Action at Hooked or Twisted-Hooked DNA Juxtapositions Rationalizes Unlinking Preference of Type-2 Topoisomerases

The mathematical basis of the hypothesis that type-2 topoisomerases recognize and act at specific DNA juxtapositions has been investigated by coarse-grained lattice polymer models, showing that selective segment passages at hooked juxtapositions can result in dramatic reductions in catenane and knot populations. The lattice modeling approach is here extended to account for the narrowing of variance of linking number (Lk) of DNA circles by type-2 topoisomerases. In general, the steady-state variance of Lk resulting from selective segment passages at a specific juxtaposition geometry j is inversely proportional to the average linking number, 〈Lk〉_j, of circles with the given juxtaposition. Based on this formulation, we demonstrate that selective segment passages at hooked juxtapositions reduce the variance of Lk. The dependence of this effect on model DNA circle size is remarkably similar to that observed experimentally for type-2 topoisomerases, which appear to be less capable in narrowing Lk variance for small DNA circles than for larger DNA circles. This behavior is rationalized by a substantial cancellation of writhe in small circles with hook-like juxtapositions. During our simulations, we uncovered a twisted variation of the hooked juxtaposition that has an even more dramatic effect on Lk variance narrowing than the hooked juxtaposition. For an extended set of juxtapositions, we detected a significant correlation between the Lk narrowing potential and the logarithmic decatenating and unknotting potentials for a given juxtaposition, a trend reminiscent of scaling relations observed with experimental measurements on type-2 topoisomerases from a variety of organisms. The consistent agreement between theory and experiment argues for type-2 topoisomerase action at hooked or twisted-hooked DNA juxtapositions.     

Inferring global topology from local juxtaposition geometry: interlinking polymer rings and ramifications for topoisomerase action

Lattice modeling is applied to investigate how the configurations of local chain juxtapositions may provide information about whether two ring polymers (loops) are topologically linked globally. Given a particular juxtaposition, the conditional probability that the loops are linked is determined by exact enumeration and extensive Monte Carlo sampling of conformations satisfying excluded volume constraints. A discrimination factor fL, defined as the ratio of linked to unlinked probabilities, varies widely depending on which juxtaposition is presumed. /log fL/s that are large for small loop size n tend to decrease, signaling diminishing topological information content of the juxtapositions, with increasing n. However, some juxtaposition geometries can impose sufficient overall conformational biases such that /log fL/ remains significant for large n. Notably, for two loops as large as n=200 in the model, the probability that passing the segments of a hooked juxtaposition would unlink an originally linked configuration is remarkably high, approximately 85%. In contrast, segment-passage of a free juxtaposition would link the loops from an originally unlinked configuration more than 90% of the time. The statistical mechanical principles emerging from these findings suggest that it is physically possible for DNA topoisomerases to decatenate effectively by acting selectively on juxtapositions with specific "hooked" geometries.     

Local selection rules that can determine specific pathways of DNA unknotting by type II DNA topoisomerases

We performed numerical simulations of DNA chains to understand how local geometry of juxtaposed segments in knotted DNA molecules can guide type II DNA topoisomerases to perform very efficient relaxation of DNA knots. We investigated how the various parameters defining the geometry of inter-segmental juxtapositions at sites of inter-segmental passage reactions mediated by type II DNA topoisomerases can affect the topological consequences of these reactions. We confirmed the hypothesis that by recognizing specific geometry of juxtaposed DNA segments in knotted DNA molecules, type II DNA topoisomerases can maintain the steady-state knotting level below the topological equilibrium. In addition, we revealed that a preference for a particular geometry of juxtaposed segments as sites of strand-passage reaction enables type II DNA topoisomerases to select the most efficient pathway of relaxation of complex DNA knots. The analysis of the best selection criteria for efficient relaxation of complex knots revealed that local structures in random configurations of a given knot type statistically behave as analogous local structures in ideal geometric configurations of the corresponding knot type.     

How topoisomerase IV can efficiently unknot and decatenate negatively supercoiled DNA molecules without causing their torsional relaxation

Freshly replicated DNA molecules initially form multiply interlinked right-handed catenanes. In bacteria, these catenated molecules become supercoiled by DNA gyrase before they undergo a complete decatenation by topoisomerase IV (Topo IV). Topo IV is also involved in the unknotting of supercoiled DNA molecules. Using Metropolis Monte Carlo simulations, we investigate the shapes of supercoiled DNA molecules that are either knotted or catenated. We are especially interested in understanding how Topo IV can unknot right-handed knots and decatenate right-handed catenanes without acting on right-handed plectonemes in negatively supercoiled DNA molecules. To this end, we investigate how the topological consequences of intersegmental passages depend on the geometry of the DNA-DNA juxtapositions at which these passages occur. We observe that there are interesting differences between the geometries of DNA-DNA juxtapositions in the interwound portions and in the knotted or catenated portions of the studied molecules. In particular, in negatively supercoiled, multiply interlinked, right-handed catenanes, we detect specific regions where DNA segments belonging to two freshly replicated sister DNA molecules form left-handed crossings. We propose that, due to its geometrical preference to act on left-handed crossings, Topo IV can specifically unknot supercoiled DNA, as well as decatenate postreplicative catenanes, without causing their torsional relaxation.     

DNA disentangling by type-2 topoisomerases

A type-2 topoisomerase cleaves a DNA strand, passes another through the break, and then rejoins the severed ends. Because it appears that this action is as likely to increase as to decrease entanglements, the question is: how are entanglements removed? We argue that type-2 topoisomerases have evolved to act at "hooked" juxtapositions of strands (where the strands are curved toward each other). This type of juxtaposition is a natural consequence of entangled long strands. Our model accounts for the observed preference for unlinking and unknotting of short DNA plasmids by type-2 topoisomerases and well explains experimental observations.     

Generation of supercoils in nicked and gapped DNA drives DNA unknotting and postreplicative decatenation

Due to the helical structure of DNA the process of DNA replication is topologically complex. Freshly replicated DNA molecules are catenated with each other and are frequently knotted. For proper functioning of DNA it is necessary to remove all of these entanglements. This is done by DNA topoisomerases that pass DNA segments through each other. However, it has been a riddle how DNA topoisomerases select the sites of their action. In highly crowded DNA in living cells random passages between contacting segments would only increase the extent of entanglement. Using molecular dynamics simulations we observed that in actively supercoiled DNA molecules the entanglements resulting from DNA knotting or catenation spontaneously approach sites of nicks and gaps in the DNA. Type I topoisomerases, that preferentially act at sites of nick and gaps, are thus naturally provided with DNA–DNA juxtapositions where a passage results in an error-free DNA unknotting or DNA decatenation.     

3D visualization software to analyze topological outcomes of topoisomerase reactions

The action of various DNA topoisomerases frequently results in characteristic changes in DNA topology. Important information for understanding mechanistic details of action of these topoisomerases can be provided by investigating the knot types resulting from topoisomerase action on circular DNA forming a particular knot type. Depending on the topological bias of a given topoisomerase reaction, one observes different subsets of knotted products. To establish the character of topological bias, one needs to be aware of all possible topological outcomes of intersegmental passages occurring within a given knot type. However, it is not trivial to systematically enumerate topological outcomes of strand passage from a given knot type. We present here a 3D visualization software (TopoICE-X in KnotPlot) that incorporates topological analysis methods in order to visualize, for example, knots that can be obtained from a given knot by one intersegmental passage. The software has several other options for the topological analysis of mechanisms of action of various topoisomerases.     

Direct measurement of DNA bending by type IIA topoisomerases: implications for non-equilibrium topology simplification

Type IIA topoisomerases modify DNA topology by passing one segment of duplex DNA (transfer or T–segment) through a transient double-strand break in a second segment of DNA (gate or G–segment) in an ATP-dependent reaction. Type IIA topoisomerases decatenate, unknot and relax supercoiled DNA to levels below equilibrium, resulting in global topology simplification. The mechanism underlying this non-equilibrium topology simplification remains speculative. The bend angle model postulates that non-equilibrium topology simplification scales with the bend angle imposed on the G–segment DNA by the binding of a type IIA topoisomerase. To test this bend angle model, we used atomic force microscopy and single-molecule Förster resonance energy transfer to measure the extent of bending imposed on DNA by three type IIA topoisomerases that span the range of topology simplification activity. We found that Escherichia coli topoisomerase IV, yeast topoisomerase II and human topoisomerase IIα each bend DNA to a similar degree. These data suggest that DNA bending is not the sole determinant of non-equilibrium topology simplification. Rather, they suggest a fundamental and conserved role for DNA bending in the enzymatic cycle of type IIA topoisomerases.     

Simulations of action of DNA topoisomerases to investigate boundaries and shapes of spaces of knots

The configuration space available to randomly cyclized polymers is divided into subspaces accessible to individual knot types. A phantom chain utilized in numerical simulations of polymers can explore all subspaces, whereas a real closed chain forming a figure-of-eight knot, for example, is confined to a subspace corresponding to this knot type only. One can conceptually compare the assembly of configuration spaces of various knot types to a complex foam where individual cells delimit the configuration space available to a given knot type. Neighboring cells in the foam harbor knots that can be converted into each other by just one intersegmental passage. Such a segment-segment passage occurring at the level of knotted configurations corresponds to a passage through the interface between neighboring cells in the foamy knot space. Using a DNA topoisomerase-inspired simulation approach we characterize here the effective interface area between neighboring knot spaces as well as the surface-to-volume ratio of individual knot spaces. These results provide a reference system required for better understanding mechanisms of action of various DNA topoisomerases.     

Does topoisomerase II specifically recognize and cleave hairpins,cruciforms and crossovers of DNA?

DNA topoisomerase II is an enzyme that specializes in DNA disentanglement. It catalyzes the interconversion of DNA between different topological states. This event requires the passage of one duplex through another one via a transient double-strand break. Topoisomerase II is able to process any type of DNA, including structures such as DNA juxtapositions (crossovers), DNA hairpins or cruciforms, which are recognized with high specificity. In this review, we focused our attention on topoisomerase II recognizing DNA substrates that possess particular geometries. A strong cleavage site, as we identified in pBR322 DNA in the presence of ellipticine (site 22), appears to be characterized by a cruciform structure formed from two stable hairpins. The same sequence could also constitute a four-way junction structure stabilized by interactions involving ATC sequences. The latter have been shown to be able to promote Holliday junctions. We reviewed the recent literature that deals with the preferential recognition of crossovers by various topoisomerases. The single molecule relaxation experiments have demonstrated the differential abilities of the topoisomerases to recognize crossovers. It appears that enzymes, which distinguish the chirality of the crossovers, possess specialized domains dedicated to this function. We also stress that the formation of crossovers is dependent on the presence of adequate stabilizing sequences. Investigation of the impact of such structures on enzyme activity is important in order to both improve our knowledge of the mechanism of action of the topoisomerase II and to develop new inhibitors of this enzyme.     

DNA supercoiling inhibits DNA knotting

Despite the fact that in living cells DNA molecules are long and highly crowded, they are rarely knotted. DNA knotting interferes with the normal functioning of the DNA and, therefore, molecular mechanisms evolved that maintain the knotting and catenation level below that which would be achieved if the DNA segments could pass randomly through each other. Biochemical experiments with torsionally relaxed DNA demonstrated earlier that type II DNA topoisomerases that permit inter- and intramolecular passages between segments of DNA molecules use the energy of ATP hydrolysis to select passages that lead to unknotting rather than to the formation of knots. Using numerical simulations, we identify here another mechanism by which topoisomerases can keep the knotting level low. We observe that DNA supercoiling, such as found in bacterial cells, creates a situation where intramolecular passages leading to knotting are opposed by the free-energy change connected to transitions from unknotted to knotted circular DNA molecules.     

Condensin minimizes topoisomerase II‐mediated entanglements of DNA in vivo

The juxtaposition of intracellular DNA segments, together with the DNA‐passage activity of topoisomerase II, leads to the formation of DNA knots and interlinks, which jeopardize chromatin structure and gene expression. Recent studies in budding yeast have shown that some mechanism minimizes the knotting probability of intracellular DNA. Here, we tested whether this is achieved via the intrinsic capacity of topoisomerase II for simplifying the equilibrium topology of DNA; or whether it is mediated by SMC (structural maintenance of chromosomes) protein complexes like condensin or cohesin, whose capacity to extrude DNA loops could enforce dissolution of DNA knots by topoisomerase II. We show that the low knotting probability of DNA does not depend on the simplification capacity of topoisomerase II nor on the activities of cohesin or Smc5/6 complexes. However, inactivation of condensin increases the occurrence of DNA knots throughout the cell cycle. These results suggest an in vivo role for the DNA loop extrusion activity of condensin and may explain why condensin disruption produces a variety of alterations in interphase chromatin, in addition to persistent sister chromatid interlinks in mitotic chromatin.     

Asymmetric removal of supercoils suggests how topoisomerase II simplifies DNA topology

Type-IIA topoisomerases consume ATP as they catalyse the interconversion of DNA topoisomers by transporting one DNA segment through a transient break in another. It remains unclear how their activity simplifies the topology of DNA below equilibrium values. Here we report that eukaryotic topoisomerase II narrows the thermal distribution of DNA supercoils, by mainly removing negative DNA crossings. Surprisingly, this asymmetry in supercoil removal is not due to deformation of the DNA before strand passage. Topoisomerase II neither bends nor alters the helical conformation of the interacting DNA. Rather, it appears to interact with a third DNA segment, in addition to the gated and the transported segments. Remarkably, the simultaneous interaction with three DNA segments accounts for the asymmetric removal of supercoils in relaxed DNA and gives a clue to how topoisomerase II simplifies the topology of DNA against the thermal drive.     

Topoisomerase II, not topoisomerase I, is the proficient relaxase of nucleosomal DNA

Eukaryotic topoisomerases I and II efficiently remove helical tension in naked DNA molecules. However, this activity has not been examined in nucleosomal DNA, their natural substrate. Here, we obtained yeast minichromosomes holding DNA under (+) helical tension, and incubated them with topoisomerases. We show that DNA supercoiling density can rise above +0.04 without displacement of the histones and that the typical nucleosome topology is restored upon DNA relaxation. However, in contrast to what is observed in naked DNA, topoisomerase II relaxes nucleosomal DNA much faster than topoisomerase I. The same effect occurs in cell extracts containing physiological dosages of topoisomeraseI and II. Apparently, the DNA strand-rotation mechanism of topoisomerase I does not efficiently relax chromatin, which imposes barriers for DNA twist diffusion. Conversely, the DNA cross-inversion mechanism of topoisomerase II is facilitated in chromatin, which favor the juxtaposition of DNA segments. We conclude that topoisomerase II is the main modulator of DNA topology in chromatin fibers. The nonessential topoisomerase I then assists DNA relaxation where chromatin structure impairs DNA juxtaposition but allows twist diffusion.     

Computational analysis of DNA gyrase action

DNA gyrase introduces negative supercoiling into circular DNA by catalyzing the passage of one DNA segment through another. The efficiency of the reaction is many times higher than that of other topological transformations. We analyze, by a computer simulation, the reaction selectivity for a model of DNA gyrase action that assumes existence of a free loop between the G- and T- DNA segments participating in the reaction. A popular model of this type assumed that the selectivity can be provided by the conformation of the DNA segment wrapped around the enzyme into the right-handed helix turn (G-segment). We simulated the distribution of the reaction products for this model. Equilibrium sets of DNA conformations with one segment of the double helix wrapped around the enzyme were constructed. From these sets we selected conformations that had a second segment properly juxtaposed with the first one. Assuming that the juxtapositions result in the strand-passing reaction, we calculated the reaction products for all these conformations. The results show that different products have to be formed if the enzyme acts according to the model. This conclusion can be extended for any model with a free loop between the G- and T-segments. An alternative model that is consistent with the major experimental observations and the computational analysis, is suggested.     

Solution structure of ADO1, a toxin extracted from the saliva of the assassin bug, Agriosphodrus dohrni

ADO1 is a toxin purified from the saliva of the assassin bug, Agriosphodrus dohrni. Because of its similarity in sequence to Ptu1 from another assassin bug, we did not assess its pharmacologic target. Here, we demonstrate by electrophysiologic means that ADO1 targets the P/Q-type voltage-sensitive calcium channel. We also determine the solution structure of ADO1 using two-dimensional NMR techniques, followed by distance geometry and molecular dynamics. The structure of ADO1 belongs to the inhibitory cystine knot (ICK) structural family (i.e., a compact disulfide-bonded core from which four loops emerge). ADO1 contains a two-stranded, antiparallel beta-sheet structure. We compare the structure of ADO1 with other voltage-sensitive calcium-channel blockers, analyze the topologic juxtaposition of key functional residues, and conclude that the recognition of voltage-sensitive calcium channels by toxins belonging to the ICK structural family requires residues located on two distinct areas of the molecular surface of the toxins.     

The mechanism of negative DNA supercoiling: A cascade of DNA-induced conformational changes prepares gyrase for strand passage

DNA topoisomerases inter-convert different DNA topoisomers in the cell. They catalyze the introduction or relaxation of DNA supercoils, as well as catenation and decatenation. Members of the type I topoisomerase family cleave a single strand of their double-stranded DNA substrate, whereas enzymes of the type II family cleave both DNA strands. Bacterial DNA gyrase, a type II topoisomerase, catalyzes the introduction of negative supercoils into DNA in an ATP-dependent reaction. Gyrase is not present in humans, and constitutes an attractive drug target for the treatment of bacterial and parasite infections. DNA supercoiling by gyrase is believed to occur by a strand passage mechanism, in which one segment of the double-stranded DNA substrate is passed through a (transient) break in a second segment. This mechanism requires the coordinated opening and closing of three protein interfaces, so-called gates, to ensure the directionality of strand passage toward negative supercoiling.Single molecule fluorescence resonance energy transfer experiments are ideally suited to investigate conformational changes during the catalytic cycle of DNA topoisomerases. In this review, we summarize the current knowledge on the cascade of DNA- and nucleotide-induced conformational changes in gyrase that lead to strand passage and negative supercoiling of DNA. We discuss how these conformational changes couple ATP hydrolysis to DNA supercoiling in gyrase, and how the common mechanistic principle of coordinated gate opening and closing is modulated to allow for the catalysis of different reactions by different type II topoisomerases.     

Reprint of “The mechanism of negative DNA supercoiling: A cascade of DNA-induced conformational changes prepares gyrase for strand passage”

DNA topoisomerases inter-convert different DNA topoisomers in the cell. They catalyze the introduction or relaxation of DNA supercoils, as well as catenation and decatenation. Members of the type I topoisomerase family cleave a single strand of their double-stranded DNA substrate, whereas enzymes of the type II family cleave both DNA strands. Bacterial DNA gyrase, a type II topoisomerase, catalyzes the introduction of negative supercoils into DNA in an ATP-dependent reaction. Gyrase is not present in humans, and constitutes an attractive drug target for the treatment of bacterial and parasite infections. DNA supercoiling by gyrase is believed to occur by a strand passage mechanism, in which one segment of the double-stranded DNA substrate is passed through a (transient) break in a second segment. This mechanism requires the coordinated opening and closing of three protein interfaces, so-called gates, to ensure the directionality of strand passage toward negative supercoiling.Single molecule fluorescence resonance energy transfer experiments are ideally suited to investigate conformational changes during the catalytic cycle of DNA topoisomerases. In this review, we summarize the current knowledge on the cascade of DNA- and nucleotide-induced conformational changes in gyrase that lead to strand passage and negative supercoiling of DNA. We discuss how these conformational changes couple ATP hydrolysis to DNA supercoiling in gyrase, and how the common mechanistic principle of coordinated gate opening and closing is modulated to allow for the catalysis of different reactions by different type II topoisomerases.     

Eukaryotic topoisomerases recognize nucleic acid topology by preferentially interacting with DNA crossovers.

Eukaryotic topoisomerases recognize DNA topology and preferentially react with positively or negatively supercoiled molecules over relaxed substrates. To elucidate the mechanism of this recognition, we examined the interaction of topoisomerases with DNA by electron microscopy. Under all conditions employed, approximately 90% of the bound type I or II enzyme was observed at points of helix--helix juxtaposition on negatively supercoiled plasmids which contained as few as four crossovers. Recognition was independent of torsional stress, as enzyme molecules were also found at crossovers on linear DNA. Since juxtaposed helices are more prevalent in supercoiled compared with relaxed nucleic acids, we propose that eukaryotic topoisomerases I and II recognize underwound or overwound substrates by interacting preferentially with DNA crossovers. This may represent a general mechanism for the recognition of DNA topology by proteins.     

Direct observation of strand passage by DNA-topoisomerase and its limited processivity

Type-II DNA topoisomerases resolve DNA entanglements such as supercoils, knots and catenanes by passing one segment of DNA duplex through a transient enzyme-bridged double-stranded break in another segment. The ATP-dependent passage reaction has previously been demonstrated at the single-molecule level, showing apparent processivity at saturating ATP. Here we directly observed the strand passage by human topoisomerase IIα, after winding a pair of fluorescently stained DNA molecules with optical tweezers for 30 turns into an X-shaped braid. On average 0.51 ± 0.33 μm (11 ± 6 turns) of a braid was unlinked in a burst of reactions taking 8 ± 4 s, the unlinked length being essentially independent of the enzyme concentration between 0.25-37 pM. The time elapsed before the start of processive unlinking decreased with the enzyme concentration, being ~100 s at 3.7 pM. These results are consistent with a scenario where the enzyme binds to one DNA for a period of ~10 s, waiting for multiple diffusional encounters with the other DNA to transport it across the break ~10 times, and then dissociates from the binding site without waiting for the exhaustion of transportable DNA segments.