DNA Relaxation Research Articles

Biological evaluation of a halogenated triterpenoid, 2α-bromo-dihydrobelulonic acid as inhibitor of human topoisomerase IIα and HeLa cell proliferation

BackgroundThe pentacyclic lupane-type (6-6-6-6-5 type) triterpenoid, Betulinic acid (BA) is a potent inhibitor of topoisomerases and is of immense interest as anticancer drugs. However, the compound being highly lipophilic, has limited in vivo uptake capacity. BA derivatives with halogen substituent at C-2 have improved membrane permeability and cytotoxicity against cancer cells. AimThe halogenated triterpenoid, 2α-bromo-dihydrobetulonic acid (B1) was synthesized from betulinic acid (BA) isolated from Bischofia javanica. Aim of the study was to determine whether B1 could act as a more efficient inhibitor of Topo IIα activity and HeLa cell proliferation, in comparison to BA. ResultB1 displayed efficient inhibition of DNA relaxation activity of topoisomerase IIα and the inhibitory effect was markedly improved upon pre-incubation of the compound with enzyme. Topoisomerase IIα inhibition by B1 was relieved in presence of increasing concentrations of DNA suggesting the compound as a reversible catalytic inhibitor. Subsequent UV and fluorescence spectroscopy studies indicated that B1 interacts and intercalates with DNA at concentrations signicantly greater than that required for topoisomerase IIα inhibition. The compound showed cytotoxic activity against HeLa cells with significantly lower IC50 value (7.5 μM) as compared to that of BA (30 μM) and had very low damaging/cytotoxic effect on normal cells. Treatment of B1 impaired HeLa cell proliferation by inducing Go-G1 arrest through lowered expression of cyclin D1 and PCNA polypeptides, and enhanced expression of p21. B1 treatment also increased the accumulation of early and late apoptotic cells in a concentration dependent manner as indicated by annexin V-FITC/PI binding assay.

Relaxation of DNA supercoiling leads to increased invasion of epithelial cells and protein secretion by Campylobacter jejuni.

Invasion of intestinal epithelial cells by Campylobacter jejuni is a critical step during infection of the intestine by this important human pathogen. In this study we investigated the role played by DNA supercoiling in the regulation of invasion of epithelial cells and the mechanism by which this could be mediated. A significant correlation between more relaxed DNA supercoiling and an increased ability of C. jejuni strains to penetrate human epithelial cells was demonstrated. Directly inducing relaxation of DNA supercoiling in C. jejuni was shown to significantly increase invasion of epithelial cells. Mutants in the fibronectin binding proteins CadF and FlpA still displayed an increased invasion after treatment with novobiocin suggesting these proteins were not essential for the observed phenotype. However, a large increase in protein secretion from multiple C. jejuni strains upon relaxation of DNA supercoiling was demonstrated. This increase in protein secretion was not mediated by outer membrane vesicles and appeared to be dependent on an intact flagellar structure. This study identifies relaxation of DNA supercoiling as playing a key role in enhancing C. jejuni pathogenesis during infection of the human intestine and identifies proteins present in a specific invasion associated secretome induced by relaxation of DNA supercoiling.

Plant and Human MORC Proteins Have DNA-Modifying Activities Similar to Type II Topoisomerases, but Require One or More Additional Factors for Full Activity.

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.

The Impact of DNA Topology on Target Selection by a Cytosine-Specific Cas9

Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be 1) complementary to the Cas9-bound guide RNA and a short Cas9-specific sequence adjacent to protospacer, called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition in time and space of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). AceCas9 depends strictly on a 5′-NNNCC-3′ PAM and is more efficient in cleaving negative supercoils than relaxed DNA. We further showed that mismatches to the guide RNA on a supercoiled protospacer are tolerated by AceCas9, whereas the same mismatches on its relaxed form were not. The cytosine-specific and DNA topology-sensitive properties of the AceCas9 maybe explored for chromosome domain specific genome editing applications. Finally, our preliminary data showed that AceCas9 can disrupt target DNA in in vivo assays, demonstrating its utility as a genome editing enzyme.

Bridging Chromosomal Architecture and Pathophysiology of Streptococcus pneumoniae.

The chromosome of Streptococcus pneumoniae is organized into topological domains based on its transcriptional response to DNA relaxation: Up-regulated (UP), down-regulated (DOWN), nonregulated (NR), and AT-rich. In the present work, NR genes found to have highly conserved chromosomal locations (17% of the genome) were categorized as members of position-conserved nonregulated (pcNR) domains, while NR genes with a variable position (36% of the genome) were classified as members of position-variable nonregulated (pvNR) domains. On average, pcNR domains showed high transcription rates, optimized codon usage, and were found to contain only a small number of RUP/BOX/SPLICE repeats. They were also poor in exogenous genes but enriched in leading strand genes that code for proteins involved in primary metabolism with central roles within the interactome. In contrast, pvNR genes coding for cell wall proteins, paralogs, virulence factors and immunogenic candidates for protein-based vaccines were found to be overrepresented. DOWN domains were enriched in genes essential for infection. Many UP and DOWN domain genes were seen to be activated during different stages of competence, whereas pcNR genes tended to be repressed until the competence was switched off. Pneumococcal genes appear to be subject to a topology-driven selection pressure that defines the chromosomal location of genes involved in metabolism, virulence and competence. The pcNR domains are interleaved between UP and DOWN domains according to a pattern that suggests the existence of macrodomain entities. The term “topogenomics” is here proposed to describe the study of the topological rules of genomes and their relationship with physiology.

Identification of the amino acid residues responsible for stable nucleosome formation by histone H3.Y

ABSTRACTHistone H3.Y is conserved among primates. We previously reported that exogenously produced H3.Y accumulates around transcription start sites, suggesting that it may play a role in transcription regulation. The H3.Y nucleosome forms a relaxed chromatin conformation with flexible DNA ends. The H3.Y-specific Lys42 residue is partly responsible for enhancing the flexibility of the nucleosomal DNA. To our surprise, we found that H3.Y stably associates with chromatin and nucleosomes in vivo and in vitro. However, the H3.Y residues responsible for its stable nucleosome incorporation have not been identified yet. In the present study, we performed comprehensive mutational analyses of H3.Y, and determined that the H3.Y C-terminal region including amino acid residues 124–135 is responsible for its stable association with DNA. Among the H3.Y C-terminal residues, the H3.Y Met124 residue significantly contributed to the stable DNA association with the H3.Y-H4 tetramer. The H3.Y M124I mutation substantially reduced the H3.Y-H4 association in the nucleosome. In contrast, the H3.Y K42R mutation affected the nucleosome stability less, although it contributes to the flexible DNA ends of the nucleosome. Therefore, these H3.Y-specific residues, Lys42 and Met124, play different and specific roles in nucleosomal DNA relaxation and stable nucleosome formation, respectively, in chromatin.

Differences in sequences between HBV-relaxed circular DNA and covalently closed circular DNA

The hepatitis B virus (HBV) genome exists in two forms: circular covalently closed DNA (cccDNA) and relaxed circular DNA (RCDNA). Here, we investigated the presence of differences in the sequences of both forms in paired samples of serum and liver tissue. The serum and liver biopsy samples were collected at the same time from 67 chronically infected patients. The genotyping of the RCDNA and cccDNA was performed using mass spectrometry analysis. The HBV mutations located in the HBV pol (P) and the HBV basal core promoter/pre-core (BCP/PC) regions were included. The BCP/PC and P sequences of the RCDNA extracted from liver and blood samples were different in 39% and 16% of patients, respectively. Differences were also found between RCDNA and cccDNA extracted from the same liver specimen. Moreover, the cccDNA BCP/PC region sequence had an impact on various virological and clinical parameters. We demonstrated that there are differences between the RCDNA and cccDNA sequences that were extracted from the same liver tissue. However, further investigations are needed to analyze whether the mutations in the cccDNA are conserved and whether cccDNA serves as a ‘mutation storage’ pool for HBV. This result could have profound implications for the subsequent therapy choices for treatment-experienced patients.Emerging Microbes & Infections (2017) 6, e55; doi:10.1038/emi.2017.41; published online 21 June 2017

Large family cohorts of lymphoblastoid cells provide a new cellular model for investigating facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite repeat. The resulting DNA hypomethylation and relaxation of epigenetic repression leads to increased expression of the deleterious DUX4-fl mRNA encoded within the distal D4Z4 repeat. With the typical late onset of muscle weakness, prevalence of asymptomatic individuals, and an autosomal dominant mode of inheritance, FSHD is often passed on from one generation to the next and affects multiple individuals within a family. Here we have characterized unique collections of 114 lymphoblastoid cell lines (LCLs) generated from 12 multigenerational FSHD families, including 56 LCLs from large, genetically homogeneous families in Utah. We found robust expression of DUX4-fl in most FSHD LCLs and a good correlation between DNA hypomethylation and repeat length. In addition, DUX4-fl levels can be manipulated using epigenetic drugs as in myocytes, suggesting that some epigenetic pathways regulating DUX4-fl in myocytes are maintained in LCLs. Overall, these FSHD LCLs provide an alternative cellular model in which to study many aspects of D4Z4, DUX4, and FSHD gene regulation in a background of low genetic variation. Significantly, these non-adherent immortal LCLs are amenable for high-throughput screening of potential therapeutics targeting DUX4-fl mRNA or protein expression.

Design, Synthesis and in vitro Anticancer Activity of a Cu(II) Complex of Carminic Acid: A Novel Small Molecule Inhibitor of Human DNA Topoisomerase I and Topoisomerase II

AbstractThe hydroxy‐9,10‐anthraquinones in anthracyclines form semiquinones and reactive oxygen species (ROS) that help in antitumor activity. ROS generation is cytotoxic to cells but cardiotoxic as well. Carminic acid (CA) with a sugar bound to a hydroxyanthraquinone resembles anthracyclines closely. Role of sugar in anthracyclines was realized from physicochemical parameters of CA that were found to be in between those of anthracyclines and hydroxy‐9,10‐anthraquinones. A Cu(II)complex of CA was prepared and its structure attempted from powder X‐ray diffraction. Studies with DNA revealed unlike CA, the complex did not show decrease in binding constant with increase in the pH of the medium. DNA relaxation assay showed the complex as a dual inhibitor of human DNA topoisomerase I and topoisomerase II stabilizing covalent topoisomerase‐DNA adducts in vitro. Inhibition of growth of ALL‐MOLT‐4 cells was higher for the complex supporting in vitro experiments. An enzyme assay revealed less superoxide formation for the complex suggesting that it might be less cardiotoxic. Decrease in superoxide for the complex does not affect anticancer activity, which was greater than CA.

PCR mediated recombination impacts the analysis of hepatitis B Virus covalently closed circular DNA.

BackgroundThe replication of HBV involves the production of covalently closed circular DNA (cccDNA) from the HBV genome through the repair of virion relaxed circular DNA (rcDNA) in the virion. As cccDNA is the transcription template for HBV genomes, it needs to be eliminated from hepatocytes if the eradication of chronic HBV infection is to be achieved. PCR quantitation of cccDNA copy number is the technique of choice for evaluating the efficiency of treatment regimens. The PCR target commonly used to identify cccDNA spans the gapped region of rcDNA and is considered to accurately distinguish between cccDNA and rcDNA. There is however, a potentially confounding issue in that PCR can generate larger targets from collections of small DNA fragments, a phenomenon known as PCR recombination.ResultsThe impact of PCR recombination towards the amplification of this cccDNA specific target was explored by mixing three marked, yet overlapping HBV DNA fragments. Thirteen of sixteen possible recombinants were identified by sequencing with frequencies ranging from 0.6 to 23%. To confirm this finding in vivo, HBV positive sera were treated with DNase I and submitted to quantitative real-time PCR. Under these conditions, it was possible to amplify the cccDNA specific segment without difficulty. As the virion contains uniquely rcDNA, amplification of the cccDNA target resulted from PCR recombination.ConclusionsPCR quantitation of cccDNA may be more difficult than hitherto thought. Current detection protocols need to be investigated so as to help in the management of chronic HBV infection.

Inhibitory effects of lapachol on rat C6 glioma in vitro and in vivo by targeting DNA topoisomerase I and topoisomerase II.

BackgroundLapachol is a natural naphthoquinone compound that possesses extensive biological activities. The aim of this study is to investigate the inhibitory effects of lapachol on rat C6 glioma both in vitro and in vivo, as well as the potential mechanisms.MethodsThe antitumor effect of lapachol was firstly evaluated in the C6 glioma model in Wistar rats. The effects of lapachol on C6 cell proliferation, apoptosis and DNA damage were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)/ phenazinemethosulfate (PMS) assay, hoechst 33358 staining, annexin V-FITC/PI staining, and comet assay. Effects of lapachol on topoisomerase I (TOP I) and topoisomerase II (TOP II) activities were detected by TOP I and TOP II mediated supercoiled pBR322 DNA relaxation assays and molecular docking. TOP I and TOP II expression levels in C6 cells were also determined.ResultsHigh dose lapachol showed significant inhibitory effect on the C6 glioma in Wistar rats (P < 0.05). It was showed that lapachol could inhibit proliferation, induce apoptosis and DNA damage of C6 cells in dose dependent manners. Lapachol could inhibit the activities of both TOP I and II. Lapachol-TOP I showed relatively stronger interaction than that of lapachol-TOP II in molecular docking study. Also, lapachol could inhibit TOP II expression levels, but not TOP I expression levels.ConclusionThese results showed that lapachol could significantly inhibit C6 glioma both in vivo and in vitro, which might be related with inhibiting TOP I and TOP II activities, as well as TOP II expression.

Combination of baicalein and 10-hydroxy camptothecin exerts remarkable synergetic anti-cancer effects

Background10-Hydroxy camptothecin (HCPT), a naturally occurring alkaloid, is a clinical drug for cancer chemotherapy. Baicalein (BA) is a flavonoid extracted from the root of Scutellaria baicalensis. The synergistic anti-cancer effect of BA and HCPT has not been reported. PurposeTo explore whether and how BA enhances the anti-cancer effect of HCPT in BGC823 cells. MethodsCell viability was measured by MTT assay. Apoptosis and cell cycle were analyzed through flow cytometry and western blotting analysis. DNA damage was determined by a comet assay. The activity of topoisomerase I (Topo I) was detected by the plasmid DNA relaxation assay. The synergistic anti-cancer effect of BA and HCPT in vivo was tested by BGC823 xenografted tumor model. ResultsBA at non-toxic doses prominently enhanced the anti-cancer activities of HCPT in BGC823, MCF7 and SMMC7721 cells. Combination treatment of BA and HCPT induced BGC823 cells apoptosis mainly via intrinsic rather than extrinsic pathways, and preferentially arresting cell cycle in G1 and G2 phases with the aid of p21. Of note, p53, the upstream regulator of cell apoptosis and cycle, was increased by 5 folds in combination group. It helped to further trigger DNA damage and inhibit Topo I catalytic activity after combination treatment of BA and HCPT. Moreover, the BGC823 xenografted tumor growth rate in nude mice was repressed in a greater degree (P< 0.01) in the combinational group than the single-drug group. ConclusionHCPT and BA, a new and effective combination therapy, synergistically target Topo I and up-regulate p53 to induce cell apoptosis and cell cycle arrest.

Suppression of lupus nephritis and skin lesions in MRL/lpr mice by administration of the topoisomerase I inhibitor irinotecan.

BackgroundSince the precise mechanism for the pathogenesis of systemic lupus erythematosus (SLE) is unknown, no targeted therapies in addition to immunosuppression are available so far. We recently demonstrated that administration of the topoisomerase I (topo I) inhibitor irinotecan at extremely low concentrations reversed established lupus nephritis in NZB/NZW mice. While profound immunosuppression was absent, we proposed changes in DNA relaxation and anti-double-stranded (ds)DNA antibody binding as the underlying mechanism. To exclude that these effects were restricted to NZB/NZW mice, irinotecan was used in a genetically different strain of lupus-prone mice.MethodsMRL/lpr mice were treated with high- and low-dose irinotecan beginning at 8 weeks of age. Treatment was repeated every fourth week. In vitro, DNA was relaxed by recombinant topo I, and altered anti-dsDNA antibody binding was measured by enzyme-linked immunosorbent assay.ResultsAdministration of both high- and low-dose irinotecan prevented proteinuria and prolonged survival in MRL/lpr mice. Moreover, both concentrations of irinotecan significantly improved histopathology of the skin at 18 weeks of age. While only high-dose irinotecan diminished the numbers of plasmablasts and double-negative T cells, no changes in IgG-secreting cells or anti-dsDNA IgG were observed. In vitro, relaxation of DNA by topo I increased the binding of anti-dsDNA IgG but not the binding of anti-dsDNA IgM derived from the plasma of MRL/lpr mice.ConclusionThe beneficial effects of topo I inhibition in a second, genetically different strain of lupus-prone mice strongly implicate irinotecan as a new therapeutic option for human SLE.

DNA Garden: A Simple Method for Producing Arrays of Stretchable DNA for Single-Molecule Fluorescence Imaging of DNA-Binding Proteins

Abstract We developed a simple method for producing arrays of stretchable DNAs, called DNA garden, for single-molecule fluorescence measurements. The method is based on microcontact printing of biotinylated bovine serum albumin (biotin-BSA) on a coverslip coated by 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer and on the subsequent tethering of neutravidin and biotinylated DNA. Without the need for a microfabricated substrate used for DNA tethering, it facilitates single-molecule investigations of DNA and DNA-binding proteins based on fluorescence microscopic imaging. The salient advantage of DNA garden is continuous observation of DNA in the repeated cycles of extension and relaxation by flow control, enabling the characterization of processes occurring in and on the relaxed DNA. The DNA garden was applied to the detection of cleavage sites of restriction enzymes and for the observation of the sliding dynamics of a tumor suppressor, p53, along extended DNA at the single-molecule level. Furthermore, experiments based on the repetitive extension and relaxation of DNA demonstrated that p53 causes looping of DNA, probably by connecting multiple regions of the relaxed DNA. The DNA garden is expected to be a powerful tool for the single-molecule imaging assay of DNA and DNA-binding proteins.

DNA Interaction Studies of Selected Polyamine Conjugates.

The interaction of polyamine conjugates with DNA double helix has been studied. Binding properties were examined by ethidium bromide (EtBr) displacement and DNA unwinding/topoisomerase I/II (Topo I/II) activity assays, as well as dsDNA thermal stability studies and circular dichroism spectroscopy. Genotoxicity of the compounds was estimated by a comet assay. It has been shown that only compound 2a can interact with dsDNA via an intercalative binding mode as it displaced EtBr from the dsDNA-dye complex, with Kapp = 4.26 × 106 M−1; caused an increase in melting temperature; changed the circular dichroism spectrum of dsDNA; converted relaxed plasmid DNA into a supercoiled molecule in the presence of Topo I and reduced the amount of short oligonucleotide fragments in the comet tail. Furthermore, preliminary theoretical study has shown that interaction of the discussed compounds with dsDNA depends on molecule linker length and charge distribution over terminal aromatic chromophores.

Design, synthesis and biological evaluation of lapachol derivatives possessing indole scaffolds as topoisomerase I inhibitors

A series of novel lapachol derivatives possessing indole scaffolds was designed and synthesized. The in vitro anti-proliferative activity of these novel compounds was evaluated in Eca109 and Hela cell lines. Almost all the tested compounds showed manifested potent inhibitory activity against the two tested cancer cell lines. Topo I-mediated DNA relaxation activity indicated that these novel compounds have potent Topoisomerase I inhibition activity. The most potent compounds 4n and 4k demonstrated more cytotoxicity than camptothecin and was comparable to camptothecin in inhibitory activities on Topoisomerase I in our biological assay. In addition, the Hoechst 33342 staining method also showed that the complex can induce Hela cell apoptosis.

Synthesis and biological evaluation of imidazopyridinyl-1,3,4-oxadiazole conjugates as apoptosis inducers and topoisomerase IIα inhibitors

A series of imidazopyridinyl-1,3,4-oxadiazole conjugates were synthesized and investigated for their cytotoxic activity and some compounds showed promising cytotoxic activity. Compound 8q (NSC: 763639) exhibited notable growth inhibition that satisfies threshold criteria at single dose (10μM) on all human cancer cell lines. This compound was further evaluated at five dose levels (0.01, 0.1, 1, 10 and 100μM) to obtain GI50 values ranging from 1.30 to 5.64μM. Flow cytometric analysis revealed that compound 8q arrests the A549 cells in sub G1 phase followed by induction of apoptosis which was further confirmed by Annexin-V-FITC, Hoechst nuclear staining, caspase 3 activation, measurement of mitochondrial membrane potential and ROS generation. Topo II mediated DNA relaxation assay results showed that conjugate 8q could significantly inhibit the activity of topo II. Moreover, molecular docking studies also indicated binding to the topoisomerase enzyme (PDBID 1ZXN).

DNA gyrase with a single catalytic tyrosine can catalyze DNA supercoiling by a nicking-closing mechanism.

The topological state of DNA is important for replication, recombination and transcription, and is regulated in vivo by DNA topoisomerases. Gyrase introduces negative supercoils into DNA at the expense of ATP hydrolysis. It is the accepted view that gyrase achieves supercoiling by a strand passage mechanism, in which double-stranded DNA is cleaved, and a second double-stranded segment is passed through the gap, converting a positive DNA node into a negative node. We show here that gyrase with only one catalytic tyrosine that cleaves a single strand of its DNA substrate can catalyze DNA supercoiling without strand passage. We propose an alternative mechanism for DNA supercoiling via nicking and closing of DNA that involves trapping, segregation and relaxation of two positive supercoils. In contrast to DNA supercoiling, ATP-dependent relaxation and decatenation of DNA by gyrase lacking the C-terminal domains require both tyrosines and strand passage. Our results point towards mechanistic plasticity of gyrase and might pave the way for finding novel and specific mechanism-based gyrase inhibitors.

The Coordinated Positive Regulation of Topoisomerase Genes Maintains Topological Homeostasis in Streptomyces coelicolor.

ABSTRACTMaintaining an optimal level of chromosomal supercoiling is critical for the progression of DNA replication and transcription. Moreover, changes in global supercoiling affect the expression of a large number of genes and play a fundamental role in adapting to stress. Topoisomerase I (TopA) and gyrase are key players in the regulation of bacterial chromosomal topology through their respective abilities to relax and compact DNA. Soil bacteria such as Streptomyces species, which grow as branched, multigenomic hyphae, are subject to environmental stresses that are associated with changes in chromosomal topology. The topological fluctuations modulate the transcriptional activity of a large number of genes and in Streptomyces are related to the production of antibiotics. To better understand the regulation of topological homeostasis in Streptomyces coelicolor, we investigated the interplay between the activities of the topoisomerase-encoding genes topA and gyrBA. We show that the expression of both genes is supercoiling sensitive. Remarkably, increased chromosomal supercoiling induces the topA promoter but only slightly influences gyrBA transcription, while DNA relaxation affects the topA promoter only marginally but strongly activates the gyrBA operon. Moreover, we showed that exposure to elevated temperatures induces rapid relaxation, which results in changes in the levels of both topoisomerases. We therefore propose a unique mechanism of S. coelicolor chromosomal topology maintenance based on the supercoiling-dependent stimulation, rather than repression, of the transcription of both topoisomerase genes. These findings provide important insight into the maintenance of topological homeostasis in an industrially important antibiotic producer.IMPORTANCE We describe the unique regulation of genes encoding two topoisomerases, topoisomerase I (TopA) and gyrase, in a model Streptomyces species. Our studies demonstrate the coordination of topoisomerase gene regulation, which is crucial for maintenance of topological homeostasis. Streptomyces species are producers of a plethora of biologically active secondary metabolites, including antibiotics, antitumor agents, and immunosuppressants. The significant regulatory factor controlling the secondary metabolism is the global chromosomal topology. Thus, the investigation of chromosomal topology homeostasis in Streptomyces strains is crucial for their use in industrial applications as producers of secondary metabolites.

Importin β Can Bind Hepatitis B Virus Core Protein and Empty Core-Like Particles and Induce Structural Changes.

Hepatitis B virus (HBV) capsids are found in many forms: immature single-stranded RNA-filled cores, single-stranded DNA-filled replication intermediates, mature cores with relaxed circular double-stranded DNA, and empty capsids. A capsid, the protein shell of the core, is a complex of 240 copies of core protein. Mature cores are transported to the nucleus by a complex that includes both importin α and importin β (Impα and Impβ), which bind to the core protein’s C-terminal domains (CTDs). Here we have investigated the interactions of HBV core protein with importins in vitro. Strikingly, empty capsids and free core protein can bind Impβ without Impα. Cryo-EM image reconstructions show that the CTDs, which are located inside the capsid, can extrude through the capsid to be bound by Impβ. Impβ density localized on the capsid exterior near the quasi-sixfold vertices, suggested a maximum of 30 Impβ per capsid. However, examination of complexes using single molecule charge-detection mass spectrometry indicate that some complexes include over 90 Impβ molecules. Cryo-EM of capsids incubated with excess Impβ shows a population of damaged particles and a population of “dark” particles with internal density, suggesting that Impβ is effectively swallowed by the capsids, which implies that the capsids transiently open and close and can be destabilized by Impβ. Though the in vitro complexes with great excess of Impβ are not biological, these results have implications for trafficking of empty capsids and free core protein; activities that affect the basis of chronic HBV infection.