Condensin pinches a short negatively supercoiled DNA loop during each round of ATP usage.

Condensin, an SMC (structural maintenance of chromosomes) protein complex, extrudes DNA loops using an ATP-dependent mechanism that remains to be elucidated. Here, we show how condensin activity alters the topology of the interacting DNA. High condensin concentrations restrain positive DNA supercoils. However, in experimental conditions of DNA loop extrusion, condensin restrains negative supercoils. Namely, following ATP-mediated loading onto DNA, each condensin complex constrains a DNA linking number difference (∆Lk) of -0.4. This ∆Lk increases to -0.8 during ATP binding and resets to -0.4 upon ATP hydrolysis. These changes in DNA topology do not involve DNA unwinding, do not spread outside the condensin-DNA complex and can occur in the absence of the condensin subunit Ycg1. These findings indicate that during ATP binding, a short DNA domain delimited by condensin is pinched into a negatively supercoiled loop. We propose that this loop is the feeding segment of DNA that is subsequently merged to enlarge an extruding loop. Such a "pinch and merge" mechanism implies that two DNA-binding sites produce the feeding loop, while a third site, plausibly involving Ycg1, might anchor the extruding loop.

Telling Your Right Hand from Your Left: The Effects of DNA Supercoil Handedness on the Actions of Type II Topoisomerases.

Type II topoisomerases are essential enzymes that modulate the topological state of DNA supercoiling in all living organisms. These enzymes alter DNA topology by performing double-stranded passage reactions on over- or underwound DNA substrates. This strand passage reaction generates a transient covalent enzyme-cleaved DNA structure known as the cleavage complex. Al-though the cleavage complex is a requisite catalytic intermediate, it is also intrinsically dangerous to genomic stability in biological systems. The potential threat of type II topoisomerase function can also vary based on the nature of the supercoiled DNA substrate. During essential processes such as DNA replication and transcription, cleavage complex formation can be inherently more dangerous on overwound versus underwound DNA substrates. As such, it is important to understand the profound effects that DNA topology can have on the cellular functions of type II topoisomerases. This review will provide a broad assessment of how human and bacterial type II topoisomerases recognize and act on their substrates of various topological states.

Transcription rate and transcript length drive formation of chromosomal interaction domain boundaries.

Chromosomes in all organisms are highly organized and divided into multiple chromosomal interaction domains, or topological domains. Regions of active, high transcription help establish and maintain domain boundaries, but precisely how this occurs remains unclear. Here, using fluorescence microscopy and chromosome conformation capture in conjunction with deep sequencing (Hi-C), we show that in Caulobacter crescentus, both transcription rate and transcript length, independent of concurrent translation, drive the formation of domain boundaries. We find that long, highly expressed genes do not form topological boundaries simply through the inhibition of supercoil diffusion. Instead, our results support a model in which long, active regions of transcription drive local decompaction of the chromosome, with these more open regions of the chromosome forming spatial gaps in vivo that diminish contacts between DNA in neighboring domains. These insights into the molecular forces responsible for domain formation in Caulobacter likely generalize to other bacteria and possibly eukaryotes.

Direct observation of positive supercoils introduced by reverse gyrase through atomic force microscopy.

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.

Capillary Gel Electrophoresis (CGE) for Quality Control of Plasmid DNA in Gene Therapy: Quality Control of 20 Years Stored GMP-Grade Plasmid DNA.

Plasmid DNA in any form (plasmid DNA, minicircle, miniplasmid) does experience renewed and increasing attention for use in gene therapy and DNA vaccination. For such applications, stability analyses and quality control are essential prerequisites for clinical use. In this context we analyzed the stability of good manufacturing practice (GMP)-grade pCMVß reporter plasmid DNA by capillary gel electrophoresis. The plasmid DNA was produced for a clinical gene transfer study for treatment of malignant melanoma. The pCMVß plasmid DNA was stored at -20 °C for 20 years under continuous, controlled monitoring. Another plasmid., pCMV-Luc, stored for 15 years, served as reference. The stability of plasmid DNA was analyzed by capillary gel electrophoresis (CGE) and functionally tested in vitro by LacZ functional assay. In this chapter we provide the detailed description of CGE and functional analysis of the GMP-grade pCMVß and also pCMV-Luc plasmid DNA. By this the proportion of open circular and supercoiled or covalently closed circular forms of plasmid DNA is analyzed. Functionality of the plasmid was tested by in vitro transfection and LacZ functional assay. In result of this, the 20-year-old plasmid DNA showed topology and expression performance, which revealed significant alterations in topology while maintaining functionality regarding transgene expression. Therefore, stable storage conditions are effective to mainly preserve the integrity of the plasmid DNA as important parameter for long-term storage of, for example, reference samples.

The interaction of proanthocyanidins with DNA molecules studied by atomic force microscopy and spectroscopic method.

The interactions of the naturally available botanicals with DNA molecules have received considerable attention owing to the potential to develop for medicinal agents. In this study, the interaction of proanthocyanidins with DNA molecules was studied by atomic force microscopy (AFM) and spectroscopic method. The AFM observation indicated that proanthocyanidins induced DNA molecules from double helix chains to the thick rope and toroids. The heights of the formed DNA structures are more than eight times than that of DNA double helix. Spectroscopic measurement results revealed that proanthocyanidins intercalated between the base pairs of DNA in the intercalative binding mode, which resulted in unwinding the DNA helix, twisting the DNA strands and finally transforming into the supercoiled DNA structures. All these results implied that DNA molecule is an important interaction target of proanthocyanidins, and the formed compact DNA structures have biological significance on the gene expression and regulation.

Morphological Determinants of Carbon Nanomaterial-Induced Amyloid Peptide Self-Assembly.

Hybridizing carbon nanomaterials (CNMs) with amyloid fibrils-the ordered nanostructures self-assembled by amyloidogenic peptides-has found promising applications in bionanotechology. Understanding fundamental interactions of CNMs with amyloid peptides and uncovering the determinants of their self-assembly structures and dynamics are, therefore, pivotal for enriching and enabling this novel class of hybrid nanomaterials. Here, we applied atomistic molecular dynamics simulations to investigate the self-assembly of two amyloid peptides-the amyloidogenic core residues 16-22 of amyloid-ß (Aß16-22) and the non-amyloid-ß core of α-synuclein (NACore68-78)-on the surface of carbon nanotubes (CNT) with different sizes and chirality. Our computational results showed that with small radial CNTs, both types of peptides could form ß-sheets wrapping around the nanotube surface into a supercoiled morphology. The angle between ß-strands and nanotube axes in the supercoil structure depended mainly on the peptide sequence and CNT radius, but also weakly on the CNT chirality. Large radial CNTs and the extreme case of the flat graphene nanosheet, on the other hand, could nucleate amyloid fibrils perpendicular to the surface. Our results provided new insights of hybridizing CNMs with amyloid peptides and also offered a novel approach to manipulate the morphology of CNM-induced amyloid assembly by tuning the surface curvature, peptide sequence, and molecular ratio between peptides and available CNM surface area, which may be useful in engineering nanocomposites with high-order structures.

Direct single-molecule observations of DNA unwinding by SV40 large tumor antigen under a negative DNA supercoil state.

Superhelices, which are induced by the twisting and coiling of double-helical DNA in chromosomes, are thought to affect transcription, replication, and other DNA metabolic processes. In this study, we report the effects of negative supercoiling on the unwinding activity of simian virus 40 large tumor antigen (SV40 TAg) at a single-molecular level. The supercoiling density of linear DNA templates was controlled using magnetic tweezers and monitored using a fluorescent microscope in a flow cell. SV40 TAg-mediated DNA unwinding under relaxed and negative supercoil states was analyzed by the direct observation of both single- and double-stranded regions of single DNA molecules. Increased negative superhelicity stimulated SV40 TAg-mediated DNA unwinding more strongly than a relaxed state; furthermore, negative superhelicity was associated with an increased probability of SV40 TAg-mediated DNA unwinding. These results suggest that negative superhelicity helps to regulate the initiation of DNA replication.

DNA sequence encodes the position of DNA supercoils.

The three-dimensional organization of DNA is increasingly understood to play a decisive role in vital cellular processes. Many studies focus on the role of DNA-packaging proteins, crowding, and confinement in arranging chromatin, but structural information might also be directly encoded in bare DNA itself. Here, we visualize plectonemes (extended intertwined DNA structures formed upon supercoiling) on individual DNA molecules. Remarkably, our experiments show that the DNA sequence directly encodes the structure of supercoiled DNA by pinning plectonemes at specific sequences. We develop a physical model that predicts that sequence-dependent intrinsic curvature is the key determinant of pinning strength and demonstrate this simple model provides very good agreement with the data. Analysis of several prokaryotic genomes indicates that plectonemes localize directly upstream of promoters, which we experimentally confirm for selected promotor sequences. Our findings reveal a hidden code in the genome that helps to spatially organize the chromosomal DNA.

Research on ribosome-inactivating proteins from angiospermae to gymnospermae and cryptogamia.

Ribosome-inactivating Proteins (RIPs) are a group of cytotoxin proteins that usually contain a RNA N-glycosidase domain, which irreversibly inactivates ribosome, thus inhibiting protein synthesis. During the past 14 years (1990-2004), the studies conducted in our laboratory had been focusing on the structure and enzymatic mechanism of several PIPs. Herein, we briefly described a summary of the studies conducted mainly in our laboratory on RIPs from angiospermae to gymnospermae and cryptogamia as follows. (1) Cinnamomin is a novel type II RIP isolated from mature seeds of camphor tree. Like ricin, it specifically removes the adenine at A4324 in rat liver 28S rRNA. We systematically studied this low-toxic RIP in term of its enzymatic mechanism, the primary and crystal structure and the nucleotide sequence of its gene, the genetic expression, and its physiological role in the seed cell and the toxicity to human cancer cells and insect larvae. The cleavage of supercoiled double-stranded DNA was its intrinsic property of cinnamomin A-chain, its N- and C-terminal regions were found to be required for deadenylation of rRNA and also necessary for deadenylation of supercoiled double-stranded circular DNA. These results strongly excluded the possibility that cleavage of supercoiled DNA was due to nuclease contamination. (2) Trichosanthin, an abortifacient protein, was purified from the Chinese medicinal herb, Tian-hua-fen, obtained from root tubers of Chinese trichosanthes plant. We proved that trichosanthin was a RNA N-glycosidase, inactivating eukaryotic ribosome by hydrolyzing the N-C glycosidic bond of the adenose at site 4324 in rat 28S rRNA, and inhibited protein synthesis in vitro. (3) A unique Biota orientalis RNase (RNase Bo) was extracted from the mature seeds of the cypress cypress tree (Oriental arborvita), which was gymnospermae plant. It cleaved only a specific phosphodiester bond between C4453 and A4454 of 28S RNA in rat ribosomes, producing a small RNA-fragment (S-fragment), thus inhibiting protein synthesis and belonging to RNase-like RIP, similar to α-sarcin, a special RIP. (4) Lamjapin, the first RIP purified from kelp, the marine cryptogamia algal plant, was shown to be the first single-chained RNA N-glycosidase from marine plant to date. It hydrolyzed rat ribosomal 28S RNA to produce meanly a rather smaller RNA, shorter than the diagnostic R-fragment under the restricted condition. The significance of existence of type I RIP in the lower marine algal plant was briefly discussed.

Species-specific supercoil dynamics of the bacterial nucleoid.

Bacteria organize DNA into self-adherent conglomerates called nucleoids that are replicated, transcribed, and partitioned within the cytoplasm during growth and cell division. Three classes of proteins help condense nucleoids: (1) DNA gyrase generates diffusible negative supercoils that help compact DNA into a dynamic interwound and multiply branched structure; (2) RNA polymerase and abundant small basic nucleoid-associated proteins (NAPs) create constrained supercoils by binding, bending, and forming cooperative protein-DNA complexes; (3) a multi-protein DNA condensin organizes chromosome structure to assist sister chromosome segregation after replication. Most bacteria have four topoisomerases that participate in DNA dynamics during replication and transcription. Gyrase and topoisomerase I (Topo I) are intimately involved in transcription; Topo III and Topo IV play critical roles in decatenating and unknotting DNA during and immediately after replication. RNA polymerase generates positive (+) supercoils downstream and negative (-) supercoils upstream of highly transcribed operons. Supercoil levels vary under fast versus slow growth conditions, but what surprises many investigators is that it also varies significantly between different bacterial species. The MukFEB condensin is dispensable in the high supercoil density (σ) organism Escherichia coli but is essential in Salmonella spp. which has 15 % fewer supercoils. These observations raise two questions: (1) How do different species regulate supercoil density? (2) Why do closely related species evolve different optimal supercoil levels? Control of supercoil density in E. coli and Salmonella is largely determined by differences encoded within the gyrase subunits. Supercoil differences may arise to minimalize toxicity of mobile DNA elements in the genome.

α/ß coiled coils.

Coiled coils are the best-understood protein fold, as their backbone structure can uniquely be described by parametric equations. This level of understanding has allowed their manipulation in unprecedented detail. They do not seem a likely source of surprises, yet we describe here the unexpected formation of a new type of fiber by the simple insertion of two or six residues into the underlying heptad repeat of a parallel, trimeric coiled coil. These insertions strain the supercoil to the breaking point, causing the local formation of short ß-strands, which move the path of the chain by 120° around the trimer axis. The result is an α/ß coiled coil, which retains only one backbone hydrogen bond per repeat unit from the parent coiled coil. Our results show that a substantially novel backbone structure is possible within the allowed regions of the Ramachandran space with only minor mutations to a known fold.

4-Aminoquinoline derivatives as novel Mycobacterium tuberculosis GyrB inhibitors: Structural optimization, synthesis and biological evaluation.

Mycobacterial DNA gyrase B subunit has been identified to be one of the potentially underexploited drug targets in the field of antitubercular drug discovery. In the present study, we employed structural optimization of the reported GyrB inhibitor resulting in synthesis of a series of 46 novel quinoline derivatives. The compounds were evaluated for their in vitro Mycobacterium smegmatis GyrB inhibitory ability and Mycobacterium tuberculosis DNA supercoiling inhibitory activity. The antitubercular activity of these compounds was tested over Mtb H37Rv strain and their safety profile was checked against mouse macrophage RAW 264.7 cell line. Among all, three compounds (23, 28, and 53) emerged to be active displaying IC50 values below 1 µM against Msm GyrB and were found to be non-cytotoxic at 50 µM concentration. Compound 53 was identified to be potent GyrB inhibitor with 0.86 ± 0.16 µM and an MIC (minimum inhibitory concentration) of 3.3 µM. The binding affinity of this compound towards GyrB protein was analysed by differential scanning fluorimetry which resulted in a positive shift of 3.3 °C in melting temperature (Tm) when compared to the native protein thereby reacertaining the stabilization effect of the compound over protein.

Safety and efficacy of DNA vaccines: plasmids vs. minicircles.

While DNA vaccination using plasmid vectors is highly attractive, there is a need for further vector optimization regarding safety, stability, and efficiency. In this commentary, we review the minicircle vector (MC), which is an entity devoid of plasmid bacterial sequences, as an alternative to the traditional plasmid construct. The commentary highlights the recent discovery by Stenler et al. (2014) that the small size of an MC enables improved resistance to the shearing forces associated with e.g. pneumatic delivery methods. This observation may have implications for the regulatory agencies' requirement of plasmid integrity and quality.

Assessing sensitivity to antibacterial topoisomerase II inhibitors.

Both prokaryotes and eukaryotes have two major classes of topoisomerases that make transient single- or double-strand cuts in DNA. While these enzymes play critical roles in cellular processes, they are also important targets of therapeutic agents. This unit describes assays to use in characterizing topoisomerase II-targeting agents in vitro and in bacterial cells. It provides protocols for characterizing the action of small molecules against bacterial type II topoisomerases in vitro and the in vivo effects of putative topoisomerase II-targeting antibiotics, as well as for measuring trapped enzyme/DNA covalent complexes, the major cytotoxic lesion induced by fluoroquinolones.