TY - JOUR. T1 - Identification of a DNA supercoiling activity in Saccharomyces cerevisiae. AU - Koo, Hyeon Sook. AU - Lau, Kawai. AU - Wu, Hai Young. AU - Liu, Leroy-Fong. PY - 1992/10/11. Y1 - 1992/10/11. N2 - A relaxed plasmid DNA is shown to become positively supercoiled in cell extracts from top1 strains of Saccharomyces cerevisiae. This positive supercoiling activity is dependent on the presence of bacterial DNA topoisomerase I and ATP (or dATP), and the positive supercoils generated in this reaction are not constrained by protein(s). Non-hydrolyzable ATP analogs cannot substitute for ATP in this supercoiling reaction, and the supercoiling activity is not due to RNA synthesis. The presence of an ARS sequence in the DNA does not alter the activity. Furthermore, this activity is equally active against UV irradiated or intact DNA. Extracts prepared from rad50 and rad52 mutant cells exhibited the same activity. Partial purification of this activity suggests that a protein factor with a native ...
DNA supercoiling plays essential role in maintaining proper chromosome structure, as well as the equilibrium between genome dynamics and stability under specific physicochemical and physiological conditions. In mesophilic organisms, DNA is negatively supercoiled and, until recently, positive supercoiling was considered a peculiar mark of (hyper)thermophilic archaea needed to survive high temperatures. However, several lines of evidence suggest that negative and positive supercoiling might coexist in both (hyper)thermophilic and mesophilic organisms, raising the possibility that positive supercoiling might serve as a regulator of various cellular events, such as chromosome condensation, gene expression, mitosis, sister chromatid cohesion, centromere identity and telomere homoeostasis.. ...
When HeLa cells are lysed in solutions containing a non-ionic detergent and 0.75 M-NaCl, structures are released that retain many of the morphological features of nuclei. These nucleoids contain all the nuclear DNA, RNA and the core histones, but few other proteins characteristic of chromatin. Their DNA is intact. The core histones dissociate on raising the salt concentration. We have probed the structure of nucleoid-histone complexes using the intercalating dye, ethidium, or the RNA polymerase of Escherichia coli. Both have a higher affinity for superhelical DNA than they do for relaxed DNA. The binding of ethidium is measured fluorometrically, and using this probe we find that the DNA of nucleoids containing all the core histones behaves as if it were supercoiled slightly positively. As the salt concentration is increased, free energy characteristic of negative supercoiling appears between 0.92 M and 0.95 M-NaCl. This transition, which is reversible in the presence of the arginine-rich ...
The "upstream regions of the human CYP11A and bovine CYP11B genes [have] a distal promoter in each gene. The distal promoters are located at −1.8 to −1.5 kb in the upstream region of the CYP11A gene and −1.5 to −1.1 kb in the upstream region of the CYP11B gene."[11] "Using cloned chicken βA-globin genes, either individually or within the natural chromosomal locus, enhancer-dependent transcription is achieved in vitro at a distance of 2 kb with developmentally staged erythroid extracts. This occurs by promoter derepression and is critically dependent upon DNA topology. In the presence of the enhancer, genes must exist in a supercoiled conformation to be actively transcribed, whereas relaxed or linear templates are inactive. Distal protein-protein interactions in vitro may be favored on supercoiled DNA because of topological constraints."[12] Distal promoter regions may be a relatively small number of nucleotides, fairly close to the TSS such as (-253 to -54)[13] or several regions of ...
In article ,33F84BF9.1DB0 at box-d.nih.gov,, Darren A. Natale ,dnatale at box-d.nih.gov, wrote: riginally REMOVED negative supercoils, taking it away will again , ADD , negative supercoils. , , D. Natale , dnatale at box-d.nih.gov Ya, guys and gals, are too sophisticated for your own good - kinda hard to be a-keeping track of them supercoils ,G,. The easy way to fix the nicked DNA is to reinoculate (yes, and retransform if needed) the e. coli and isolate the DNA, right way this time, so it is not nicked. Good luck with transfection with DNA ligated in presence of EtBr. Yours, Pointless peasant Baldrick ...
Transcription by RNA polymerase in E. coli. To synthesize an RNAstrand complementary to one of the two DNA strands in a double helix, the DNA is transientlyunwound. (a) About 17 bp is unwound at any given time. The transcription bubblemoves from left to right as shown, keeping pace with RNA synthesis. The DNA isunwound ahead and rewound behind as RNA is transcribed. Red arrows show the directionin which the DNA and the short RNA-DNA hybrid must rotate to permit this process.(b) Supercoiling of DNA brought about by transcription. Positive supercoils form ahead ofthe transcription bubble, and negative supercoils form behind. Reprinted from D. L. Nelsonand M. M. Cox, Lehninger Principles of Biochemistry, 3rd ed. (Worth Publishers, NewYork, N.Y., 2000), with permission from the publisher. ...
Bacterial genomes have been shown to be partitioned into several kilobases long chromosomal domains that are topologically independent from each other, meaning that change of DNA superhelicity in one domain does not propagate to neighbors. Both in vivo and in vitro experiments have been performed to question the nature of the topological barriers at play, leading to several predictions on possible molecular actors. Here, we address the question of topological barriers using polymer models of supercoiled DNA chains. More specifically, we determine under which conditions DNA-bridging proteins may act as topological barriers. To this end, we developed a coarse-grained bead-and-spring model and investigated its properties through Brownian dynamics simulations. As a result, we find that DNA-bridging proteins must exert rather strong constraints on their binding sites: they must block the diffusion of the excess of twist through the two binding sites on the DNA molecule and, simultaneously, prevent ...
Chromosome of supercoiled DNA, conceptual image. Computer artwork of a human chromosome, representing how DNA (deoxyribonucleic acid) is supercoiled (spirals) to be packaged within it. Chromosomes are composed of DNA strands that contain sections, called genes, which encode the bodys genetic information. Each chromosome consists of two chromatids joined at their centres by the centromere, which is involved in cell division. - Stock Image C016/8433
Affinity chromatography Arginine monolith Composite Central Face design Design of experiments HPV-16 E6/E7 vaccine Supercoiled plasmid DNA
Effect of Topology on Diversity ofSpatially-Structured Evolutionary AlgorithmsM. De Felice - Energy and Environment Modelling [email protected], Rome, Italy…
Publication date: Available online 27 February 2020Source: Biochimica et Biophysica Acta (BBA) - Gene Regulatory MechanismsAuthor(s): Cristina S.D. Palma, Vinodh Kandavalli, Mohamed N.M. Bahrudeen, Marco Minoia, Vatsala Chauhan, Suchintak Dash, Andre S. Ribeiro...
BCA-1 Human Recombinant produced in E.Coli is a single, non-glycosylated polypeptide chain topological domain containing 110 amino acids (23-109 a.a).
Khturner 10:19, 7 December 2006 (EST): The first couple of times you try this, it might be tough to get a feel for the viscosity of the sample at each stage, but once you get the method to work, it pretty much eliminates the need to optimize your ligation and transformation. Sean Moore Feb. 5, 2007 Is this necessary? This adds a whole day to the screening procedure? You could have done PCR on the colonies in a few hours from the original plate if you needed to screen for colonies with the correct insert. I dont see more colonies on the "vector + insert" plate than the "vector only" plate, I usually dont even bother screening and assume something is wrong with the fragments. I have never "optimized" ligations. Also, some replication origins cause significant catenation so analysis in the supercoiled form is impractical/unreliable. *Khturner 16:51, 15 April 2007 (EDT): You know, youre probably right. It used to help me when I had problems cloning and a successful construct was rare, but now ...
Fig. 2 Left shows that incubation of RecA-covered 94-mers with dsDNA containing one copy of the same sequence resulted in limited unwinding of the duplex DNA. The most abundant topoisomers produced upon pairing had their linking number decreased by 6 compared with the most abundant topoisomers produced in corresponding control reactions without RecA-covered 94-mers. This result was independent of the type of enzyme (topoisomerase or ligase) used in the assay whereby the 5:1 or 10:1 molar ratios of oligonucleotides to target plasmids produced the same unwinding (data not shown). Control reactions containing plasmids with no homology to the RecA-covered 94-mers showed that there was no appreciable unwinding (Fig. 2 Right).. When we started this project, we considered two possible outcomes of the unwinding assay. If the incoming single-strand pairs with its complement while the displaced strand is extrahelical (D-loop formation), one should observe a complete unwinding of dsDNA in the region of ...
Since the mobile hosts in a MANET keep moving, topological changes take place frequently in the network. This causes routing information kept by the nodes
I will tell you my experience. Plasmids have different conformations. Either it is supercoiled, half coiled, relaxed state or linear form. I just transform the plasmid into E.coli cells. And i guess this applies either you are using Agrobacterium or yeast. Maybe you can try it out and check how efficient is the transformation ...
Nuestra investigación trata de explicar la base estructural de la regulación de distintos procesos mitocondriales. La técnica principal que empleamos es el análisis por cristalografía de rayos X de las proteínas involucradas, de sus complejos con otras proteínas y/o con DNA. Para obtener una visión más completa del fenómeno, estos estudios se complementan mediante análisis bioquímicos y biofísicos.. Para ver el artículo completo, pulse aquí ...
Best1 relieves DNA torsional stress generated during replication and transcription by nicking supercoiled DNA allowing relaxation to proceed via handled rotation [3], and re-ligating the nick [4] to revive the DNA duplex. CPT forms a ternary complicated with Best1 as well as the nicked DNA leading to formation of the Best1 cleavage complicated (Best1cc) that, […]. ...
Chemical and enzymatic probing methods are powerful techniques for examining details of sequence-dependent structure in DNA and RNA. Reagents that cleave nucleic acid molecules in a structure-specific, but relatively sequence-non-specific manner, such as hydroxyl radical or DNase I, have been used widely to probe helical geometry in nucleic acid structures, nucleic acid-drug complexes, and in nucleoprotein assemblies. Application of cleavage-based techniques to structures present in superhelical DNA has been hindered by the fact that the cleavage pattern attributable to supercoiling-dependent structures is heavily mixed with non-specific cleavage signals that are inevitable products of multiple cleavage events. We present a rigorous mathematical procedure for extracting the cleavage pattern specific to supercoiled DNA and use this method to investigate the hydroxyl radical cleavage pattern in a cruciform DNA structure formed by a 60 bp inverted repeat sequence embedded in a negatively ...
DNA replication requires the activity of a class of enzymes called the topoisomerases. Topoisomerase II (DNA gyrase) relaxes supercoiled DNA molecules and initiates transient breakages and rejoins phosphodiester bonds in superhelical turns of closed-circular DNA of bacteria. This allows the DNA strand to be replicated by DNA or RNA polymerases. Quinolones are a key group of antibiotics that specifically interfere with bacterial topoisomerase II and not mammalian topoisomerases. Fluoroquinolones, second-generation quinolones that include levofloxacin, norfloxacin, and ciprofloxacin, are active against both Gram-negative and Gram-positive bacteria. Inhibitors that are effective against mammalian topoisomerases, such as irinotecan and etoposide, are used as antineoplastic drugs to kill cancer cells.

Some antibiotics specifically interfere with RNA synthesis by inhibiting RNA polymerases. Rifampicin blocks initiation of RNA synthesis by specifically inhibiting bacterial RNA polymerase. It does not
Hi Ronan, for pBR322 I use 1 to 15 ug/ml chloroquin in gel and running buffer. Topoisomers of pBR isolated from E. coli are clearly separated at 10 ug/ml chloroquin, except when the DNA is extremely relaxed (like when you add a gyrase inhibitor), then you need to add less chloroquin. Extremely supercoiled DNA needs more chloroquin to pull the bands apart. but not too much, or youll get positively writhed plasmids on top of the negative ones. Good luck, Rogier * Sent from RemarQ http://www.remarq.com The Internets Discussion Network * The fastest and easiest way to search and participate in Usenet - Free ...
Analysis and computational tools and modeling capability for high-temperature electrolysis, low-temperature electrolysis, photoelectrochemical, and solar thermochemical technologies at Sandia National Laboratories
DNA gyrase is a bacterial enzyme that catalyzes the ATP-dependent negative supercoiling of double-stranded, closed-circular DNA, according to the Critical Review of Biochemical and Molecular Biology....
The high-throughput screen has also uncovered 21 structurally novel inhibitors of vTopo that had IC90 values for the ribonuclease reaction of less than 10 μM. Only 2 of these 21 compounds failed to show comparable inhibition in the supercoil relaxation assay (Fig. 3A). The remaining 19 compounds showed potencies of inhibition against DNA relaxation that were comparable with or even significantly enhanced compared with their potencies in the ribonuclease screen. It is not surprising that there are differences between the inhibitory effects of the same compound in the two assays. If the compound interacts with the covalent complex and not the free enzyme, as we have generally found (see below), the presence of a 2′ hydroxyl with the ribonuclease substrate could have a differential effect on the interaction of the inhibitor compared with the 2′ hydrogen in DNA. Nevertheless, the results here show that any differences in inhibitor interactions between these two substrates do not prevent the ...
PubMed comprises more than 30 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.
In gel electrophoresis it was observed in Ref. [1] that underwound DNA mini-rings migrate slower than overwound DNA mini-rings in EDTA solution. Motivated with the novel observation we have evaluated the diffusion constant of a closed ladder-shaped polymer in solution via Brownian dynamics with hydrodynamic interaction. It gives a model of a supercoiled DNA mini-ring consisting of DNA double strands. Here, the topology of the whole molecule such as the linking number (Lk) of the double DNA strands is conserved in time. By introducing the bending rigidity, we have found that the diffusion constant of a ladder-shaped molecule with base flipping becomes smaller and much more dependent on the linking number, Lk [2]. We thus suggest that the numerical observations should explain the different migration sppeds between the underwound and overwound DNA mini-rings. Here we also recall an interesting observation that for knotted DNAs the migration speeds in gel are proportional to th e diffusion constants ...
Non-covalent polymer-protein conjugation is emerging as a potential route to improve pharmacokinetics and pharmacodynamics of protein therapeutics. In this study, a family of structurally related block copolymers of mPEG2k - poly(glutamic acid) with linear A-B (mPEG2k-lin-polyGA) and miktoarm A-B3 ((mPEG2k-mik-(polyGA)3) structure was synthesised by N-carboxyanhydride (NCA) ring-opening polymerisation to assess the effect of macromolecular topology of the copolymers on polymer-protein complexation. The data illustrate that the synthesised copolymers are capable of complexing a model protein, lysozyme, at optimal pH conditions through non-covalent interactions, with complexation efficiencies depending on the copolymers composition and molecular architecture. In native gel electrophoresis experiments, linear mPEG2k-lin-GA10 copolymer, possessing a short polyanionic polyGA block, shows a low level of complexation, which does not change when the number of polyGA branches of the same size is ...
Hi, in this agarose gel photo (70 volts, 3.5uL Gel Red in 30ml of TBE, 1% Agarose, 1 hour run), Ive checked the digestion of a large plasmid (15Kbp, one cut site). The theory says that the digested plasmid (linearized) should migrate slower than the supercoiled form of the uncut one (bigger band of the Undigested Plasmid). In my case the digested plasmid is faster than the undigested one. Ive found that my digested plasmid could be single-stranded (http://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/). Do you think that could be the case?. ...
plasmid digest - posted in Molecular Biology: Hi, I tried to digest my pBSK(-) vector with EcoRV but failed. No linearized vector band on my agarose gel, just supercoiled + circular (nicked) DNA. I used 20 units of newly bought enzyme in the appropriate buffer + BSA, digested for 3 h at 37 degrees - should be more than sufficient. The vector is easily and completely digested by HaeIII in 1h. If the vector DNA from a Maxi-Prep is poor quality, it shouldn t be digested by HaeIII either, rig...
Everyone could have their reasons for wanting to hold tight to a chunk of their DNA, and while many may just be creeped out by the whole premise, DNA Direct is...
The incorporation, regulation and winding up of co-operative societies (other than those operating in more than one State) is a State subject and is governed by the State laws on co-operative societies. In the case of co-operatives with objects not confined to one State, their incorporation, regulation and winding up fall in the central domain and are governed by the Multi-State Co-operative Societies Act, 2002. ...
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Transcription has the capacity to mechanically modify DNA topology, DNA structure and nucleosome arrangement. Resulting from ongoing transcription, these modifications in turn may provide instant feedback to the transcription machinery. To substantiate the connection between transcription and DNA dy …
We reveal an intriguing manifestation of topology, which appears in the depletion rate of topological states of matter in response to an external drive. This phenomenon is presented by analyzing the response of a generic two-dimensional (2D) Chern insulator subjected to a circular time-periodic perturbation. Because of the systems chiral nature, the depletion rate is shown to depend on the orientation of the circular shake; taking the difference between the rates obtained from two opposite orientations of the drive, and integrating over a proper drive-frequency range, provides a direct measure of the topological Chern number (ν) of the populated band: This "differential integrated rate" is directly related to the strength of the driving field through the quantized coefficient η0 = ν/ℏ(2), where h = 2π ℏ is Plancks constant ...
The research found that, in left-handed people, the language areas of the left and right sides of the brain are more coordinated in their communication.
SFZ11 Series 220kv 63000kva Three Phase Three Winding Power Transformer, US $ 80,000 - 100,000 / Set, Beijing, China (Mainland), Daelim, SFZ11.Source from Beijing Daelim Green EP Tech Co., Ltd. on Alibaba.com.
we obtain a representation containing only "discrete" spectrum, but parameterized by a "non-discrete" subset of the unitary dual.. It is nevertheless true that the discrete spectrum is discrete in the unitary dual, in the automorphic case. I am sure this is well-known. (Unless the topology on the unitary dual is much weirder than I expect; my expertise in this respect is quite limited, but I remember asking A. Venkatesh who told me that the pathologies of the unitary dual topology are basically irrelevant as far as the automorphic case is concerned). I think this must be well-known, but I dont remember seeing it mentioned anywhere (hence this post…). Here is the argument, at least over number fields, and for ...
An electrosurgical generator is disclosed, which includes an RF output stage connected to a DC power supply and first and second connections. The first connection includes a first switching component and a first parallel inductor-capacitor resonant circuit. The second connection includes a second switching components and a second parallel inductor-capacitor resonant circuit. The first and second switching components are configured to open and close at a predetermined frequency based on a phase-correlated dual drive signal emitted by a driver and are in a 180 degree out-of-phase relationship. The first parallel inductor-capacitor resonant circuit is further configured to produce a first half-sinusoidal waveform and the second parallel inductor-capacitor resonant circuit is configured to produce a second half-sin half-sinusoidal waveform. The RF output stage further includes a transformer having a primary winding and a secondary winding and a series inductor-capacitor resonant circuit, which are
Even if you sew primarily on a machine, being able to sew well by hand will allow you to finish every project you make in the cleanest, most professional looking...
Inside the Poloidal Field Coils Winding Facility, around 60 metres of conductor length have already been submitted to the series of operations that will ultima[...]
BioAssay record AID 502675 submitted by ChEMBL: Induction of DNA cleavage activity in Escherichia coli pBR322 assessed as supercoiled circular form after 10 mins by gel electrophoresis in presence of 0.1 M NaN3 solution.
37 Figure 1 , Relaxation of DNA supercoiling by TOP1-mediated DNA cleavage complexes, and the trapping of TOP1 cleavage complexes by drugs, DNA modifications and during apoptosis. c , An expanded view of DNA relaxation by a TOP1 cleavage complex (TOP1cc). The first step (left) is a transesterification reaction whereby the catalytic tyrosine (Y) becomes linked to the 3′ DNA end (nicking step). In the second step (middle), the torsional strain that results from DNA supercoiling drives the rotation of the 5′ end of the nicked DNA strand around the intact strand. TOP1 encircles the rotating nicked DNA and slows its rotation (FIG. 3a). This process is referred to as controlled rotation. In the last step (right), the 5′ end of the nicked DNA is realigned with the corresponding 3′ end, which enables DNA religation (the closing step of the nicking-closing reaction). TOP1ccs are normally transient because the closing step is much faster than the nicking step. Drugs and DNA lesions inhibit ...
A type II topoisomerase that negatively supercoils closed circular double-stranded (ds) DNA in an ATP-dependent manner to modulate DNA topology and maintain chromosomes in an underwound state. Negative supercoiling favors strand separation, and DNA replication, transcription, recombination and repair, all of which involve strand separation. Also able to catalyze the interconversion of other topological isomers of dsDNA rings, including catenanes and knotted rings. Type II topoisomerases break and join 2 DNA strands simultaneously in an ATP-dependent manner.
The bacterial chromosome is compacted in a manner optimal for DNA transactions to occur. The degree of compaction results from the level of DNA-supercoiling and the presence of nucleoid-binding proteins. DNA-supercoiling is homeostatically maintained by the opposing activities of relaxing DNA topoisomerases and negative supercoil-inducing DNA gyrase. DNA-supercoiling acts as a general cis regulator of transcription, which can be superimposed upon other types of more specific trans regulatory mechanism. Transcriptomic studies on the human pathogen Streptococcus pneumoniae, which has a relatively small genome (~2 Mb) and few nucleoid-binding proteins, have been performed under conditions of local and global changes in supercoiling. The response to local changes induced by fluoroquinolone antibiotics, which target DNA gyrase subunit A and/or topoisomerase IV, involves an increase in oxygen radicals which reduces cell viability, while the induction of global supercoiling changes by novobiocin (a DNA gyrase
DNA gyrase subunit A; A type II topoisomerase that negatively supercoils closed circular double-stranded (ds) DNA in an ATP-dependent manner to modulate DNA topology and maintain chromosomes in an underwound state. Negative supercoiling favors strand separation, and DNA replication, transcription, recombination and repair, all of which involve strand separation. Also able to catalyze the interconversion of other topological isomers of dsDNA rings, including catenanes and knotted rings. Type II topoisomerases break and join 2 DNA strands simultaneously in an ATP-dependent manner (923 aa ...
Noma and colleagues have previously described how two protein complexes called condensin and cohesin mediate the formation of functional genome-organizing structures called topological domains by establishing contacts that bring distantly located DNA regions closer together. Specifically, cohesin mediates local contacts, forming small topological chromatin domains, whereas condensin drives longer-range contacts, organizing larger domains.. In the new study, the lab applied similar genomic methodology to dissect the condensation and de-condensation of chromatin in topological domains over time, following the formation and decay of chromatin contacts throughout the different phases of the cell cycle. They discovered that the larger domains mediated by condensin are formed during mitosis, whereas the smaller, local domains mediated by cohesin remain stable throughout the whole cycle.. Contrary to what was generally assumed in the field, we find that condensation and de-condensation of the ...
DNA topoisomerase I (EC 5.99.1.2) [1,2,3,4] is one of the two types of enzyme that catalyze the interconversion of topological DNA isomers. Type I topoisomerases act by catalyzing the transient breakage of DNA, one strand at a time, and the subsequent rejoining of the strands. When a prokaryotic type I topoisomerase breaks a DNA backbone bond, it simultaneously forms a protein-DNA link where the hydroxyl group of a tyrosine residue is joined to a 5-phosphate on DNA, at one end of the enzyme-severed DNA strand. Prokaryotic organisms, such as Escherichia coli, have two type I topoisomerase isozymes: topoisomerase I (gene topA) and topoisomerase III (gene topB). Eukaroytes also contain homologs of prokaryotic topoisomerase III. There are a number of conserved residues in the region around the active site tyrosine; we used this region as a signature pattern. Last update: December 2004 / Pattern and text revised. ...
Topoisomerases are highly specialized nuclear enzymes that remove superhelical tension on chromosomal DNA that allows replication and transcription of DNA. Many cancer chemotherapeutic drugs used in the clinics inhibit tumor growth by targeting topoisomerase functions resulting in DNA damage and cancer cell death. Cryptolepine, a major alkaloid isolated from Cryptolepis sanguinolenta plants roots, has shown anti-malarial, anti-bacterial, anti-fungal, and anti-inflammatory activities. In the present study, we examined the therapeutic effect of cryptolepine on non-melanoma skin cancer cells (NMSCC), SCC-13 and A431 as an in vitro model, and underlying mechanism of action with special emphasis on topoisomerases and DNA damage check points. Western blot analysis and enzyme activity evaluation assays demonstrated that SCC-13 and A431 cells express comparatively higher levels of topoisomerases and higher enzymatic activities compared with normal human epidermal keratinocytes (NHEK). Topoisomerase ...
Central component of the condensin complex, a complex required for conversion of interphase chromatin into mitotic-like condense chromosomes. The condensin complex probably introduces positive supercoils into relaxed DNA in the presence of type I topoisomerases and converts nicked DNA into positive knotted forms in the presence of type II topoisomerases.