subset of a quadratic hypersurface. This paper also suggests that the terminal twist induced writhe is due to the elasticity and the clamped-end boundary conditions of the DNA rods.

Single-molecule DNA nanomanipulation: Improved resolution through use of shorter DNA fragments

Single-molecule nanomanipulation of supercoiled DNA permits measurement, in real time, of spatial and temporal parameters of protein-DNA interactions that affect DNA topology 1–7 . In this method, a double-stranded DNA molecule containing at least one target for the protein of interest is attached at one end to a magnetic bead and at the other end to a glass surface. The experimental setup and the monitoring of the end-to-end extension (l) of the stretched, supercoiled DNA molecule is diagramed in Figure 1a. The protein of interest is introduced into the system, and protein-dependent changes in DNA linking number (Lk) or DNA twist (Tw) are detected as changes in the number of plectonemic supercoils (changes in DNA writhe, Wr; Lk = Tw + Wr; ref. 8) and corresponding changes in l (Fig. 1b–d). This approach has been applied to analysis of supercoil formation and relaxation by topoisomerases 1–5 and to promoter unwinding by bacterial RNA polymerase (RNAP) 6,7 . The spatial and temporal resolution of the method is expected to increase with decreasing length of the supercoiled DNA segment (equations in ref. 6). (Reducing the length of the supercoilable DNA segment should have no effect on the amplitude of changes in DNA extension resulting from protein-dependent changes in DNA topology (signal), but should reduce the amplitude of random fluctuations in DNA extension (noise), thereby resulting in improvement in the signal-to-noise ratio.) Previous work has involved supercoiled DNA segments 4–44 kilobases (kb) in length 1–7 . Here, we describe preparation of DNA molecules with 2-kb supercoilable DNA segments and document superior resolution in analysis of promoter unwinding and DNA compaction by bacterial RNAP.

Writhe of DNA induced by a terminal twist

This paper considers the three-dimensional structure of B-form DNA. The molecule may be open or covalently closed. For the former, its two ends are not allowed to move or rotate freely in space unless the molecule is under the influence of rigid body motions of the ambient space. Implied by the elastic rod model for DNA, the molecule writhes immediately when subject to a terminal twist as long as its axis is none of the following curves: lines, circular arcs, circular helices. This result is remarkably different from well-known results about DNA of other conformations. For example, if a DNA is regarded as an elastic rod whose axis is a circle, then it has no induced writhe when subject to a terminal twist until the latter meets a critical extent.

Colworth memorial lecture. Pathways for remodelling chromatin.

The alteration of chromatin structure plays an integral role in gene regulation. One means by which eukaryotes manipulate chromatin structure involves the use of ATP-dependent chromatin-remodelling enzymes. It appears likely that these enzymes play a widespread role in the regulation of many nuclear processes. Recently, significant progress has been made in defining the alterations to chromatin structure that these enzymes generate. The ability to alter nucleosome positioning may be a common feature of all ATP-dependent remodelling enzymes, but the spectrum of positions to which nucleosomes are relocated varies. Mounting evidence supports the ability of remodelling enzymes to translocate along DNA. This provides a means by which they could alter both the twist and writhe of DNA on the surface of nucleosomes, and so accelerate nucleosome repositioning.

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 consistence 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.

Helical repeat and linking number of surface-wrapped DNA.

The geometric properties of duplex DNA are systematically altered when the DNA is wrapped on a protein surface. The linking number of surface-wrapped closed circular DNA is the sum of two integers: the winding number, phi, a function of the helical repeat; and the surface linking number, SLk, a newly defined geometric constant that accounts for the effects of surface geometry on the twist and writhe of DNA. Changes in the helical repeat, h, and in the winding number can be deduced solely from surface geometry and superhelix density, sigma. This treatment relates the theoretically important properties twist and writhe to the more experimentally accessible quantities phi, h, SLk, and sigma. The analysis is applied to three biologically important cases: interwinding of DNA in a plectonemic superhelix, catenated DNA, and minichromosomes.

Interaction of the Escherichia coli HU protein with DNA : Evidence for formation of nucleosome-like structures with altered DNA helical pitch

Nuclease digestion studies of DNA bound to the histone-like protein HU show that cuts in each strand of the DNA double helix are made with a periodicity of 8.5 base-pairs. By contrast, similar digestions of DNA in eukaryotic nucleosomes show a repeat of 10.4 base-pairs. This and other results (including circular dichroism studies) are consistent with the proposal that the pitch of the DNA double helix in the HU complex is reduced from a repeat length of 10.5 to 8.5 base-pairs per helical turn. Simultaneously, the DNA in the HU-DNA complex containing two dimers of HU per 60 base-pairs has its linking number decreased by 1.0 turn per 290 base-pairs. From these changes it is calculated that HU imposes a DNA writhe of 1.0 per three to four monomers of HU. The results suggest a model in which DNA is coiled in left-handed toroidal supercoils on the HU complex, having a stoichiometry resembling that of the half-nucleosome of eukaryotic chromatin. An important distinction is that HU complexes can restrain the same number of DNA superhelical turns as eukaryotic nucleosomes, yet the DNA retains more negative torsional tension, just as is observed in prokaryotic chromosomes in vivo. Another distinction is that HU-DNA complexes are less stable, having a dissociation half-life of 0.6 min in 50 m m-NaCl. This last property may explain prior difficulties in detecting prokaryotic nucleosome-like structures.

Studies of DNA organization in hydrated spermidine-condensed DNA toruses and spermidine-DNA fibres.

We have investigated in vitro spermidine-condensed DNA preparations by both biochemical and freeze-etch electron microscopic approaches. These studies lead us to the conclusion that the reversibly condensed preparations, qualitatively described by Manning's counterion condensation theory, contain disk-like torus structures largely comprised of unidirectional, circumferentially wrapped DNA. Stereoscopic measurements on stereomicrographs of DNA torus and fibre objects have demonstrated the feasibility of directly measuring DNA writhe or, for that matter, mapping any secondary, tertiary or quaternary structure features of a hydrated macromolecular array in which the features can be differentially highlighted by low replica metal shadow levels.