Mechanism of double-stranded supercoiled DNA cleavage induced by RNA N-glycosidase

Plant RNA N-glycosidase specifically hydrolyzes the N-C glycosidic bond of a conserved adenosine in the sarcin/ricin domain of the largest RNA in ribosome, releasing an adenine base and thus inhibiting protein synthesis. This substrate specificity was challenged later by discovery that various RNA derivatives and DNAs, especially the double-stranded supercoiled DNA could be used as substrate by RNA N-glycosidase. Thus, it was argued whether the DNA-cleaving activity was an intrinsic feature of RNA N-glycosidase or it was contaminated by DNase. In this article, several lines of evidence are presented to show that RNA N-glycosidase can really release the adenine base from the double-stranded supercoi/ed DNA. It was proposed that the cleavage mechanism of supercoiled DNA was the phosphodiester bonds in enzymatically deadenylated regions of the supercoiled DNA would become fragile and liable to produce nicked or linear form owing to the existence of tension in the supercoiled DNA molecule, not direct result of enzymatic action on the phosphodiester bond.

Supercoiled DNA Minicircles under Double-strand Breaks

Understanding how supercoiled DNA releases intramolecular stress is essential for its functional realization.However,the molecular mechanism underlying the relaxation process remains insufficiently explored.Here we employed MD simulations based on the oxDNA2 model to investigate the relaxation process of a 336-base pair supercoiled minicircular DNA under double-strand breaks with two fixed endpoints.Our simulations show that the conformational changes in the DNA occur continuously,with intramolecular stress release happening abruptly only when the DNA chain traverses the breakage site.The relaxation process is influenced not only by the separation distance between the fixed ends but also their angle.Importantly,we observe an inhibitory effect on the relaxation characterized by small angles,where short terminal loops impede DNA conformational adjustments,preserving the supercoiled structure.These findings elucidate the intricate interplay between DNA conformational change,DNA motion and intramolecular stress release,shedding light on the mechanisms governing the relaxation of supercoiled DNA at the molecular level.

Visualization of interaction between ribosome-inactivating proteins and supercoiled DNA with an atomic force microscope

The interaction between ribosome-inactivating proteins (RIPs) and supercoiled DNA was observed with an atomic force microscope (AFM). It was found that RIPs can bind to both supercoiled DNA and the unwound double stranded loop region in supercoiled DNA. The RIPs hound to the supercoils can induce the conformational change of supercoiled DNA. Furthermore, the supercoiled DNA was relaxed and cleaved into nick or linear form by RIPs. It indicated that RIP seemed to be a supercoil-dependent DNA binding protein and exhibited the activity of su-percoil-dependent DNA endonuclease.

Dark-field Imaging of Supercoiled DNA

With the discovery and further understanding of topoisomerases, it becomes clear that the supercoiling of double stranded DNA plays an important role in DNA replication, RNA transcription and even in the control of gene expression. Although the small circular DNA can be separated by means of density gradient centrifugation or gel electrophoresis and

Direct Observation of Histone-Induced DNA Shortening

We construct a system of magnetic tweezers and apply it to study the interaction between histones and DNA. The condensation of DNA by purified histones at low ionic strengths is directly monitored by recording the length of the DNA as a function of elapsed time. It is found that DNA condensates in a dynamic manner. The binding of hist, ones to DNA is energetically favoured, but the ten,sion applied on DNA tends to unravel the DNA-histone complex, The competition between the two processes determiners the rate of the DNA condensation.

Comparison of CIM~ C4-HLD monolithic column with Sartobind phenyl membrane column for pIDKE2 purification

The main component of the Center for Genetic Engineering and Biotechnology(CIGB)candidate vaccine against Hepatitis C virus(HCV)is the pIDKE2 plasmid.The current designed downstream process for the production of pIDKE2 fulfils all regulatory requirements and renders the required quantities of pharmaceuticalgrade plasmid DNA(pDNA)with 95%purity.The advantages of this procedure include high plasmid purity and the elimination of undesirable additives,such as toxic organic extractants and animal-derived enzymes.However,yields and consequently the productivity of the process are low.Previous work demonstrated that the most critical step of the process is the reverse phase chromatography,where conventional porous particle resins are used.Therefore,to increase the process productivity,alternative technologies such as membranes and chromatographic monoliths were tested as alternative options for this critical step.Here,a comparison between the behaviors of CIM~ C4-HLD and Sartobind phenyl matrices was performed.To obtain higher productivities and purities,the dynamic binding capacities and selectivities were evaluated.The results showed that both matrices had a similar capacity for pIDKE2 plasmid,but the separation of pDNA isoforms using CIM~ technology was much better than that with Sartobind.Additionally,the optimal conditions for loading plasmid DNA on a CIMC4-HLD 800-mL monolithic column in a real production process were determined.These optimizations will allow production levels to satisfy the high plasmid consumption demanded by clinical trials.

Helical Repeats of Left-Handed DNA

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.

<i>In-Vitro</i>Inhibitory Effect of Methanol Extracts of Chinese Herbal Drugs on Supercoiling Activity of Bacterial DNA Gyrase

A large number of Chinese herbal drugs (CHDs) exhibit antibacterial activities both in vivo and in vitro, but until now little is known regarding their inhibitory mechanisms. Bacterial DNA gyrase is a proven target for antibacterial agents. Aim of this study was to investigate the in-vitro inhibitory effect of methanol extracts of CHDs against supercoiling activity of bacterial DNA gyrase. Fifteen CHDs were selected and extracted with methanol, respectively. Inhibitory effect of the extracts on DNA gyrase was tested using gel-based DNA supercoiling assay. Among fifteen CHDs tested, methanol extracts of Lonicerae Japonicae Flos (S2), Taraxaci Herba (S7), Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (S8) demonstrated an obvious inhibitory effect against supercoiling activity of DNA gyrase, and the others were either less active or could not be determined with the present method. Moreover, it was likely that S7 and S8 inhibit gyrase in a concentration-dependent manner. In conclusion, DNA supercoiling assay is a promising method to study the inhibitory activity of CHDs on bacterial DNA gyrase. Some CHDs do have gyrase-inhibitory activity as proposed. Further investigations are needed to elucidate the inhibition mechanism of these CHDs on supercoiling activity of gyrase.

Temperature regulates negative supercoils to modulate meiotic crossovers and chromosome organization

Crossover recombination is a hallmark of meiosis that holds the paternal and maternal chromosomes(homologs)together for their faithful segregation,while promoting genetic diversity of the progeny.The pattern of crossover is mainly controlled by the architecture of the meiotic chromosomes.Environmental factors,especially temperature,also play an important role in modulating crossovers.However,it is unclear how temperature affects crossovers.Here,we examined the distribution of budding yeast axis components(Red1,Hop1,and Rec8)and the crossover-associated Zip3 foci in detail at different temperatures,and found that both increased and decreased temperatures result in shorter meiotic chromosome axes and more crossovers.Further investigations showed that temperature changes coordinately enhanced the hyperabundant accumulation of Hop1 and Red1 on chromosomes and the number of Zip3 foci.Most importantly,temperature-induced changes in the distribution of axis proteins and Zip3 foci depend on changes in DNA negative supercoils.These results suggest that yeast meiosis senses temperature changes by increasing the level of negative supercoils to increase crossovers and modulate chromosome organization.These findings provide a new perspective on understanding the effect and mechanism of temperature on meiotic recombination and chromosome organization,with important implications for evolution and breeding.

Synthesis, DNA and Photocleavage Studies of Ru（ll） Polypyridyl Complexes： [Ru（dppz）（pyz）4]（ClO4）2 and [Ru（dppz）（dmpyz）4]（ClO4）2 Complexes

In view of the growing interest for the synthesis of metal complexes and their interaction with DNA, we have synthesized and characterized two complexes containing ruthenium as metal center. The complexes are of the type [Ru（dppz）L4]（C104）2 where L are biologically important ligands such as pyrazole and dimethylpyrazole. The characterization of these complexes is done by 1 H NMR, 13C NMR, elemental analysis and mass spectroscopy. The interaction of these complexes with CT DNA was monitored and binding constants were determined using absorption and fluorescence spectroscopy. The mode of binding was found to be intercalative for both complexes and was determined using hydrodynamic viscosity studies. The complexes were further studied for photocleavage studies with supercoiled plasmid pBR322 DNA.

Transcriptomic and proteomic analyses on the supercooling ability and mining of antifreeze proteins of the Chinese white wax scale insect

The Chinese white wax scale insect, Ericerus pela, can survive at extremely low temperatures, and some overwintering individuals exhibit supercooling at tempera- tures below -30℃. To investigate the deep supercooling ability ofE. pela, transcriptomic and proteomic analyses were performed to delineate the major gene and protein families responsible for the deep supercooling ability of overwintering females. Gene Ontology （GO） classification and Kyoto Encyclopedia of Genes and Genomes （KEGG） analysis indicated that genes involved in the mitogen-activated protein kinase, calcium, and PI3K-Akt signaling pathways and pathways associated with the biosynthesis of soluble sugars, sugar alcohols and free amino acids were dominant. Proteins responsible for low-temperature stress, such as cold acclimation proteins, glycerol biosynthesis-related enzymes and heat shock proteins （HSPs） were identified. However, no antifreeze proteins （AFPs） were identified through sequence similarity search methods. A random forest approach identified 388 putative AFPs in the proteome. The AFP gene ep-afp was expressed in Escherichia coli, and the expressed protein exhibited a thermal hysteresis activity of 0.97℃, suggesting its potential role in the deep supercooling ability ofE. pela.

Effect of DNA binding protein Ssh12 from hyperthermophilic archaeon Sulfolobus shibatae on DNA supercoiling

An 11.5-ku DNA binding protein, designated as Sshl2, was purified from the hyperthermophilic archaeon Sulfolobus shibatae by column chromatography in SP Sepharose, DNA cellulose and phosphocellulose. Sshl2 accounts for about 4 % of the total cellular protein. The protein is capable of binding to both negatively supercoiled and relaxed DNAs. Nick closure analysis revealed that Sshl2 constrains negative supercoils upon binding to DNA. While the ability of the protein to constrain supercoils is weak at 22℃ , it is enhanced substantially at temperatures higher than 37℃ . Both the cellular content and supercoil-constraining ability of Sshl2 suggest that the protein may play an important role in the organization and stabilization of the chromosome of S. shibatae.

Global Genomic Arrangement of Bacterial Genes Is Closely Tied with the Total Transcriptional Efficiency

The availability of a large number of sequenced bacterial genomes allows researchers not only to derive functional and regulation information about specific organisms but also to study the fundamental properties of the organization of a genome. Here we address an important and chal- lenging question regarding the global arrangement of operons in a bacterial genome： why operons in a bacterial genome are arranged in the way they are. We have previously studied this question and found that operons of more frequently activated pathways tend to be more clustered together in a genome. Specifically, we have developed a simple sequential distance-based pseudo energy func- tion and found that the arrangement of operons in a bacterial genome tend to minimize the clus- teredness function （C value） in comparison with artificially-generated alternatives, for a variety of bacterial genomes. Here we extend our previous work, and report a number of new observations： （a） operons of the same pathways tend to group into a few clusters rather than one; and （b） the global arrangement of these operon clusters tend to minimize a new ＂energy＂ function （C＋ value） that reflects the efficiency of the transcriptional activation of the encoded pathways. These obser- vations provide insights into further study of the genomic organization of genes in bacteria.

SPATIAL WRITHING OF DNA LOOPS RESULTING FROM AXIAL TWIST

In vivo DNA often appears in supercoiled states. This phenomenon is related to the constraints imposed on DNA structures, such as co-valenced ring closure, protein binding. When a certain amount of supercoiling isconfined in a DNA, the conformation of the molecule may responds in two different ways: either the supercoiling is completely absorbed by the twist of the double helix leaving an unaltered spatial shape: or the supercoiling

Chemical microenvironment mediated formation of organic nanostructures from self-assembly of melamine and barbituric acid derivatives

The recent progresses on constructing organic nanostructures from the self-assembly of melamine and barbituric acid derivatives are reviewed. By mediating the chemical microenvironment during the self-assembly, the information contained in the molecular components can be expressed at different levels, thus resulting in the formation of different organic nanostructures. When the assembly is carried out in anhydrous chloroform, a kind of asymmetric layered structure with a d value of 4.1 nm is obtained. When a little amount of polar solvent such as alcohol is contained in the chloroform, organic nanotubes with diameter of 6 nm and length of several hundreds of nanometers are observed. After being treated by appropriate polar solvents, the nanotubes are induced into supercoils with diameter of about 300 nm and length of several tens of microns. The sensitivity of the self-assembly process origins from the weak noncovalent intermolecular interactions between the molecular components. The enthalpy change of such interactions is pretty small, so slight change of the molecular structure or microenvironment could affect the primary equilibrium, resulting in the rearrangement and transformation of the supramolecular structure.