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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.

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.

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.

Bacillus cereus topoisomerase IIIbeta (bcTopo IIIbeta) has been cloned, overexpressed and biochemically characterized. This enzyme exhibits 64% and 33% sequence identity to Bacillus subtilis topoisomerase III (bsTopo III) and Escherichia coli topoisomerase III (ecTopo III) respectively. The enzymatic properties of bcTopo IIIbeta differ substantially from other bacterial type IA topoisomerases, including E. coli type IA topoisomerases and B. cereus topoisomerase I (bcTopo I) and IIIalpha (bcTopo IIIalpha). bcTopo IIIbeta only partially relaxes negatively supercoiled DNA and appears incapable of generating fully relaxed topoisomers. In contrast to ecTopo III and bcTopo IIIalpha, bcTopo IIIbeta is not a decatenase. bcTopo IIIbeta is unable to compensate the loss of ecTopo III in vivo. Therefore, bcTopo IIIbeta is a unique prokaryotic type IA topoisomerase that is different from previously characterized topoisomerases.

Changes in DNA supercoiling might be essential to generate the response of cellular machinery to temperature stress. The heat-induced structural transition for a topoisomer depends on the value of its specific linking difference. We detect only less negatively supercoiled DNA and an abundance of alternative irregular DNA forms at culture temperatures close to the growth limit of Escherichia coli. We show that the irregular forms are derived from regular plasmid DNAs and their population in the cells is temperature-dependent. Here, we show that it is possible to isolate and characterize individual DNA topoisomers directly from cells without a topoisomerase treatment. Temperature gradient gel electrophoresis (TGGE) and atomic force microscopy (AFM) were used to study the effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability.

DNA branched junctions are analogues of Holliday junction recombination intermediates. Partially mobile junctions contain a limited amount of homology flanking the branch point. A partially mobile DNA branched junction has been incorporated into a synthetic double-stranded circular DNA molecule. The junction is flanked by four homologous nucleotide pairs, so that there are five possible locations for the branch point. Two opposite arms of the branched junction are joined to form the circular molecule, which contains 262 nucleotides to the base of the junction. This molecule represents a system whereby torque applied to the circular molecule can have an impact on the junction, by relocating its branch point. Ligation of the molecule produces two topoisomers; about 87% of the product is a relaxed molecule, and the rest is a molecule with one positive supercoil. The position of the branch point is assayed by cleaving the molecule with endonuclease VII. We find that the major site of the branch point in the relaxed topoisomer is at the maximally extruded position in the relaxed molecule. Upon the addition of ethidium, the major site of the branch point migrates to the minimally extruded position. © 1998 John Wiley & Sons, Inc. Biopoly 45: 69–83, 1998

Background: The RecA protein is a central player in bacterial homologous recombination. It promotes two key events: the search for homology between two DNA molecules and the subsequent formation of the synaptic complex composed of RecA and three DNA strands (two from one duplex and one single strand). In spite of numerous studies, the architecture of the synaptic complex is still far from clear. Results: We have exploited two approaches to study the structure of Escherichia coli RecA protein-mediated DNA synapsis: chemical modification with potassium permanganate and treatment of synaptic complexes of different lengths with topoisomerase I. The linking number difference values, obtained after separation of the individual sets of topoisomers in an agarose gel, were used to determine the number of bases per helical turn. We were able to show that the topology of the three-stranded complexes containing RecA is quite different from that expected for deproteinized D-loops. The original duplex in the synaptic complex is unwound, but not necessarily unpaired, to a structure topologically equivalent to DNA with approximately 27 bp per turn. Despite the fact that the patterns of reactivity towards potassium permanganate cannot be interpreted unambiguously, the results of chemical footprinting can be explained in terms of a synaptic complex as an extended and unwound three-stranded helical structure. Conclusions: This work provides the first quantitative topological parameters for the RecA protein-mediated three-stranded synaptic complex. Furthermore, the structure of synaptic complexes is different from that of a simple D-loop.

We started ethidium DNA agarose gel electrophoresis when our ultracentrifuge broke down and we needed an alternative method to check the quality of our mitochondrial DNA preparations. Agarose proved convenient for sizing DNA; ethidium in gel and buffer allowed visualization of DNA bands immediately after the run and improved the separation of the closed and open duplex forms of mitochondrial DNA circles. At smaller gel pore size mitochondrial DNA circles were excluded from the gel, whereas long linear DNAs were not. We concluded that the linear DNAs 'crawl like snakes head on through the gel'. This paper reviews some of the early experiments preceding the introduction of ethidium agarose gel electrophoresis.

Using dynamic light scattering (DLS), we have measured the diffusion coefficient and internal motions of narrow topoisomer distributions of pUCl8 DNA (2687 base pairs) as a function of linking number. The topoisomer distributions were prepared by C-18 reversed phase high performance liquid chromatography separation of topoisomerase I/ethidium-generated topoisomer families. The measured diffusion coefficients agree well with those predicted by a Monte Carlo model for the generation of equilibrium ensembles of DNA topoisomers of defined linking number. The only parameters used in the model were the bending persistence length, torsional rigidity, and hydrodynamic radius of DNA known independently from other techniques. Two different values for the torsional rigidity, α = 4 · 10−12 and 8.8 · 10−12 dyn cm, were used. Intrachain interactions in the DNA were taken into account by using an “effective DNA radius,” which was varied between rDNA = 1–8 nm. The best agreement between the measured and calculated values was obtained for α = 4 · 10−12 dyn cm, rDNA = 4 nm. The internal motions of the DNA topoisomers were characterized by the amplitude of the fast relaxation of the DLS autocorrelation function. Our earlier result that supercoiling leads to a decrease in the amplitude of internal motion was confirmed. In addition, we see a characteristic maximum of the internal motion amplitude at a superhelix density of σ = −0.03. The maximum occurs in the same range as a structural transition in pUC8 dimers previously described by L. Song et al. (1990 J. Mol. Biol. 214, 307–326). © 1994 John Wiley & Sons, Inc.

The effect of various non-intercalating minor groove binders on closed circular DNA in the presence of topoisomerase I has been studied by means of agarose gel electrophoresis. Analogues of the netropsin series (lexitropsins) and SN-6999 can effectively produce positive supercoils, as indicated by analysis of the topoisomers in the presence of chloroquine and the evaluated linking number changes. Analogues of the distamycin series are less effective, and bisquaternary ammonium heterocycles, as well as DAPI and pentamidine, were found to be ineffective ligands. The large differences observed in the ability of minor groove binders to induce positive supercoils are discussed.

Molecular interactions underlie the whole of chemistry and biology. This tutorial review illustrates the use of rotameric folding molecules, topoisomers, atropoisomers, and tautomers as molecular balances for quantifying non-covalent interactions. This intramolecular approach enables a wide variety of interactions to be examined with a degree of geometric control that is difficult to achieve in supramolecular complexes. Synthetic variation of molecular balances allows the fundamental physicochemical origins of molecular recognition to be systematically examined by providing insights into the interplay of geometry and solvation on non-covalent interactions.

Gel electrophoresis is a convenient technique for sepa- rating fragments of linear DNA differing in length or for separating the topoisomers of a supercoiled DNA from a mixture.'-4 A number of theoretical attempts have been made in order to understand the physical basis underlying the process of gel electrophoresis of linear DNA, employing mostly the idea of reptation?-" However, in spite of con- siderable advances in the understanding of gel electro- phoresis of linear DNA, the physical mechanism behind the resolution of topoisomers by gel electrophoresis has been inadequately studied. The purpose of this commu- nication is to show that the reptation model may be suit- ably extended to describe the gel electrophoretic mobility of the topoisomers of supercoiled DNA molecules also. The reptation mechanism was originally developed to study the diffusion of macromolecules in a polymer net- work." The polymer segments of the network surrounding the chain molecule restrict the motion of the chain, ef- fectively confining it to a tube.I3 Lateral motion is con- strained and the chain molecule can diffuse only longi- tudinally along the tube. During diffusion it is assumed that the head, i.e., the leading end of the chain, creates a path through the network and the rest of the chain follows the head in a snake-like fashion. For describing the gel electrophoresis of linear DNA on the basis of the reptation mechanism, Lumpkin and Zimm' assumed that the DNA chain, which is essentially a polyion, moves through a tube in the gel matrix under the influence of the applied electric field. On its basis, they obtained a simple expression for the steady state mobility of the DNA chain as where

Electrophoretic properties have been measured for a DNA molecule whose helix axes have the connectivity of a cube. This molecule is a topologically bonded complex of six cyclic 80-mer molecules, in which each cycle corresponds to a face of the cube. Each cyclic molecule is doubly catenated to each of its four neighbors. Substructures of this molecule include a 5-cycle structure lacking one strand, two topoisomers of 4-cycle structures and two topoisomers of 3-cycle structures. One 4-cycle structure is a cyclic belt around the cube, lacking a top and a bottom, whereas the other lacks two catenated strands, such as the top and the front. One 3-cycle structure is a linear belt of three cycles, and the other corresponds to the three cycles that surround a corner. Each of these molecules is separable from the others under appropriate gel conditions. We have measured mobilities and calculated Ferguson plots for each of these molecules on polyacrylamide gels under both native and denaturing conditions. The measurements have been made with 1.25, 2.5, and 5% crosslinking of the gels. The data show that the higher-symmetry 3-cycle and 4-cycle structures migrate more slowly than their lower-symmetry isomers, under conditions where their Ferguson plots are parallel.

Whether or not DNA gyrase can supercoil DNA so that alternative structures will arise in it is the major question of this work. We have shown gyrase to produce in pAO3 DNA a superhelix density sufficient for cruciform formation. However, the transition does not take place because of too slow kinetics. A change of ionic conditions in favour of more intense DNA supercoiling by gyrase shifts the midpoint of the equilibrium transition to the cruciform structure toward more supercoiled topoisomers. The width of the equilibrium transition to the cruciform as a function of linking number has been revealed to be an order of magnitude larger in buffers containing magnesium and spermidine than in buffers with monovalent cations only. We ascribe this effect to the influence that the counter ions surrounding the DNA molecule have on its elasticity, the coefficient of elasticity being dependent on superhelix density σ. Thus, the free energy of supercoiling (a) depends on the ionic conditions and (b) is not a quadratic function of σ in the physiological range of parameters. We propose a description of DNA as a system of links that can be either closed or open; we also introduce a new concept of the DNA linking potential akin to the chemical and electric potentials. The linking potential is a suitable parameter for describing the equilibrium distribution of links in heterogeneous DNA, the coexistence of various DNA structures, the equilibrium input and output of DNA links by enzymes, and the nonequilibrium movement of links along DNA chains. Within the framework of this approach DNA gyrase is considered as the source of the DNA linking potential.

The mean-square radius 〈S2〉N of gyration of the DNA topoisomer with the linking number N is evaluated as a function of N and chain length L on the basis of a (circular) twisted wormlike chain, i.e., a special case of the helical wormlike chain. Evaluation of 〈S2〉N and also of the moment 〈Wr2〉 of the writhe Wr is carried out over a wide range of L, following the Monte Carlo procedure of Frank-Kamenetskii et al. It is found that the present Monte Carlo values of 〈Wr2〉 for large L are appreciably larger than the known Monte Carlo values for freely jointed chains. Thus, the empirical interpolation formula for 〈Wr2〉 previously constructed on the basis of the theoretical values for small L along with the latter Monte Carlo values for large L is revised with the present Monte Carlo values. By the use of the revised formula, a reanalysis of the experimental data for the distribution of topoisomers is made, and it is found that the present estimates of the torsional constant and the stiffness parameter are equal to and somewhat larger than the previous ones, respectively. It is shown that 〈S2〉N decreases with increasing |ΔN|, where ΔN = N − N, with N the number of helix turns in the linear DNA chain in its undeformed state. The mean-square radii of gyration 〈S2〉Wr and 〈S2〉 of the original circular Kratky–Porod wormlike chain with and without Wr fixed are also evaluated.

The alternating DNA sequence d(CA/GT)n is known to adopt the left-handed Z-form in negatively supercoiled DNA in vitro. This element represents a significant fraction of the highly repeated DNA sequences found in eukaryotic genomes. We have cloned an alternating d(CA/GT)30 sequence into SV40 minichromosomes at the unique HpaII restriction site which occurs in the transcriptional leader region of the viral late genes. By comparing the linkage differences of topoisomers obtained from viral DNA with or without the d(CA/GT)30 insert, at various stages of the lytic cycle, we conclude that this sequence does not predominate in the Z-form in vivo. Furthermore, we find that the d(CA/GT)30 sequence is packaged into nucleosomal core particles and the region of the minichromosomes which contain the d(CA/GT)30 sequence is organized as nucleosomes.

Electrophoretic resolution of topoisomers was used to compare the in vivo superhelicity of recombinant plasmids containing a fragment of cDNA for an immunoglobulin light chain, cloned in the two possible orientations into the BamHI site of pBR313 or pBR322. Previously, frequent transpositions of IS1 or IS5 were observed into the sequence upstream to the cloned fragment in recombinants in one orientation [(+) plasmids] but not in recombinants in the opposite, (−) orientation [(1982) Nucleic Acids Res. 10, 4525-4542]. The results of the present analyses show that, on average, (−) plasmids are less negatively supercoiled than (+) plasmids, or pBR322. These results suggest that primary sequence rearrangements in plasmids could affect their in vivo topological state, and consequently, perhaps, their effectiveness as recipients of transposable elements.