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The topology of DNA is a critical quality attribute for plasmid-based pharmaceuticals, making quantification of trace levels of plasmid topoisomers an important analytical priority. An automated and cost-effective method based on capillary gel electrophoresis laser-induced fluorescence detection is described. The method outlined in this report is significant because it is easily implemented by any laboratory for which routine analyses of plasmid topology are critical for the development of new plasmid-based therapies as well as for quality control of gene therapies utilizing supercoiled DNA. Detection of topoisomers was achieved by incorporating ethidium bromide in the separation medium. The detector response was improved by 3 orders of magnitude by utilizing a 605-nm optical filter with a 15-nm bandwidth. Separations of linear, open circle, supercoiled, and multimer DNA plasmids ranging from 4.2 to 10.5 kbp were accomplished in under 6 min using an unmodified fused silica capillary (50-μm internal diameter). The background electrolyte was comprised of 0.5% gel, which was hydroxypropylmethyl cellulose, 1 mM ethylenediaminetetraacetic acid, and 50 mM N-(2-acetamido)-2-aminoethanesulfonic acid (pH of 6.25). The separations, which balanced the bulk electroosmotic flow, the electrophoretic mobility of the DNA, and gel sieving were dependent upon the pH of the electrolyte and the gel concentration. Reproducibility was dependent upon the procedure used to prepare the gel as well as other factors including the ethidium bromide concentration and capillary conditioning. A single unmodified capillary operated for more than 150 runs had an across-day migration time precision of 1% relative standard deviation and percent area precision of 10% relative standard deviation.

New forms of molecular nanocarbons particularly looped polyarenes adopting various topologies contribute to the fundamental science and practical applications. Here we report on the synthesis of an infinity-shaped polycyclic aromatic hydrocarbon, infinitene 1 (cyclo[c.c.c.c.c.c.e.e.e.e.e.e]dodecakisbenzene) comprising consecutively fused 12-benzene rings forming an enclosed loop with a strain energy of 60.2 kcal·mol-1. Infinitene 1 represents a topoisomer of still-hypothetical [12]circulene, and its scaffold can be formally visualized as the outcome of the “stitching” of two homochiral [6]helicene subunits by their both ends. The synthetic strategy encompasses transformation of a rationally designed dithiacyclophane to cyclophadiene through the Stevens rearrangement and pyrolysis of the corresponding S,S′-bis(oxide) followed by the UV-light mediated twofold photocyclization. The structure of infinitene 1 is a unique hybrid of helicene and circulene with a molecular formula C48H24, which can be regarded as an isomer for kekulene, [6,6]carbon nanobelt ([6,6]CNB), [12]cyclacene, and tetrabenzo[8]circulene as well. Infinitene 1 is a bench-stable yellow solid with green fluorescence, and soluble to common organic solvents. The figure-eight molecular structure of 1 was unambiguously confirmed by X-ray crystallography. The scaffold of 1, reminiscent of a squeezed spring, stem from its enclosed, fully-fused architecture, is significantly compressed as manifested by a remarkably shortened distance (3.152–3.192 Å) between the centroids of two π-π stacked central benzene rings and the closest C···C distance of 2.920 Å. Combined lamellar and herringbone-like crystal packing suggested three-dimensional electronic inter-actions. Fundamental photophysical properties of infinitene 1 were thoroughly elucidated by means of UV-vis absorption and fluorescence spectroscopic studies as well as density functional theory (DFT) calculations. Its configurational stability enabled separation of the corresponding enantiomers (P,P) and (M,M) by a chiral HPLC. Circular dichroism (CD) and circularly polarized luminescence (CPL) measurements revealed that 1 has moderate |gCD| and |gCPL| values.

A twisted small-molecule synthesis Some molecules are easy to draw on paper, whereas others contain rings and contortions that require one to think in three dimensions. Reisberg et al. set out to synthesize the bicyclic small molecule tryptorubin A but found that their initial attempt produced a molecule with the right bonds but the wrong molecular shape, a form of noncanonical atropisomerism. The authors then devised a synthesis where they locked in the correct isomer before forming the second ring, which produced a product indistinguishable from the authentic natural product. Such structural isomers may be lurking when working with complex small molecules with constrained rotation. Science , this issue p. 458

Lasso peptides belong to the natural product superfamily of ribosomally synthesized and post-translationally modified peptides (RiPPs). They are defined by an N-terminal macrolactam ring that is threaded by the C-terminal tail. In class II lasso peptides, this fold is maintained only through steric hindrance. Nonetheless, this fold can often withstand prolonged incubation at highly elevated temperatures. However, some lasso peptides will irreversibly unthread into their branched-cyclic counterparts upon heating. In recent years, an increasing number of research studies have focused on studying the factors that govern the thermal stability (or the lack thereof) of lasso peptides by using in vitro stability assays, mutational analysis, and molecular dynamics simulations. In this review, the current state of understanding the physicochemical parameters deciding the fate of a lasso peptide at elevated temperatures is discussed, and an overview is given of the techniques developed to streamline the separation and discrimination of lasso peptides from their branched-cyclic topoisomers.

How type 2 Topoisomerase (TopoII) proteins relax and simplify the topology of DNA molecules is one of the most intriguing open questions in genome and DNA biophysics. Most of the existing models neglect the dynamics of TopoII which is characteristics for proteins searching their targets via facilitated diffusion. Here, we show that dynamic binding of TopoII speeds up the topological relaxation of knotted substrates by enhancing the search of the knotted arc. Intriguingly, this in turn implies that the timescale of topological relaxation is virtually independent of the substrate length. We then discover that considering binding biases due to facilitated diffusion on looped substrates steers the sampling of the topological space closer to the boundaries between different topoisomers yielding an optimally fast topological relaxation. We discuss our findings in the context of topological simplification in vitro and in vivo.

We discuss recent observations of polymorphic chromatin packaging at the oligonucleosomal level and compare them with computer simulations. Our computations reveal two topologically different families of two-start 30-nm fiber conformations distinguished by the linker length L; fibers with L ≈ 10n and L ≈ 10n+5 basepairs have DNA linking numbers per nucleosome of ΔLk ≈ −1.5 and −1.0, respectively (where n is a natural number). Although fibers with ΔLk ≈ −1.5 were observed earlier, the topoisomer with ΔLk ≈ −1.0 is novel. These predictions were confirmed experimentally for circular nucleosome arrays with precisely positioned nucleosomes. We suggest that topological polymorphism of chromatin may play a role in transcription, with the {10n+5} fibers producing transcriptionally competent chromatin structures. This hypothesis is consistent with available data for yeast and, partially, for fly. We show that both fiber topoisomers (with ΔLk ≈ −1.5 and −1.0) have to be taken into account to interpret experimental data obtained using new techniques: genome-wide Micro-C, Hi-CO, and RICC-seq, as well as self-association of nucleosome arrays in vitro. The relative stability of these topoisomers is likely to depend on epigenetic histone modifications modulating the strength of internucleosome interactions. Potentially, our findings may reflect a general tendency of functionally distinct parts of the genome to retain topologically different higher-order structures.

The topoisomerase II inhibitor idarubicin (Ida) is an effective anticancer anthracycline drug and has been used for clinical therapies of multiple cancers. It is well-known that Ida and its analogues can induce DNA double strand breakage (DSB) by inhibiting topoisomer II and kill tumor cells. To date, it remains unknown whether they alter DNA epigenomes. Here, we show that Ida significantly stimulates the oxidation of a key epigenetic mark DNA 5-methyl-2'-deoxycytidine (5mC), which results in elevation of 5-hydroxymethyl-2'-deoxycytidine (5hmC) in four tested cell lines. Similarly, Ida analogues also display elevated 5hmC. DSB-causing topoisomer II inhibitor etopside fails to induce 5hmC change even at very high dose, which suggests the independence of the DSB. Moreover, the structure comparison supports that the histone eviction-associated amino sugar moiety is a characteristic of the anthracyclines required to promote the 5hmC elevation. Noteworthy, we also found that the 5mC oxidation is also cell-cycle dependent and mainly occurs during the S and G2/M phases. TET2 depletion diminishes the observed 5hmC elevation, which suggests that the Ida stimulation of 5hmC formation is mainly TET2-dependent. Deep-sequencing shows that 5hmC increases in all regions of the tested genome of T47D cells. The observation of a novel effect of Ida as well as other anthracycline compounds on epigenetic DNA modifications may help to further elucidate their biological and clinical effects.

Lasso peptides are a class of bioactive ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked topology, where the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. BI-32169 is a class III lasso peptide containing one disulfide bond that further stabilizes the lasso structure. In contrast to its branched-cyclic analog, BI-32169 has higher stability and is known to exert a potent inhibitory activity against the human glucagon receptor. In the present work, tandem mass spectrometry, using collision-induced dissociation (CID) and electron capture dissociation (ECD), and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) experiments were carried out to evidence specific structural signatures of the two topologies. CID experiments showed similar fragmentation patterns for the two topoisomers, where a part of the C-terminal tail remains covalently linked to the macrolactam ring by the disulfide bond, which cannot clearly constitute a signature of the lasso topology. ECD experiments of BI-32169 showed an increase of hydrogen migration events in the loop region when compared with those of its branched-cyclic topoisomer evidencing specific structural signatures for the lasso topology. The high mobility resolving power of TIMS resulted in the identification of multiple conformations for the protonated species but did not allow the clear differentiation of the two topologies in mixture. When in complex with cesium metal ions, a reduced number of conformations led to a clear identification of the two structures. Experiments reducing and alkylating the disulfide bond of BI-32169 showed that the lasso structure is preserved and heat stable and the associated conformational changes provide new insights about the role of the disulfide bond in the inhibitory activity against the human glucagon receptor. Graphical abstract ᅟ.

A wider application of minicircle (MC) vectors in gene therapy research depends critically on the ability to purify supercoiled (sc) MC from related miniplasmid (MP) and parental plasmid (PP) impurities. This protocol describes a purification strategy that combines the in vitro enzymatic relaxation of sc MP and PP impurities by a nicking endonuclease, and topoisomer separation and RNA clearance by hydrophobic interaction chromatography. The time required to follow the full protocol, from production to isolation of sc MC, is approximately 50 h. The process delivers sc MCs that are virtually free from MP, PP, RNA, and protein impurities.

Over the past 35 years, DNA has been used to produce various nanometer-scale constructs, nanomechanical devices, and walkers. Construction of complex DNA nanostructures relies on the creation of rigid DNA motifs. Paranemic crossover (PX) DNA is one such motif that has played many roles in DNA nanotechnology. Specifically, PX cohesion has been used to connect topologically closed molecules, to assemble a three-dimensional object, and to create two-dimensional DNA crystals. Additionally, a sequence-dependent nanodevice based on conformational change between PX and its topoisomer, JX2, has been used in robust nanoscale assembly lines, as a key component in a DNA transducer, and to dictate polymer assembly. Furthermore, the PX motif has recently found a new role directly in basic biology, by possibly serving as the molecular structure for double-stranded DNA homology recognition, a prominent feature of molecular biology and essential for many crucial biological processes. This review discusses the many attrib...

Lasso peptides are a fascinating class of bioactive ribosomal natural products characterized by a mechanically interlocked topology. In contrast to their branched-cyclic forms, lasso peptides have higher stability and have become a scaffold for drug development. However, the identification and separation of lasso peptides from their unthreaded topoisomers (branched-cyclic peptides) is analytically challenging since the higher stability is based solely on differences in their tertiary structures. In the present work, a fast and effective workflow is proposed for the separation and identification of lasso from branched cyclic peptides based on differences in their mobility space under native nanoelectrospray ionization-trapped ion mobility spectrometry-mass spectrometry (nESI-TIMS-MS). The high mobility resolving power ( R) of TIMS resulted in the separation of lasso and branched-cyclic topoisomers ( R up to 250, 150 needed on average). The advantages of alkali metalation reagents (e.g., Na, K, and Cs salts) as a way to increase the analytical power of TIMS is demonstrated for topoisomers with similar mobilities as protonated species, efficiently turning the metal ion adduction into additional separation dimensions.

The sequence dependence of the conformational distribution of DNA under various levels of torsional stress is an important unsolved problem. Combining theory and coarse-grained simulations shows that the DNA sequence and a structural correlation due to topology constraints of a circle are the main factors that dictate the 3D structure of a 336 bp DNA minicircle under torsional stress. We found that DNA minicircle topoisomers can have multiple bend locations under high torsional stress and that the positions of these sharp bends are determined by the sequence, and by a positive mechanical correlation along the sequence. We showed that simulations and theory are able to provide sequence-specific information about individual DNA minicircles observed by cryo-electron tomography (cryo-ET). We provided a sequence-specific cryo-ET tomogram fitting of DNA minicircles, registering the sequence within the geometric features. Our results indicate that the conformational distribution of minicircles under torsional stress can be designed, which has important implications for using minicircle DNA for gene therapy.

Human O6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts in single-stranded and duplex DNAs. The search for these lesions, through a vast excess of competing, unmodified genomic DNA, is a mechanistic challenge that may limit the repair rate in vivo. Here, we examine influences of DNA secondary structure and twist on protein–protein interactions in cooperative AGT complexes formed on lesion-free DNAs that model the unmodified parts of the genome. We used a new approach to resolve nearest neighbor (nn) and long-range (lr) components from the ensemble-average cooperativity, ωave. We found that while nearest-neighbor contacts were significant, long-range interactions dominated cooperativity and this pattern held true whether the DNA was single-stranded or duplex. Experiments with single plasmid topoisomers showed that the average cooperativity was sensitive to DNA twist, and was strongest when the DNA was slightly underwound. This suggests that AGT proteins are optimally juxtaposed when the DNA is near its torsionally-relaxed state. Most striking was the decline of binding stoichiometry with linking number. As stoichiometry and affinity differences were not correlated, we interpret this as evidence that supercoiling occludes AGT binding sites. These features suggest that AGT's lesion-search distributes preferentially to sites containing torsionally-relaxed DNA, in vivo.

Agarose gel electrophoresis is one of the most straightforward techniques that can be used to differentiate between topoisomers of closed circular DNA molecules. Generally, the products of reactions that monitor the interconversion of DNA between negatively supercoiled and relaxed DNA or positively supercoiled and relaxed DNA can be resolved by one-dimensional gel electrophoresis. However, in more complex reactions that contain both positively and negatively supercoiled DNA, one-dimensional resolution is insufficient. In these cases, a second dimension of gel electrophoresis is necessary. This chapter describes the technique of two-dimensional agarose gel electrophoresis and how it can be used to resolve a spectrum of DNA topoisomers.

The study of mitochondrial DNA (mtDNA) integrity and how replication, transcription, repair, and degradation maintain mitochondrial function has been hampered due to the inability to identify mtDNA structural forms. Here we describe the use of 2D intact mtDNA agarose gel electrophoresis, or 2D-IMAGE, to identify up to 25 major mtDNA topoisomers such as double-stranded circular mtDNA (including supercoiled molecules, nicked circles, and multiple catenated species) and various forms containing single-stranded DNA (ssDNA) structures. Using this modification of a classical 1D gel electrophoresis procedure, many of the identified mtDNA species have been associated with mitochondrial replication, damage, deletions, and possibly transcription. The increased resolution of 2D-IMAGE allows for the identification and monitoring of novel mtDNA intermediates to reveal alterations in genome replication, transcription, repair, or degradation associated with perturbations during mitochondrial stress.

There is an increased interest in plasmid DNA as therapeutics. This is evident in the number of ongoing clinical trials involving the use of plasmid DNA. In order to be an effective therapeutic, high yield and high level of supercoiling are required. From the bioprocessing point of view, the supercoiling level potentially has an impact on the ease of downstream processing. We approached meeting these requirements through plasmid engineering. A 7.2 kb plasmid was developed by the insertion of a bacteriophage Mu strong gyrase-binding sequence (Mu-SGS) to a 6.8 kb pSV&beta, Gal and it was used to transform four different E. coli strains, and cultured in order to investigate the Mu-SGS effect and dependence on strain. There was an increase of over 20% in the total plasmid yield with pSV&beta, Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The extent of supercoiling was examined using superhelical density (&sigma, ) quantification with pSV&beta, Gal398 maintaining a superhelical density of &minus, 0.022, and pSV&beta, Gal &minus, 0.019, in both strains. This study has shown that plasmid modification with the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing.

Genomic DNA is vastly longer than the space allotted to it in a cell. The molecule must fold with a level of organization that satisfies the imposed spatial constraints as well as allow for the processing of genetic information. Key players in this organization include the negative supercoiling of DNA, which facilitates the unwinding of the double-helical molecule, and the associations of DNA with proteins, which partition the DNA into isolated loops, or domains. In order to gain insight into the principles of genome organization and to visualize the folding of spatially constrained DNA, we have developed new computational methods to identify the preferred three-dimensional pathways of protein-mediated DNA loops and to characterize the topological properties of these structures. Here, we focus on the levels of supercoiling and the spatial arrangements of DNA in model nucleoprotein systems with two topological domains. We construct these systems by anchoring DNA loops in opposing orientations on a common protein–DNA assembly, namely the Lac repressor protein with two bound DNA operators. The linked pieces of DNA form a covalently closed circle such that the protein attaches to two widely spaced sites along the DNA. We examine the effects of operator spacing, loop orientation, and long-range contacts on overall chain configuration and topology, and discuss our findings in the context of classic experiments on the effects of supercoiling and operator spacing on Lac repressor-mediated looping and recent work on the role of proteins as barriers that divide genomes into independent topological domains.

Review and analyses of the experimental data indicate that in nearly all cases bending elastic constants of the effective springs between bp of DNA actually undergo a net increase with increasing T from 278 to 315 K. The exceptions to this rule are bending elastic constants obtained from equilibrium topoisomer distributions of a 2686 bp pUC19 DNA by assuming a fixed T-independent value of the torsion elastic constant. When the same data are analyzed using measured T-dependent values of the torsion elastic constant, which decline with increasing T, a modest increase in bending elastic constant with increasing T is obtained. After revising the torsion elastic constants of the previously formulated two-state cooperative transition model to account for additional data, that model is fitted to the bending elastic constants reckoned from the aforementioned topoisomer distributions to determine the best-fit values for each state. The rather good fit implies a strong negative linear correlation between the inverse bending and inverse torsion elastic constants as T is varied. Predictions of the resulting two-state model, wherein each state has fixed bending and torsion elastic constants, agree surprisingly well with single-molecule relative extension and torque data. The same model also yields good agreement with numerous other experimental data. With increasing T the equilibrium is shifted from the (longer, torsionally stiffer, flexurally softer) b-state toward the (shorter, torsionally softer, flexurally stiffer) a-state. This transition is suggested to be the origin of the so-called broad pre-melting transition exhibited by many, but not all, DNAs.

We discuss various models of spatial organization of the 30-nm fiber that remains enigmatic despite 40 years of intensive studies. Using computer simulations, we found two topologically different families of the fiber conformations distinguished by the linker length, L: the fibers with L = {10n} and {10n+5} bp have DNA linking numbers per nucleosome DLk » –1.5 and –1.0, respectively. The fibers with DLk » –1.5 were observed earlier, while the topoisomer with DLk » –1.0 is novel. These predictions were confirmed for circular nucleosome arrays with precisely positioned nucleosomes. We suggest that topological polymorphism of chromatin fibers may play a role in the process of transcription, which is known to generate different levels of DNA supercoiling upstream and downstream from RNA polymerase. In particular, the {10n+5} DNA linkers are likely to produce transcriptionally competent chromatin structures. This hypothesis is consistent with available data for several eukaryotes, from yeast to human. We also analyzed two recent studies of chromatin fibers – on the nucleosome crosslinking in vitro, and on radioprobing DNA folding in human cells. In both cases, we show that the novel topoisomer with DLk » –1.0 has to be taken into account to interpret experimental data. This is yet another evidence for occurrence of two distinct fiber topoisomers. Potentially, our findings may reflect a general tendency of chromosomal domains with different levels of transcription to retain topologically different higher-order structures.

DNA catenanes are characterized by their flexible and dynamic motions and have been regarded as one of the key players in sophisticated DNA-based molecular machines. There, the linking number (Lk) between adjacent interlocked rings is one of the most critical factors, since it governs the feasibility of dynamic motions. However, there has been no established way to synthesize catenanes in which Lk is controlled to a predetermined value. This paper reports a new methodology to selectively synthesize Lk 1 catenanes composed of single-stranded DNA rings, in which these rings can most freely rotate each other due to minimal inter-ring interactions. To the mixture for the synthesis, two holder strands (oligonucleotides of 18–46 nt) were added, and the structure of the quasi-catenane intermediate was interlocked through Watson–Crick base pairings into a favorable conformation for Lk 1 catenation. The length of the complementary part between the two quasi-rings was kept at 10 bp or shorter. Under these steric constraints, two quasi-rings were cyclized with the use of T4 DNA ligase. By this simple procedure, the formation of undesired topoisomers (Lk ≥ 2) was almost completely inhibited, and Lk 1 catenane was selectively prepared in high yield up to 70 mole%. These Lk 1 catenanes have high potentials as dynamic parts for versatile DNA architectures.

It is well accepted that the introduction of negative supercoils locally unwinds the DNA double helix, influencing thus the activity of proteins. Despite the use of recent methods of molecular dynamics simulations to model the DNA supercoiling-induced DNA deformation, the precise extent and location of unpaired bases induced by the negative supercoiling have never been investigated at the nucleotide level. Our goals in this study were to use radiolabeled double-stranded DNA mini-circles (dsMCs) to locate the unpaired bases on dsMCs whose topology ranged from relaxed to hyper-negatively supercoiled states, and to characterize the binding of proteins involved in the DNA metabolism. Our results show that the Nuclease SI is nearly ten times more active on hyper-negatively supercoiled than relaxed DNA. The structural changes responsible for this stimulation of activity were mapped for the first time with a base pair resolution and shown to be subtle and distributed along the entire sequence. As divalent cations modify the DNA topology, our binding studies were conducted with or without magnesium. Without magnesium, the dsMCs topoisomers mostly differ by their twist. Under these conditions, the Escherichia coli topoisomerase I weakly binds relaxed dsMCs and exhibits a stronger binding on negatively and hyper-negatively supercoiled dsMCs than relaxed dsMCs, with no significant difference in the binding activity among the supercoiled topoisomers. For the human replication protein A (hRPA), the more negatively supercoiled is the DNA, the better the binding, illustrating the twist-dependent binding activity for this protein. The presence of magnesium permits the dsMCs to writhe upon introduction of negative supercoiling and greatly modifies the binding properties of the hRPA and Escherichia coli SSB on dsMCs, indicating a magnesium-dependent DNA binding behavior. Finally, our experiments that probe the topology of the DNA in the hRPA-dsMC complexes show that naked and hRPA-bound dsMCs have the same topology.

Catenation is the process by which cyclic strands are combined like the links of a chain, whereas knotting changes the linking properties of a single strand. In the cell, topoisomerases catalyzing strand passage operations enable the knotting and catenation of DNA so that single- or double-stranded segments can be passed through each other. Here, we use a system of closed DNA structures involving a paranemic motif, called PX-DNA, to bind double strands of DNA together. These PX-cohesive closed molecules contain complementary loops whose linking by Escherichia coli topoisomerase 1 (Topo 1) leads to various types of catenated and knotted structures. We were able to obtain specific DNA topological constructs by varying the lengths of the complementary tracts between the complementary loops. The formation of the structures was analyzed by denaturing gel electrophoresis, and the various topologies of the constructs were characterized using the program Knotilus.

The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample.

Objective Agarose gel electrophoresis has been the mainstay technique for the analysis of DNA samples of moderate size. In addition to separating linear DNA molecules, it can also resolve different topological forms of plasmid DNAs, an application useful for the analysis of the reactions of DNA topoisomerases. However, gel electrophoresis is an intrinsically low-throughput technique and suffers from other potential disadvantages. We describe the application of the QIAxcel Advanced System, a high-throughput capillary electrophoresis system, to separate DNA topoisomers, and compare this technique with gel electrophoresis. Results We prepared a range of topoisomers of plasmids pBR322 and pUC19, and a 339 bp DNA minicircle, and compared their separation by gel electrophoresis and the QIAxcel System. We found superior resolution with the QIAxcel System, and that quantitative analysis of topoisomer distributions was straightforward. We show that the QIAxcel system has advantages in terms of speed, resolution and cost, and can be applied to DNA circles of various sizes. It can readily be adapted for use in compound screening against topoisomerase targets.

Metal ions can play a significant role in a variety of important functions in protein systems including cofactor for catalysis, protein folding, assembly, structural stability and conformational change. In the present work, we examined the influence of alkali (Na, K and Cs), alkaline earth (Mg and Ca) and transition (Co, Ni and Zn) metal ions on the conformational space and analytical separation of mechanically interlocked lasso peptides. Syanodin I, sphingonodin I, caulonodin III and microcin J25, selected as models of lasso peptides, and their respective branched-cyclic topoisomers were submitted to native nESI trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). The high mobility resolving power of TIMS permitted to group conformational families regardless of the metal ion. The lower diversity of conformational families for syanodin I as compared to the other lasso peptides supports that syanodin I probably forms tighter binding interactions with metal ions limiting their conformational space in the gas-phase. Conversely, the higher diversity of conformational families for the branched-cyclic topologies further supports that the metal ions probably interact with a higher number of electronegative groups arising from the fully unconstraint C-terminal part. A correlation between the lengths of the loop and the C-terminal tail with the conformational space of lasso peptides becomes apparent upon addition of metal ions. It was shown that the threaded C-terminal region in lasso peptides allows only for distinct interactions of the metal ion with either residues in the loop or tail region. This limits the size of the interacting region and apparently leads to a bias of metal ion binding in either the loop or tail region, depending whichever section is larger in the respective lasso peptide. For branched-cyclic peptides, the non-restricted C-terminal tail allows metal coordination by residues throughout this region, which can result in gas-phase structures that are sometimes even more compact than the lasso peptides. The high TIMS resolution also resulted in the separation of almost all lasso and branched-cyclic topoisomer metal ions (r ∼ 2.1 on average). It is also shown that the metal incorporation (e.g., doubly cesiated species) can lead to the formation of a simplified IMS pattern (or preferential conformers), which results in baseline analytical separation and discrimination between lasso and branched-cyclic topologies using TIMS-MS.

International audience; Lasso peptides constitute a structurally unique class of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked structure in which the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Tandem mass spectrometry using collision induced dissociation (CID) and electron capture dissociation (ECD) have shown previously different fragmentation patterns for capistruin, microcin J25 and their corresponding branched-cyclic forms in which the C-terminal tail is unthreaded. In order to develop general rules that unambiguously discriminate the lasso and branched-cyclic topologies, this report presents experimental evidence for a set of twenty-one lasso peptides analyzed by CID and electron transfer dissociation (ETD). CID experiments on lasso peptides specifically yielded mechanically interlocked species with associated bi and yj fragments. For class II lasso peptides, these lasso-specific fragments were observed only for peptides in which the loop, located above the macrolactam ring, was strictly longer than four amino acid residues. For class I and III lasso peptides, part of the C-terminal tail remains covalently linked to the macrolactam ring by disulfide bonds; associated bi and yj fragments therefore do not clearly constitute a signature of the lasso topology. ETD experiments of lasso peptides showed a significant increase of hydrogen migration events in the loop region when compared to their branched-cyclic topoisomers, leading to the formation of specific ci˙/z′j fragments for all lasso peptides, regardless of their class and loop size. Our experiments enabled us to establish general rules for obtaining structural details from CID and ETD fragmentation patterns, obviating the need for structure determination by NMR or X-ray crystallography.

10 p.-7 fig.6 tab. Cebrián, Jorge et al. Este artículo forma parte de tesis doctoral https://digital.csic.es/handle/10261/120925, We systematically varied conditions of twodimensional(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., Spanish Ministerio de Economía y Competitividad [BFU2011-22489 to J.B.S.]; Leverhulme Trust [RP2013-K-017 to A.S.]. Funding for open access charge: Spanish Downloaded from http://nar.oxfordjournals.org/ at Centro de Información y Documentación Científica on February 3, 2015 Nucleic Acids Research, 2014 9 Ministerio de Economía y Competitividad [BFU2011-22489 to J.B.S.]; Leverhulme Trust [RP2013-K-017 to A.S.].

11 páginas, 8 figuras, -- PAGS nros.656-666, To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter-TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive DeltaLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide., PM 9701-38 PGC PB98048 from the Spanish CICYT, 99/0850 from the Spanish FIS, 08.6/0016/2007 from CAM, and from the Swiss National Science Foundation to JMS (31-63418), and A.S. (31-61636)and (31-58841).

Reverse gyrase is a hyperthermophilic enzyme that can introduce positive supercoiling in substrate DNA. It is showed in our studies that positive DNA supercoils were induced in both pBR322 vector and an artificially synthesized mini-plasmid DNA by reverse gyrase. The left-handed structures adopted by positively supercoiled DNA molecules could be identified from their right-handed topoisomers through atomic force microscopic examination. Additional structural comparisons revealed that positively supercoiled DNA molecule AFM images exhibited increased contour lengths. Moreover, enzymatic assays showed that the positively supercoiled DNA could not be cleaved by T7 endonuclease. Together, this suggests that the overwound structure of positive supercoils could prevent genomic duplex DNA from randomly forming single-stranded DNA regions and intra-stranded secondary structures.

DNA is generally assumed as a right-handed double helix and Z-DNA is a special kind of left-handed DNA infrequently found in nature. However, the finding of a zero linking number topoisomer supports a hypothesis that the two strands of DNA are winding ambidextrously, rather than plectonemically. It logically leads to a notion that the left-handed DNA is as common as right-handed DNA and the amount of left-handed DNA in a positively supercoiled plasmid prevails that of the right-handed DNA. In this report, the helical repeat of left-handed DNA, 12 bp per turn, was determined by a new method. How the positively supercoiled DNA was generated in hyperthermophiles and why their DNA can withstand the extreme high temperature are answered from an alternative theory.

The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.

Guanine-rich DNAs can fold into four-stranded structures that contain stacks of G-quartets. Bioinformatics studies have revealed that G-rich sequences with the potential to adopt these structures are unevenly distributed throughout genomes, and are especially found in gene promoter regions. With the exception of the single-stranded telomeric DNA, all genomic G-rich sequences will always be present along with their C-rich complements, and quadruplex formation will be in competition with the corresponding Watson-Crick duplex. Quadruplex formation must therefore first require local dissociation (melting) of the duplex strands. Since negative supercoiling is known to facilitate the formation of alternative DNA structures, we have investigated G-quadruplex formation within negatively supercoiled DNA plasmids. Plasmids containing multiple copies of (G3T)n and (G3T4)n repeats, were probed with dimethylsulphate, potassium permanganate, and S1 nuclease. While dimethylsulphate footprinting revealed some evidence for G-quadruplex formation in (G3T)n sequences, this was not affected by supercoiling, and permanganate failed to detect exposed thymines in the loop regions. (G3T4)n sequences were not protected from DMS and showed no reaction with permanganate. Similarly, both S1 nuclease and two-dimensional gel electrophoresis of DNA topoisomers did not detect any supercoil-dependent structural transitions. These results suggest that negative supercoiling alone is not sufficient to drive G-quadruplex formation.

Plasmid DNA may exist in three isoforms, the linear, open-circular (oc, "nicked"), and covalently closed circular (ccc, "supercoiled") form. We have recently reported on the chromatographic separation of supercoiled plasmid topoisomers on cinchona-alkaloid modified silica-based stationary phases. Herein, we present a selectivity switching mechanism to achieve separation of isoforms and/or supercoiled topoisomers using the very same chromatographic column and system. While salt gradient elution facilitates topoisomer separation, the supercoiled species are eluting as a single peak upon elution by a mixed pH and organic modifier gradient, still well separated from the other isoforms. We have found that a mobile phase pH value near the pI of the zwitterionic adsorbent surface leads to full recovery of all plasmid DNA isoforms, which is a major issue when using anion exchange-based resins. Furthermore, the observed elution pattern, oc < linear < ccc, is constant upon changes of mobile phase composition, gradient slope, and plasmid size. The remarkable isoform selectivity found on quinine-based selectors is explained by van't Hoff plots, revealing a different binding mechanism between the supercoiled plasmid on one hand and the oc and linear isoforms on the other hand.

To elucidate one or more mechanisms through which microrchidia (MORC) proteins impact immunity, epigenetic gene silencing, and DNA modifications, the enzymatic activities of plant MORCs were characterized. Previously, we showed that plant MORC1s have ATPase and DNA endonuclease activities. Here, we demonstrate that plant MORCs have topoisomerase type II (topo II)-like activities, as they i) covalently bind DNA, ii) exhibit DNA-stimulated ATPase activity, iii) relax or nick supercoiled DNA, iv) catenate DNA, and v) decatenante kinetoplast DNA. Mutational analysis of tomato SlMORC1 suggests that a K loop-like sequence is required to couple DNA binding to ATPase stimulation as well as for efficient SlMORC1’s DNA relaxation and catenation activities and in planta suppression of INF1-induced cell death, which is related to immunity. Human MORCs were found to exhibit the same topo II-like DNA modification activities as their plant counterparts. In contrast to typical topo IIs, SlMORC1 appears to require one or more accessory factors to complete some of its enzymatic activities, since addition of tomato extracts were needed for ATP-dependent, efficient conversion of supercoiled DNA to nicked/relaxed DNA and catenanes and for formation of topoisomer intermediates. Both plant and human MORCs bind salicylic acid; this suppresses their decatenation but not relaxation activity.

International audience; Lasso peptides are characterized by a mechanically interlocked structure, where the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Their compact and stable structures have a significant impact on their biological and physical properties and make them highly interesting for drug development. Ion mobility - mass spectrometry (IM-MS) has shown to be effective to discriminate the lasso topology from their corresponding branched-cyclic topoisomers in which the C-terminal tail is unthreaded. In fact, previous comparison of the IM-MS data of the two topologies has yielded three trends that allow differentiation of the lasso fold from the branched-cyclic structure: (1) the low abundance of highly charged ions, (2) the low change in collision cross sections (CCS) with increasing charge state and (3) a narrow ion mobility peak width. In this study, a three-dimensional plot was generated using three indicators based on these three trends: (1) mean charge divided by mass (ζ), (2) relative range of CCS covered by all protonated molecules (ΔΩ/Ω) and (3) mean ion mobility peak width (δΩ). The data were first collected on a set of twenty one lasso peptides and eight branched-cyclic peptides. The indicators were obtained also for eight variants of the well-known lasso peptide MccJ25 obtained by site-directed mutagenesis and further extended to five linear peptides, two macrocyclic peptides and one disulfide constrained peptide. In all cases, a clear clustering was observed between constrained and unconstrained structures, thus providing a new strategy to discriminate mechanically interlocked topologies.

International audience; Characterizing the conformation of biomolecules by mass spectrometry still represents a challenge. With their knotted structure involving a N-terminal macrolactam ring where the C-terminal tail of the peptide is threaded and sterically trapped, lasso peptides constitute an attractive model for developing methods for characterizing gas-phase conformation, through comparison with their unknotted topoisomers. Here, the kinetics of electron capture dissociation (ECD) of a lasso peptide, capistruin, was investigated by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry and compared to that of its branched-cyclic topoisomer, lactam-capistruin. Both peptides produced rather similar ECD spectra but showed different extent of H(*) transfer from c(i)' to z(j)(*) ions. Time-resolved double-resonance experiments under ECD conditions were performed to measure the formation rate constants of typical product ions. Such experiments showed that certain product ions, in particular those related to H(*) transfer, proceeded through long-lived complexes for capistruin, while fast dissociation processes predominated for lactam-capistruin. The formation rate constants of specific ECD product ions enabled a clear differentiation of the lasso and branched-cyclic topoisomers. These results indicate that the formation kinetics of ECD product ions constitute a new way to explore the conformation of biomolecules and distinguish between topoisomers and, more generally, conformers.

Microcin J25 is the archetype of a growing class of bacterial ribosomal peptides possessing a knotted topology (lasso peptides). It consists of an eight-residue macrolactam ring through which the C-terminal tail is threaded. It is biosynthesized as a precursor that is processed by two maturation enzymes (McjB/McjC). Insights into the mechanism of microcin J25 biosynthesis have been provided previously by mutagenesis of the precursor peptide in vivo. In this study we have demonstrated distinct functions of McjB and McjC in vitro for the first time, based on the detection of reaction intermediates. McjB was characterized as a new ATP-dependent cysteine protease, whereas McjC was confirmed to be a lactam synthetase. The two enzymes were functionally interdependent, likely forming a structural complex. Their substrate preference was directly investigated with the aid of mutated precursor peptides. Depending on the substitutions, microcin J25 variants with either a lasso or branched-cyclic topology could be generated in vitro.

We recently discovered the molecular recognition capability of a quinine carbamate ligand attached to silica as a powerful chemoaffinity material for the chromatographic separation of circular plasmid topoisomers of different linking numbers. In this paper we develop structure-selectivity relationship studies to figure out the essential structural features for topoisomer recognition. By varying different moieties of the original cinchonan-derived selector, it was shown that intercalation by the quinoline moiety of the ligand as assumed initially as the working hypothesis is not an essential feature for topoisomer recognition during chromatography. We found that the key elements for topoisomer selectivity are the presence of a rigid weak anion-exchange site and a H-donor site separated from each other in a defined distance by a 4-atom spacer. Additionally, incorporation of the weak anion-exchange site into a cyclic ring structure provides greater rigidity of the ligand molecule and turned out to be advantageous, if not mandatory, for (close to) baseline separation.

Plasmid DNA (pDNA) holds great potential for use in genetherapy and DNA vaccines. Therefore much attention hasbeen paid to the biotechnological production of novel drugsof this type as they enter clinical evaluation. Herein wepresent a unique liquid-chromatography-based method forthe efficient separation of topoisomers of the covalentlyclosedcircular(ccc)plasmidDNA,whichisconsideredbytheU.S. Food and Drug Administration to be therapeutically themost active isoform.

Crossover motifs are integral components for designing DNA-based nanostructures and nanomechanical devices due to their enhanced rigidity compared to the normal B-DNA. Although the structural rigidity of the double helix B-DNA has been investigated extensively using both experimental and theoretical tools, to date there is no quantitative information about structural rigidity and the mechanical strength of parallel crossover DNA motifs. We have used fully atomistic molecular dynamics simulations in explicit solvent to get the force-extension curve of parallel DNA nanostructures to characterize their mechanical rigidity. In the presence of monovalent Na(+) ions, we find that the stretch modulus (gamma(1)) of the paranemic crossover and its topoisomer JX DNA structure is significantly higher (similar to 30%) compared to normal B-DNA of the same sequence and length. However, this is in contrast to the original expectation that these motifs are almost twice as rigid compared to the double-stranded B-DNA. When the DNA motif is surrounded by a solvent with Mg(2+) counterions, we find an enhanced rigidity compared to Na(+) environment due to the electrostatic screening effects arising from the divalent nature of Mg(2+) ions. To our knowledge, this is the first direct determination of the mechanical strength of these crossover motifs, which can be useful for the design of suitable DNA for DNA-based nanostructures and nanomechanical devices with improved structural rigidity.

DNA flexibility controls packaging, looping and in some cases sequence specific protein binding. Molecular dynamics simulations carried out with a computationally efficient implicit solvent model are potentially a powerful tool for studying larger DNA molecules than can be currently simulated when water and counterions are represented explicitly. In this work we compare DNA flexibility at the base pair step level modelled using an implicit solvent model to that previously determined from explicit solvent simulations and database analysis. Although much of the sequence dependent behaviour is preserved in implicit solvent, the DNA is considerably more flexible when the approximate model is used. In addition we test the ability of the implicit solvent to model stress induced DNA disruptions by simulating a series of DNA minicircle topoisomers which vary in size and superhelical density. When compared with previously run explicit solvent simulations, we find that while the levels of DNA denaturation are similar using both computational methodologies, the specific structural form of the disruptions is different.

Selective binding of the wild type tumor suppressor protein p53 to negatively and positively supercoiled (sc) DNA was studied using intercalative drugs chloroquine (CQ), ethidium bromide, acridine derivatives and doxorubicin as a modulators of the level of DNA supercoiling. The p53 was found to lose gradually its preferential binding to negatively scDNA with increasing concentrations of intercalators until the DNA negative superhelix turns were relaxed. Formation of positive superhelices (due to further increasing intercalator concentrations) rendered the circular duplex DNA to be preferentially bound by the p53 again. CQ at concentrations modulating the closed circular DNA topology did not prevent the p53 from recognizing a specific target sequence within topologically unconstrained linear DNA. Experiments with DNA topoisomer distributions differing in their superhelix densities revealed the p53 to bind selectively DNA molecules possessing higher number of negative or positive superturns. Possible modes of the p53 binding to the negatively or positively supercoiled DNA and tentative biological consequences are discussed.

The process of genetic recombination involves the formation of branched four-stranded DNA structures known as Holliday junctions. The Holliday junction is known to have an antiparallel orientation of its helices, i.e., the crossover occurs between strands of opposite polarity. Some intermediates in this process are known to involve two crossover sites, and these may involve crossovers between strands of identical polarity. Surprisingly, if a crossover occurs at every possible juxtaposition of backbones between parallel DNA double helices, the molecules form a paranemic structure with two helical domains, known as PX-DNA. Model PX-DNA molecules can be constructed from a variety of DNA molecules with five nucleotide pairs in the minor groove and six, seven or eight nucleotide pairs in the major groove. A topoisomer of the PX motif is the juxtaposed JX1 molecule, wherein one crossover is missing between the two helical domains. The JX1 molecule offers an outstanding baseline molecule with which to compare the PX molecule, so as to measure the thermodynamic cost of forming a crossover in a parallel molecule. We have made these measurements using calorimetric and ultraviolet hypochromicity methods, as well as denaturing gradient gel electrophoretic methods. The results suggest that in relaxed conditions, a system that meets the pairing requirements for PX-DNA would prefer to form the PX motif relative to juxtaposed molecules, particularly for the 6:5 structure.

It is generally assumed that native deoxyribonucleic acid (DNA) is a right-handed double helix. A reasonable deduction is that during replication, the two parental strands have to unwind very quickly. However, this surmised quick unwinding is problematic and has never been proven experimentally. It is hypothesized that the two strands of DNA are winding with each other ambidextrously rather than plectonemically. The successful assembling and disassembling of a zero linking number topoisomer supports this hypothesis. It was further proven by quick separation of singly nicked DNA. The new DNA model was also verified by the “figure 8” structure, which is the annealing product of two single-stranded circular DNA with a 2 kb complementary insert in opposite directions. These experimental results are hard to be explained by the traditional Watson–Crick model. The significance of this finding in the understanding of DNA replication is briefly discussed.

The histone-like heat unstable HU protein plays a key role in the organization and regulation of the Escherichia coli genome. The non-specific nature of HU binding to DNA complicates analysis of the mechanism by which the protein contributes to the looping of DNA. Conventional models of the looping of HU-bound duplexes attribute the changes in biophysical properties of DNA brought about by the random binding of protein to changes in the effective parameters of an ideal helical wormlike chain. Here we introduce a novel Monte-Carlo approach to study the effects of non-specific HU binding on the configurational properties of DNA directly. We randomly decorate segments of an ideal double-helical DNA with HU molecules that induce the bends and other structural distortions of the double helix found in currently available X-ray structures. We find that the presence of HU at levels approximating those found in the cell reduces the persistence length by roughly threefold compared to that of naked DNA. The binding of protein has particularly striking effects on the cyclization properties of short duplexes, altering the dependence of ring closure on chain length in a way that cannot be mimicked by a simple wormlike model and accumulating at higher than expected levels on successfully closed chains. Moreover, the uptake of protein on small minicircles depends upon chain length, taking advantage of the HU-induced deformations of DNA structure to facilitate ligation. Circular duplexes with bound HU show a much greater propensity than protein-free DNA to exist as negatively supercoiled topoisomers, suggesting a potential role of HU in organizing the bacterial nucleoid. The local bending and undertwisting of DNA by HU, in combination with the number of bound proteins, provide a structural rationale for the condensation of DNA and the observed expression levels of reporter genes in vivo.

In this chapter the statistical-mechanical and transport theories of the HW chain developed so far are applied to some interesting problems of circular DNA such as cyclization of linear DNA and analysis of circular DNA topoisomers (topological isomers) or supercoiled forms. These problems may be treated theoretically by modeling duplex DNA as the KP1 chain [or sometimes the (original) KP chain], which is a spacial case of the HW chain. From the statistical-mechanical standpoint, all kinds of ring-closure probabilities for these KP chains, which do not necessarily concern DNA problems, are also considered in this chapter. Relevant experimental data are analyzed by the use of the present theories in order to determine the stiffness parameter (Kuhn segment length) and torsional force constant of duplex DNA. However, all aspects of the problem of the supercoiling of DNA are not discussed since it is beyond the scope of this book.

Molecular machines undergo large-scale conformational changes during their catalytic cycles that are linked to their biological functions. DNA topoisomerases are molecular machines that interconvert different DNA topoisomers and resolve torsional stress that is introduced during cellular processes that involve local DNA unwinding. DNA gyrase catalyzes the introduction of negative supercoils into DNA in an ATP-dependent reaction. During its catalytic cycle, gyrase undergoes large-scale conformational changes that drive the supercoiling reaction. These conformational changes can be followed by single-molecule Forster resonance energy transfer (FRET). Here, we use DNA gyrase from Bacillus subtilis as an illustrative example to present strategies for the investigation of conformational dynamics of multisubunit complexes. We provide a brief introduction into single-molecule FRET and confocal microscopy, with a focus on practical considerations in sample preparation and data analysis. Different strategies in the preparation of donor-acceptor-labeled molecules suitable for single-molecule FRET experiments are outlined. The insight into the mechanism of DNA supercoiling by gyrase gained from single-molecule FRET experiment is summarized. The general strategies described here can also be applied to investigate conformational changes and their link to biological function of other multisubunit molecular machines.

While DNA supercoiling is ubiquitous in vivo, the structure of supercoiled DNA is more challenging to study experimentally than simple linear sequences because the DNA must have a closed topology in order to sustain superhelical stress. DNA minicircles, which are closed circular double-stranded DNA sequences typically containing between 60 and 500 base pairs, have proven to be useful biochemical tools for the study of supercoiled DNA mechanics. We present detailed protocols for constructing models of DNA minicircles in silico, for performing atomistic molecular dynamics (MD) simulations of supercoiled minicircle DNA, and for analyzing the results of the calculations. These simulations are computationally challenging due to the large system sizes. However, improvements in parallel computing software and hardware promise access to improve conformational sampling and simulation timescales. Given the concurrent improvements in the resolution of experimental techniques such as atomic force microscopy (AFM) and cryo-electron microscopy, the study of DNA minicircles will provide a more complete understanding of both the structure and the mechanics of supercoiled DNA.