Human topoisomerase IIalpha rapidly relaxes positively supercoiled DNA: implications for enzyme action ahead of replication forks

Movement of the DNA replication machinery through the double helix induces acute positive supercoiling ahead of the fork and precatenanes behind it. Because topoisomerase I and II create transient single- and double-stranded DNA breaks, respectively, it has been assumed that type I enzymes relax the positive supercoils that precede the replication fork. Conversely, type II enzymes primarily resolve the precatenanes and untangle catenated daughter chromosomes. However, studies on yeast and bacteria suggest that type II topoisomerases may also function ahead of the replication machinery. If this is the case, then positive DNA supercoils should be the preferred relaxation substrate for topoisomerase IIalpha, the enzyme isoform involved in replicative processes in humans. Results indicate that human topoisomerase IIalpha relaxes positively supercoiled plasmids >10-fold faster than negatively supercoiled molecules. In contrast, topoisomerase IIbeta, which is not required for DNA replication, displays no such preference. In addition to its high rates of relaxation, topoisomerase IIalpha maintains lower levels of DNA cleavage complexes with positively supercoiled molecules. These properties suggest that human topoisomerase IIalpha has the potential to alleviate torsional stress ahead of replication forks in an efficient and safe manner.     

On the toroidal condensed state of closed circular DNA

The influence of double helix torsional elasticity on the compaction and structure of circular DNA compact form is studied theoretically in the case when the compact (globular) form has torus shape. For closed circular DNA the topological invariant, the linking number, yields a strict connection between conformation of the double helix considered as unifilar homopolymer and elastic energy of torsional twisting. The contribution of torsional elasticity to the free energy of the toruslike globule is calculated. This contribution is shown to be proportional to the square of superhelical density. Allowance of the torsional elasticity decreases the equilibrium radius of the toruslike globule formed by circular DNA. Closure of linear DNA into a ring widens the stability range of the relatively short DNA compact form and tightens it for long DNA.     

Toroidal globular state of circular DNA

The influence of torsional elasticity of the double helix on compactization and structure of circular DNA in a compact form is studied in the case when the compact (globular) particle has a torus shape. For closed circular DNA the topological invariant, linking number of two strains, yields strict connection between conformation of double helix, considered as a unifilar homopolymer, and elastic energy of torsional twisting. The contribution of torsional elasticity to free energy of the toruslike globule is calculated. This contribution is shown to be proportional to the square of superturn's density. Torsional elasticity decreases the equilibrium radius of the toruslike globule formed by circular DNA in comparison with the case of linear DNA. Closure of linear DNA into a ring widens the stability range of the relatively short DNA compact form and tightens it for long DNA.     

On the Toroidal Condensed State of Closed Circular DNA

Abstract

The influence of double helix torsional elasticity on the compaction and structure of circular DNA compact form is studied theoretically in the case when the compact (globular) form has torus shape. For closed circular DNA the topological invariant, the linking number, yields a strict connection between conformation of the double helix considered as unifilar homopolymer and elastic energy of torsional twisting. The contribution of torsional elasticity to the free energy of the toruslike globule is calculated. This contribution is shown to be proportional to the square of superhelical density. Allowance of the torsional elasticity decreases the equilibrium radius of the toruslike globule formed by circular DNA. Closure of linear DNA into a ring widens the stability range of the relatively short DNA compact form and tightens it for long DNA.     

Negative constrained DNA supercoiling in archaeal nucleosomes

Archaeal histones have significant sequence and structural similarity to their eukaryal counterparts. However, whereas DNA is wrapped in negatively constrained supercoils in eukaryal nucleosomes, it has been reported that DNA is positively supercoiled by archaeal nucleosomes. This was inferred from experiments performed at low temperature and low salt concentrations, conditions markedly different from those expected for many archaea in vivo. Here, we report that the archaeal histones HMf and HTz wrap DNA in negatively constrained supercoils in buffers containing potassium glutamate (K-Glu) above 300 mM, either at 37 degrees C or at 70 degrees C. This suggests that high salt concentrations allow an alternate archaeal nucleosome topology: a left-handed tetramer rather than the right-handed tetramer seen in low salt conditions. In contrast, the archaeal histone MkaH produces DNA negative supercoiling at all salt concentrations, suggesting that this duality of structure is not possible for this atypical protein, which is formed by the association of two histone folds in a single polypeptide. These results extend the already remarkable similarity between archaeal and eukaryal nucleosomes, as it has been recently shown that DNA can be wrapped into either positive or negative supercoils around the H3/H4 tetramer. Negative supercoiling could correspond to the predominant physiological mode of DNA supercoiling in archaeal nucleosomes.     

Topoisomer gel retardation: detection of anti-Z-DNA antibodies bound to Z-DNA within supercoiled DNA minicircles.

Small DNA fragments of approximately 350 bp in length, either with or without d(CG)n tracts, are ligated into underwound DNA minicircles to generate topoisomeric rings with different topological linking numbers, Lk. These minicircles, differing by an Lk of one, can be separated by acrylamide gel electrophoresis. Furthermore, electrophoresis can be used to reveal DNA double helix conformational changes that are induced by supercoiling, such as left-handed Z-DNA. When anti-Z-DNA antibodies are added to such minicircles, their binding leads to a selective retardation of the electrophoretic migration of the Z-DNA containing circles. This effect is not seen with relaxed minicircles and those with insufficient torsional stress to induce a conformational transition. Thus the technique of 'topoisomer gel retardation' presents a very sensitive assay for the identification of proteins that selectively bind to DNA conformations stabilized by negative DNA supercoiling.     

Negative supercoiling of DNA by eukaryotic DNA topoisomerase II and dextran sulfate.

In the presence of a molar excess of eukaryotic DNA topoisomerase II and an appropriate concentration of dextran sulfate, relaxed closed circular DNA is converted to a negatively supercoiled form. The reaction is dependent on ATP. Neither adenosine 5'-[beta,gamma-imido]-triphosphate nor adenosine 5'-[gamma-thio]triphosphate can substitute for ATP. The negative supercoils formed are relaxed by subsequent addition of DNA topoisomerase I to the supercoiling reaction mixture. Covalent closure of a nicked circular DNA in the presence of DNA topoisomerase II and dextran sulfate but in the absence of ATP causes a small decrease in the linking number. These results suggest that when an appropriate concentration of dextran sulfate is present, the binding of a molar excess of eukaryotic DNA topoisomerase II constrains a small number of negative supercoils in DNA, which in turn generate unconstrained negative supercoils at the expense of ATP.     

High positive supercoiling in vitro catalyzed by an ATP and polyethylene glycol-stimulated topoisomerase from Sulfolobus acidocaldarius

A topoisomerase able to introduce positive supercoils in a closed circular DNA, has been isolated from the archaebacterium Sulfolobus acidocaldarius. This enzyme, fully active at 75 degrees C, performed in vitro positive supercoiling either from negatively supercoiled, or from relaxed DNA in a catalytic reaction. In the presence of polyethylene glycol (PEG 6000), this reaction became very fast and highly processive, and the product was positively supercoiled DNA with a high superhelical density (form I+). Very low (5 - 10 micromoles) ATP concentrations were sufficient to support full supercoiling; the nonhydrolyzable analogue adenosine-5' -0-(3-thiotriphosphate) also sustained the production of positive supercoils, but to a lesser extent, suggesting that ATP hydrolysis was necessary for efficient activity. Nevertheless, low residual of positive supercoiling occurred, even in the absence of ATP, when the substrate was negatively supercoiled. Finally, the different ATP-driven topoisomerizations observed, i.e., relaxation of negative supercoils and positive supercoiling, in all cases increased the linking number of DNA in steps of 1, suggesting the action of a type I, rather than a type II topoisomerase.=     

Assembly of nucleosome-like structures mediated by cauliflower DNA topoisomerase

Nucleosome-like structures have been efficiently assembled in vitro by interaction of cauliflower histones, pBR322 DNA and cauliflower DNA topoisomerase, as assayed by supercoiling of relaxed circular DNA and by digestion with micrococcal nuclease. The optimum ionic strength for supercoiling was 150 mM KCl and the optimum weight ratio of histone to DNA was approximately 1.0. Four histones, H2A, H2B, H3 and H4, were necessary for the optimum assembling conditions, and the nucleosomes assembled protected DNA fragments of approximately 150 bp in length. It was found that cauliflower DNA topoisomerase acts not only as a DNA-relaxing enzyme but also as a chaperon factor for nucleosome assembly.     

Role of HU proteins in forming and constraining supercoils of chromosomal DNA inEscherichia coli

Induction of supercoiling in plasmid DNA by HU heterotypic and homotypic dimers, a mutant HU-2 (HupAN12), HBs and HB1 proteins with different DNA-binding affinities was investigated in vitro. The abilities of these proteins to induce supercoiling in DNA correlated with their affinities for DNA. Stoichiometrical analysis of HU heterodimers bound to DNA in the complex restraining the negative torsional tension of DNA showed that 12–13 dimers account for a single superhelical turn. The number of supercoils in the plasmid in vivo decreased on inhibition of DNA gyrase with coumermycin, reaching a steady-state level that indicated the existence of a compartment of restrained supercoils. The size of the restrained compartment was reduced in the absence of HU, indicating the participation of HU in constituting this fraction, and was larger on overproduction of HU-2 in the cells. An increased level of DNA gyrase, expressed from a plasmid carrying bothgyr genes, in the cells did not compensate for the deficit of the restrained supercoils caused by HU deficiency, indicating seeming distinct and unrelated action of HU and DNA gyrase in introducing and constraining supercoiling of intracellular DNA.     

DNA supercoiling during ATP-dependent DNA translocation by the type I restriction enzyme EcoAI

Type I restriction enzymes cleave DNA at non-specific sites far from their recognition sequence as a consequence of ATP-dependent DNA translocation past the enzyme. During this reaction, the enzyme remains bound to the recognition sequence and translocates DNA towards itself simultaneously from both directions, generating DNA loops, which appear to be supercoiled when visualised by electron microscopy. To further investigate the mechanism of DNA translocation by type I restriction enzymes, we have probed the reaction intermediates with DNA topoisomerases. A DNA cleavage-deficient mutant of EcoAI, which has normal DNA translocation and ATPase activities, was used in these DNA supercoiling assays. In the presence of eubacterial DNA topoisomerase I, which specifically removes negative supercoils, the EcoAI mutant introduced positive supercoils into relaxed plasmid DNA substrate in a reaction dependent on ATP hydrolysis. The same DNA supercoiling activity followed by DNA cleavage was observed with the wild-type EcoAI endonuclease. Positive supercoils were not seen when eubacterial DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I, which removes both positive and negative supercoils. Furthermore, addition of eukaryotic DNA topoisomerase I to the product of the supercoiling reaction resulted in its rapid relaxation. These results are consistent with a model in which EcoAI translocation along the helical path of closed circular DNA duplex simultaneously generates positive supercoils ahead and negative supercoils behind the moving complex in the contracting and expanding DNA loops, respectively. In addition, we show that the highly positively supercoiled DNA generated by the EcoAI mutant is cleaved by EcoAI wild-type endonuclease much more slowly than relaxed DNA. This suggests that the topological changes in the DNA substrate associated with DNA translocation by type I restriction enzymes do not appear to be the trigger for DNA cleavage.     

Role of HU proteins in forming and constraining supercoils of chromosomal DNA inEscherichia coli

Induction of supercoiling in plasmid DNA by HU heterotypic and homotypic dimers, a mutant HU-2 (HupAN12), HBs and HB1 proteins with different DNA-binding affinities was investigated in vitro. The abilities of these proteins to induce supercoiling in DNA correlated with their affinities for DNA. Stoichiometrical analysis of HU heterodimers bound to DNA in the complex restraining the negative torsional tension of DNA showed that 12–13 dimers account for a single superhelical turn. The number of supercoils in the plasmid in vivo decreased on inhibition of DNA gyrase with coumermycin, reaching a steady-state level that indicated the existence of a compartment of restrained supercoils. The size of the restrained compartment was reduced in the absence of HU, indicating the participation of HU in constituting this fraction, and was larger on overproduction of HU-2 in the cells. An increased level of DNA gyrase, expressed from a plasmid carrying bothgyr genes, in the cells did not compensate for the deficit of the restrained supercoils caused by HU deficiency, indicating seeming distinct and unrelated action of HU and DNA gyrase in introducing and constraining supercoiling of intracellular DNA.     

Modeling DNA supercoils and knots with B-spline functions

A method is offered to model the complex trajectories of closed circular DNA supercoils and knots. The trajectories are approximated by polygons and analytical expressions of the curves are generated from the polygons with B-spline functions. The resulting curves are used to evaluate the writhe and elastic energy of a series of interrelated supercoils, and to generate detailed atomic models of the deformed double helix.     

Local supercoil-stabilized DNA structures

The DNA double helix exhibits local sequence-dependent polymorphism at the level of the single base pair and dinucleotide step. Curvature of the DNA molecule occurs in DNA regions with a specific type of nucleotide sequence periodicities. Negative supercoiling induces in vitro local nucleotide sequence-dependent DNA structures such as cruciforms, left-handed DNA, multistranded structures, etc. Techniques based on chemical probes have been proposed that make it possible to study DNA local structures in cells. Recent results suggest that the local DNA structures observed in vitro exist in the cell, but their occurrence and structural details are dependent on the DNA superhelical density in the cell and can be related to some cellular processes.     

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

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

Statistical Mechanical Approach for Predicting the Transition to Non-B DNA Structures in Supercoiled DNA

Abstract

Supercoiling causes global twist of DNA structure and the supercoiled state has wide influence on conformational transition. A statistical mechanical approach was made for prediction of the transition probability to non-B DNA structures under torsional stress. A conditional partition function was defined as the sum over all possible states of the DNA sequence with basepair 1 and basepair n being in B-form helix and a recurrence formula was developed which expressed the partition function for basepair n with those for less number of pairs. This new definition permits a quick enumeration of every configuration of secondary structures. Energetic parameters of all conformations concerned, involving B-form, interior loop, cruciform and Z-form, were included in the equation. The probability of transition to each non-B conformation could be derived from these conditional partition functions. For treatment of effects of superhelicity, supercoiling energy was considered, and a twist of each conformation was determined to minimize the supercoiling energy. As the twist itself affects the transition probability, the whole scheme of equations was solved by renormalization technique. The present method permits a simultaneous treatment of serveral types of conformations under a common torsional stress.

A set of energetic parameters of DNA secondary structures has been chosen for calculation. Some DNA sequences were submitted to the calculation, and all the sequences that we submitted gave stable convergence. Some of them have been investigated the critical supercoil density for the transition to non-B DNA structures. Even though the reliability of the set of parameters was not enough, the prediction of secondary structure transition showed good agreement with reported observation. Hence, the present algorithm can estimate the probability of local conformational change of DNA under a given supercoil density, and also be employed to predict some specific sequences in which conformational change is sensitive to superhelicity.     

Formation of positive supercoiled DNA by a nuclear factor from myeloma cells.

The formation of positive supercoiled DNA by an activity from a hypermutating myeloma line is reported. This activity forms positive supercoils from negative supercoiled DNA, it does not use positive supercoils to form negative ones and does not require an exogenous source of energy. The linking number changes by steps of 1, suggesting a type-I mechanism of action, and there seems to be an upper limit to the degree of positive supercoiling that can be achieved. Positive supercoiled DNA has to be taken into account as a possible structure of DNA in vivo for those functions where torsional stress is involved.