Structural diversity of supercoiled DNA.
Ontology highlight
ABSTRACT:
SUBMITTER: Irobalieva RN
PROVIDER: S-EPMC4608029 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Single molecule experiments have demonstrated a progressive transition from a B- to an L-form helix as DNA is gently stretched and progressively unwound. The particular sequence of a DNA segment defines both base stacking and hydrogen bonding that affect the partitioning and conformations of the two phases. Naturally or artificially modified bases alter H-bonds and base stacking and DNA with diaminopurine (DAP) replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded DAP:T base pairs. Both unmodified and DAP-substituted DNA transitioned from a B- to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing and the ease of this transition may prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length than B-DNA. However, left-handed DAP-substituted DNA was twice as stiff as unmodified L-DNA. Unmodified DNA and DAP-substituted DNA have very distinct mechanical characteristics at physiological levels of negative supercoiling and tension.
| S-EPMC8599871 | biostudies-literature
Project description:Juxtaposition kinetics between specific sites in supercoiled DNA is investigated at close to physiological ionic conditions by Brownian dynamics simulations. At such conditions, supercoiled DNA is interwound, and the probability of spatial site juxtaposition is much higher than in relaxed DNA. We find, however, that supercoiling does not correspondingly increase the rate of juxtaposition at these physiological conditions. An explanation to this unexpected finding emerges on analysis of the juxtaposition dynamics. We note that although a particular site i(1) in supercoiled DNA is often in close proximity (juxtaposed) to another site i(2), the change of i(2) occurs very slowly and depends largely on internal slithering of opposite segments of the DNA superhelix. Such slithering results in long correlations between successive values of i(2); these correlations increase the average time of juxtaposition between two DNA sites. Random collisions between sites located on different superhelix branches-although increasing in importance with DNA size-contribute less substantially to site juxtaposition at high salt than slithering for DNA up to 6 kb in length.
| S-EPMC14693 | biostudies-literature
Project description:Supercoiled DNA is the relevant substrate for a large number of DNA transactions and has additionally been found to be a favorable form for delivering DNA and protein-DNA complexes to cells. We report here a facile method for stoichiometrically incorporating several different modifications at multiple, specific, and widely spaced sites in supercoiled DNA. The method is based upon generating an appropriately gapped circular DNA, starting from single-strand circular DNA from two phagemids with oppositely oriented origins of replication. The gapped circular DNA is annealed with labeled and unlabeled synthetic oligonucleotides to make a multiply nicked circle, which is covalently sealed and supercoiled. The method is efficient, robust and can be readily scaled up to produce large quantities of labeled supercoiled DNA for biochemical and structural studies. We have applied this method to generate dye-labeled supercoiled DNA with heteroduplex bubbles for a Förster resonance energy transfer (FRET) analysis of supercoiled Holliday junction intermediates in the λ integrative recombination reaction. We found that a higher-order structure revealed by FRET in the supercoiled Holliday junction intermediate is preserved in the linear recombination product. We suggest that in addition to studies on recombination complexes, these methods will be generally useful in other reactions and systems involving supercoiled DNA.
| S-EPMC2952878 | biostudies-literature
Project description:We directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmid's structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.
| S-EPMC5961182 | biostudies-literature
Project description:Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry.
| S-EPMC4827127 | biostudies-literature
Project description:We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ?15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresis.
| S-EPMC4344484 | biostudies-literature
Project description:Topoisomerase V is a type I topoisomerase without structural or sequence similarities to other topoisomerases. Although it belongs to the type I subfamily of topoisomerases, it is unrelated to either type IA or IB enzymes. We used real-time single-molecule micromechanical experiments to show that topoisomerase V relaxes DNA via events that release multiple DNA turns, employing a constrained swiveling mechanism similar to that for type IB enzymes. Relaxation is powered by the torque in the supercoiled DNA and is constrained by friction between the protein and the DNA. Although all type IB enzymes share a common structure and mechanism and type IA and type II enzymes show marked structural and functional similarities, topoisomerase V represents a different type of topoisomerase that relaxes DNA in a similar overall manner as type IB molecules but by using a completely different structural and mechanistic framework.
| S-EPMC1976220 | biostudies-literature
Project description:Although bacterial gyrase and topoisomerase IV have critical interactions with positively supercoiled DNA, little is known about the actions of these enzymes on overwound substrates. Therefore, the abilities of Bacillus anthracis and Escherichia coli gyrase and topoisomerase IV to relax and cleave positively supercoiled DNA were analyzed. Gyrase removed positive supercoils ?10-fold more rapidly and more processively than it introduced negative supercoils into relaxed DNA. In time-resolved single-molecule measurements, gyrase relaxed overwound DNA with burst rates of ?100 supercoils per second (average burst size was 6.2 supercoils). Efficient positive supercoil removal required the GyrA-box, which is necessary for DNA wrapping. Topoisomerase IV also was able to distinguish DNA geometry during strand passage and relaxed positively supercoiled substrates ?3-fold faster than negatively supercoiled molecules. Gyrase maintained lower levels of cleavage complexes with positively supercoiled (compared with negatively supercoiled) DNA, whereas topoisomerase IV generated similar levels with both substrates. Results indicate that gyrase is better suited than topoisomerase IV to safely remove positive supercoils that accumulate ahead of replication forks. They also suggest that the wrapping mechanism of gyrase may have evolved to promote rapid removal of positive supercoils, rather than induction of negative supercoils.
| S-EPMC5766186 | biostudies-literature
Project description:B-DNA becomes unstable under superhelical stress and is able to adopt a wide range of alternative conformations including strand-separated DNA and Z-DNA. Localized sequence-dependent structural transitions are important for the regulation of biological processes such as DNA replication and transcription. To directly probe the effect of sequence on structural transitions driven by torque, we have measured the torsional response of a panel of DNA sequences using single molecule assays that employ nanosphere rotational probes to achieve high torque resolution. The responses of Z-forming d(pGpC)(n) sequences match our predictions based on a theoretical treatment of cooperative transitions in helical polymers. "Bubble" templates containing 50-100 bp mismatch regions show cooperative structural transitions similar to B-DNA, although less torque is required to disrupt strand-strand interactions. Our mechanical measurements, including direct characterization of the torsional rigidity of strand-separated DNA, establish a framework for quantitative predictions of the complex torsional response of arbitrary sequences in their biological context.
| S-EPMC3341030 | biostudies-literature
Project description:The dynamics of supercoiled DNA play an important role in various cellular processes such as transcription and replication that involve DNA supercoiling. We present experiments that enhance our understanding of these dynamics by measuring the intrinsic response of single DNA molecules to sudden changes in tension or torsion. The observed dynamics can be accurately described by quasistatic models, independent of the degree of supercoiling initially present in the molecules. In particular, the dynamics are not affected by the continuous removal of the plectonemes. These results set an upper bound on the hydrodynamic drag opposing plectoneme removal, and thus provide a quantitative baseline for the dynamics of bare DNA.
| S-EPMC1924543 | biostudies-literature