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El Houdaigui, Bilal, Raphaël Forquet, Thomas Hindré, Dominique Schneider, William Nasser, Sylvie Reverchon i Sam Meyer. "Bacterial genome architecture shapes global transcriptional regulation by DNA supercoiling". Nucleic Acids Research 47, nr 11 (24.04.2019): 5648–57. http://dx.doi.org/10.1093/nar/gkz300.
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Abstract DNA supercoiling acts as a global transcriptional regulator in bacteria, that plays an important role in adapting their expression programme to environmental changes, but for which no quantitative or even qualitative regulatory model is available. Here, we focus on spatial supercoiling heterogeneities caused by the transcription process itself, which strongly contribute to this regulation mode. We propose a new mechanistic modeling of the transcription-supercoiling dynamical coupling along a genome, which allows simulating and quantitatively reproducing in vitro and in vivo transcription assays, and highlights the role of genes’ local orientation in their supercoiling sensitivity. Consistently with predictions, we show that chromosomal relaxation artificially induced by gyrase inhibitors selectively activates convergent genes in several enterobacteria, while conversely, an increase in DNA supercoiling naturally selected in a long-term evolution experiment with Escherichia coli favours divergent genes. Simulations show that these global expression responses to changes in DNA supercoiling result from fundamental mechanical constraints imposed by transcription, independently from more specific regulation of each promoter. These constraints underpin a significant and predictable contribution to the complex rules by which bacteria use DNA supercoiling as a global but fine-tuned transcriptional regulator.
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Valenti, Anna, Giuseppe Perugino, Mosè Rossi i Maria Ciaramella. "Positive supercoiling in thermophiles and mesophiles: of the good and evil". Biochemical Society Transactions 39, nr 1 (19.01.2011): 58–63. http://dx.doi.org/10.1042/bst0390058.
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DNA supercoiling plays essential role in maintaining proper chromosome structure, as well as the equilibrium between genome dynamics and stability under specific physicochemical and physiological conditions. In mesophilic organisms, DNA is negatively supercoiled and, until recently, positive supercoiling was considered a peculiar mark of (hyper)thermophilic archaea needed to survive high temperatures. However, several lines of evidence suggest that negative and positive supercoiling might coexist in both (hyper)thermophilic and mesophilic organisms, raising the possibility that positive supercoiling might serve as a regulator of various cellular events, such as chromosome condensation, gene expression, mitosis, sister chromatid cohesion, centromere identity and telomere homoeostasis.
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Geng, Yuncong, Christopher Herrick Bohrer, Nicolás Yehya, Hunter Hendrix, Lior Shachaf, Jian Liu, Jie Xiao i Elijah Roberts. "A spatially resolved stochastic model reveals the role of supercoiling in transcription regulation". PLOS Computational Biology 18, nr 9 (19.09.2022): e1009788. http://dx.doi.org/10.1371/journal.pcbi.1009788.
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In Escherichia coli, translocation of RNA polymerase (RNAP) during transcription introduces supercoiling to DNA, which influences the initiation and elongation behaviors of RNAP. To quantify the role of supercoiling in transcription regulation, we developed a spatially resolved supercoiling model of transcription. The integrated model describes how RNAP activity feeds back with the local DNA supercoiling and how this mechanochemical feedback controls transcription, subject to topoisomerase activities and stochastic topological domain formation. This model establishes that transcription-induced supercoiling mediates the cooperation of co-transcribing RNAP molecules in highly expressed genes, and this cooperation is achieved under moderate supercoiling diffusion and high topoisomerase unbinding rates. It predicts that a topological domain could serve as a transcription regulator, generating substantial transcriptional noise. It also shows the relative orientation of two closely arranged genes plays an important role in regulating their transcription. The model provides a quantitative platform for investigating how genome organization impacts transcription.
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Ahmed, Syed Moiz, i Peter Dröge. "Chromatin Architectural Factors as Safeguards against Excessive Supercoiling during DNA Replication". International Journal of Molecular Sciences 21, nr 12 (24.06.2020): 4504. http://dx.doi.org/10.3390/ijms21124504.
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Key DNA transactions, such as genome replication and transcription, rely on the speedy translocation of specialized protein complexes along a double-stranded, right-handed helical template. Physical tethering of these molecular machines during translocation, in conjunction with their internal architectural features, generates DNA topological strain in the form of template supercoiling. It is known that the build-up of transient excessive supercoiling poses severe threats to genome function and stability and that highly specialized enzymes—the topoisomerases (TOP)—have evolved to mitigate these threats. Furthermore, due to their intracellular abundance and fast supercoil relaxation rates, it is generally assumed that these enzymes are sufficient in coping with genome-wide bursts of excessive supercoiling. However, the recent discoveries of chromatin architectural factors that play important accessory functions have cast reasonable doubts on this concept. Here, we reviewed the background of these new findings and described emerging models of how these accessory factors contribute to supercoil homeostasis. We focused on DNA replication and the generation of positive (+) supercoiling in front of replisomes, where two accessory factors—GapR and HMGA2—from pro- and eukaryotic cells, respectively, appear to play important roles as sinks for excessive (+) supercoiling by employing a combination of supercoil constrainment and activation of topoisomerases. Looking forward, we expect that additional factors will be identified in the future as part of an expanding cellular repertoire to cope with bursts of topological strain. Furthermore, identifying antagonists that target these accessory factors and work synergistically with clinically relevant topoisomerase inhibitors could become an interesting novel strategy, leading to improved treatment outcomes.
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Salvador, Maria L., Uwe Klein i Lawrence Bogorad. "Endogenous Fluctuations of DNA Topology in the Chloroplast of Chlamydomonas reinhardtii". Molecular and Cellular Biology 18, nr 12 (1.12.1998): 7235–42. http://dx.doi.org/10.1128/mcb.18.12.7235.
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ABSTRACT DNA supercoiling in the chloroplast of the unicellular green algaChlamydomonas reinhardtii was found to change with a diurnal rhythm in cells growing in alternating 12-h dark–12-h light periods. Highest and lowest DNA superhelicities occurred at the beginning and towards the end of the 12-h light periods, respectively. The fluctuations in DNA supercoiling occurred concurrently and in the same direction in two separate parts of the chloroplast genome, one containing the genes psaB, rbcL, andatpA and the other containing the atpB gene. Fluctuations were not confined to transcribed DNA regions, indicating simultaneous changes in DNA conformation all over the chloroplast genome. Because the diurnal fluctuations persisted in cells kept in continuous light, DNA supercoiling is judged to be under endogenous control. The endogenous fluctuations in chloroplast DNA topology correlated tightly with the endogenous fluctuations of overall chloroplast gene transcription and with those of the pool sizes of most chloroplast transcripts analyzed. This result suggests that DNA superhelical changes have a role in the regulation of chloroplast gene expression in Chlamydomonas.
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Neguembor, Maria Victoria, Laura Martin, Álvaro Castells-García, Pablo Aurelio Gómez-García, Chiara Vicario, Davide Carnevali, Jumana AlHaj Abed i in. "Transcription-mediated supercoiling regulates genome folding and loop formation". Molecular Cell 81, nr 15 (sierpień 2021): 3065–81. http://dx.doi.org/10.1016/j.molcel.2021.06.009.
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Gilbert, Nick. "Organisation and Function of DNA Supercoiling in the Human Genome". Biophysical Journal 114, nr 3 (luty 2018): 13a. http://dx.doi.org/10.1016/j.bpj.2017.11.113.
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Alcorlo, Martín, Margarita Salas i José M. Hermoso. "In Vivo DNA Binding of Bacteriophage GA-1 Protein p6". Journal of Bacteriology 189, nr 22 (14.09.2007): 8024–33. http://dx.doi.org/10.1128/jb.01047-07.
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ABSTRACT Bacteriophage GA-1 infects Bacillus sp. strain G1R and has a linear double-stranded DNA genome with a terminal protein covalently linked to its 5′ ends. GA-1 protein p6 is very abundant in infected cells and binds DNA with no sequence specificity. We show here that it binds in vivo to the whole viral genome, as detected by cross-linking, chromatin immunoprecipitation, and real-time PCR analyses, and has the characteristics of a histone-like protein. Binding to DNA of GA-1 protein p6 shows little supercoiling dependency, in contrast to the ortholog protein of the evolutionary related Bacillus subtilis phage φ29. This feature is a property of the protein rather than the DNA or the cellular background, since φ29 protein p6 shows supercoiling-dependent binding to GA-1 DNA in Bacillus sp. strain G1R. GA-1 DNA replication is impaired in the presence of the gyrase inhibitors novobiocin and nalidixic acid, which indicates that, although noncovalently closed, the viral genome is topologically constrained in vivo. GA-1 protein p6 is also able to bind φ29 DNA in B. subtilis cells; however, as expected, the binding is less supercoiling dependent than the one observed with the φ29 protein p6. In addition, the nucleoprotein complex formed is not functional, since it is not able to transcomplement the DNA replication deficiency of a φ29 sus6 mutant. Furthermore, we took advantage of φ29 protein p6 binding to GA-1 DNA to find that the viral DNA ejection mechanism seems to take place, as in the case of φ29, with a right to left polarity in a two-step, push-pull process.
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Adkins, Melissa W., i Jessica K. Tyler. "The Histone Chaperone Asf1p Mediates Global Chromatin Disassemblyin Vivo". Journal of Biological Chemistry 279, nr 50 (26.09.2004): 52069–74. http://dx.doi.org/10.1074/jbc.m406113200.
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The packaging of the eukaryotic genome into chromatin is likely to be mediated by chromatin assembly factors, including histone chaperones. We investigated the function of the histone H3/H4 chaperones anti-silencing function 1 (Asf1p) and chromatin assembly factor 1 (CAF-1)in vivo. Analysis of chromatin structure by accessibility to micrococcal nuclease and DNase I digestion demonstrated that the chromatin from CAF-1 mutant yeast has increased accessibility to these enzymes. In agreement, the supercoiling of the endogenous 2μ plasmid is reduced in yeast lacking CAF-1. These results indicate that CAF-1 mutant yeast globally under-assemble their genome into chromatin, consistent with a role for CAF-1 in chromatin assemblyin vivo. By contrast,asf1mutants globally over-assemble their genome into chromatin, as suggested by decreased accessibility of their chromatin to micrococcal nuclease and DNase I digestion and increased supercoiling of the endogenous 2μ plasmid. Deletion ofASF1causes a striking loss of acetylation on histone H3 lysine 9, but this is not responsible for the altered chromatin structure inasf1mutants. These data indicate that Asf1p may have a global role in chromatin disassembly and an unexpected role in histone acetylationin vivo.
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Blot, Nicolas, Ramesh Mavathur, Marcel Geertz, Andrew Travers i Georgi Muskhelishvili. "Homeostatic regulation of supercoiling sensitivity coordinates transcription of the bacterial genome". EMBO reports 7, nr 7 (16.06.2006): 710–15. http://dx.doi.org/10.1038/sj.embor.7400729.
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Ivanov, Ivan E., Addison V. Wright, Joshua C. Cofsky, Kevin D. Palacio Aris, Jennifer A. Doudna i Zev Bryant. "Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling". Proceedings of the National Academy of Sciences 117, nr 11 (2.03.2020): 5853–60. http://dx.doi.org/10.1073/pnas.1913445117.
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The CRISPR-Cas9 nuclease has been widely repurposed as a molecular and cell biology tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its associated guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mechanical description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-molecule technique that can simultaneously monitor DNA unwinding with base-pair resolution and binding of fluorescently labeled macromolecules in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mechanical perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage. Consistent with this energy landscape model, in bulk experiments we observe promiscuous cleavage under physiological negative supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling.
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El Houdaigui, Bilal, i Sam Meyer. "TwisTranscripT: stochastic simulation of the transcription-supercoiling coupling". Bioinformatics 36, nr 12 (31.03.2020): 3899–901. http://dx.doi.org/10.1093/bioinformatics/btaa221.
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Abstract Summary Transcription and DNA supercoiling are involved in a complex, dynamical and non-linear coupling that results from the basal interaction between DNA and RNA polymerase. We present the first software to simulate this coupling, applicable to a wide range of bacterial organisms. TwisTranscripT allows quantifying its contribution in global transcriptional regulation, and provides a mechanistic basis for the widely observed, evolutionarily conserved and currently unexplained co-regulation of adjacent operons that might play an important role in genome evolution. Availability and implementation TwisTranscripT is freely available at https://github.com/sammeyer2017/TwisTranscripT. It is implemented in Python3 and supported on MacOS X, Linux and Windows.
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Laghi, Luigi, Ann E. Randolph, Alberto Malesci i C. Richard Boland. "Constraints imposed by supercoiling on in vitro amplification of polyomavirus DNA". Journal of General Virology 85, nr 11 (1.11.2004): 3383–88. http://dx.doi.org/10.1099/vir.0.80039-0.
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Previous attempts to identify oncogenic polyomaviruses in human cancers have yielded conflicting results, even with the application of PCR technology. Here, it was considered whether the topological features of the polyomavirus genome interfere with efficient PCR amplification. Plasmid and SV40 DNAs were used as a model system for comparing the amplification efficiency of supercoiled, circular relaxed and linear templates. It was found that detection of circular templates required 10 times more molecules than detection of identical but linear templates. Supercoiling hindered the in vitro amplification of SV40 circles by a factor of 10, and erratic amplification of supercoiled SV40 occurred with subpicogram amounts of template. Accordingly, topoisomerase I treatment of DNA improved the PCR detection of supercoiled SV40, significantly decreasing the number of false-negative samples. Previously described, yet controversial, polyomavirus presence in human tissues should be reconsidered and topoisomerase I-sensitive polyomavirus amplification might help to detect polyomavirus genomes in mammalian tissues.
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Marinov, Georgi K., Alexandro E. Trevino, Tingting Xiang, Anshul Kundaje, Arthur R. Grossman i William J. Greenleaf. "Transcription-dependent domain-scale three-dimensional genome organization in the dinoflagellate Breviolum minutum". Nature Genetics 53, nr 5 (29.04.2021): 613–17. http://dx.doi.org/10.1038/s41588-021-00848-5.
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AbstractDinoflagellate chromosomes represent a unique evolutionary experiment, as they exist in a permanently condensed, liquid crystalline state; are not packaged by histones; and contain genes organized into tandem gene arrays, with minimal transcriptional regulation. We analyze the three-dimensional genome of Breviolum minutum, and find large topological domains (dinoflagellate topologically associating domains, which we term ‘dinoTADs’) without chromatin loops, which are demarcated by convergent gene array boundaries. Transcriptional inhibition disrupts dinoTADs, implicating transcription-induced supercoiling as the primary topological force in dinoflagellates.
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Kantidze, Omar L., i Sergey V. Razin. "Weak interactions in higher-order chromatin organization". Nucleic Acids Research 48, nr 9 (20.04.2020): 4614–26. http://dx.doi.org/10.1093/nar/gkaa261.
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Abstract The detailed principles of the hierarchical folding of eukaryotic chromosomes have been revealed during the last two decades. Along with structures composing three-dimensional (3D) genome organization (chromatin compartments, topologically associating domains, chromatin loops, etc.), the molecular mechanisms that are involved in their establishment and maintenance have been characterized. Generally, protein–protein and protein–DNA interactions underlie the spatial genome organization in eukaryotes. However, it is becoming increasingly evident that weak interactions, which exist in biological systems, also contribute to the 3D genome. Here, we provide a snapshot of our current understanding of the role of the weak interactions in the establishment and maintenance of the 3D genome organization. We discuss how weak biological forces, such as entropic forces operating in crowded solutions, electrostatic interactions of the biomolecules, liquid-liquid phase separation, DNA supercoiling, and RNA environment participate in chromosome segregation into structural and functional units and drive intranuclear functional compartmentalization.
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St Germain, Commodore, Hongchang Zhao i Jacqueline H. Barlow. "Transcription-Replication Collisions—A Series of Unfortunate Events". Biomolecules 11, nr 8 (21.08.2021): 1249. http://dx.doi.org/10.3390/biom11081249.
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Transcription-replication interactions occur when DNA replication encounters genomic regions undergoing transcription. Both replication and transcription are essential for life and use the same DNA template making conflicts unavoidable. R-loops, DNA supercoiling, DNA secondary structure, and chromatin-binding proteins are all potential obstacles for processive replication or transcription and pose an even more potent threat to genome integrity when these processes co-occur. It is critical to maintaining high fidelity and processivity of transcription and replication while navigating through a complex chromatin environment, highlighting the importance of defining cellular pathways regulating transcription-replication interaction formation, evasion, and resolution. Here we discuss how transcription influences replication fork stability, and the safeguards that have evolved to navigate transcription-replication interactions and maintain genome integrity in mammalian cells.
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Ferrándiz, María-José, Pablo Hernández i Adela G. de la Campa. "Genome-wide proximity between RNA polymerase and DNA topoisomerase I supports transcription in Streptococcus pneumoniae". PLOS Genetics 17, nr 4 (30.04.2021): e1009542. http://dx.doi.org/10.1371/journal.pgen.1009542.
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Streptococcus pneumoniae is a major cause of disease and death that develops resistance to multiple antibiotics. DNA topoisomerase I (TopoI) is a novel pneumococcal drug target. TopoI is the sole type-I pneumococcal topoisomerase that regulates supercoiling homeostasis in this bacterium. In this study, a direct in vitro interaction between TopoI and RNA polymerase (RNAP) was detected by surface plasmon resonance. To understand the interplay between transcription and supercoiling regulation in vivo, genome-wide association of RNAP and TopoI was studied by ChIP-Seq. RNAP and TopoI were enriched at the promoters of 435 and 356 genes, respectively. Higher levels of expression were consistently measured in those genes whose promoters recruit both RNAP and TopoI, in contrast with those enriched in only one of them. Both enzymes occupied a narrow region close to the ATG codon. In addition, RNAP displayed a regular distribution throughout the coding regions. Likewise, the summits of peaks called with MACS tool, mapped around the ATG codon in both cases. However, RNAP showed a broader distribution towards ATG-downstream positions. Remarkably, inhibition of RNAP with rifampicin prevented the localization of TopoI at promoters and, vice versa, inhibition of TopoI with seconeolitsine prevented the binding of RNAP to promoters. This indicates a functional interplay between RNAP and TopoI. To determine the molecular factors responsible for RNAP and TopoI co-recruitment, we looked for DNA sequence motifs. We identified a motif corresponding to a -10-extended promoter for TopoI and for RNAP. Furthermore, RNAP was preferentially recruited to genes co-directionally oriented with replication, while TopoI was more abundant in head-on genes. TopoI was located in the intergenic regions of divergent genes pairs, near the promoter of the head-on gene of the pair. These results suggest a role for TopoI in the formation/stability of the RNAP-DNA complex at the promoter and during transcript elongation.
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Ross, Margery A., i Peter Setlow. "The Bacillus subtilis HBsu Protein Modifies the Effects of α/β-Type, Small Acid-Soluble Spore Proteins on DNA". Journal of Bacteriology 182, nr 7 (1.04.2000): 1942–48. http://dx.doi.org/10.1128/jb.182.7.1942-1948.2000.
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ABSTRACT HBsu, the Bacillus subtilis homolog of theEscherichia coli HU proteins and the major chromosomal protein in vegetative cells of B. subtilis, is present at similar levels in vegetative cells and spores (∼5 × 104 monomers/genome). The level of HBsu in spores was unaffected by the presence or absence of the α/β-type, small acid-soluble proteins (SASP), which are the major chromosomal proteins in spores. In developing forespores, HBsu colocalized with α/β-type SASP on the nucleoid, suggesting that HBsu could modulate α/β-type SASP-mediated properties of spore DNA. Indeed, in vitro studies showed that HBsu altered α/β-type SASP protection of pUC19 from DNase digestion, induced negative DNA supercoiling opposing α/β-type SASP-mediated positive supercoiling, and greatly ameliorated the α/β-type SASP-mediated increase in DNA persistence length. However, HBsu did not significantly interfere with the α/β-type SASP-mediated changes in the UV photochemistry of DNA that explain the heightened resistance of spores to UV radiation. These data strongly support a role for HBsu in modulating the effects of α/β-type SASP on the properties of DNA in the developing and dormant spore.
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Oram, Mark, i Martin L. Pato. "Mu-Like Prophage Strong Gyrase Site Sequences: Analysis of Properties Required for Promoting Efficient Mu DNA Replication". Journal of Bacteriology 186, nr 14 (15.07.2004): 4575–84. http://dx.doi.org/10.1128/jb.186.14.4575-4584.2004.
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ABSTRACT The bacteriophage Mu genome contains a centrally located strong gyrase site (SGS) that is required for efficient prophage replication. To aid in studying the unusual properties of the SGS, we sought other gyrase sites that might be able to substitute for the SGS in Mu replication. Five candidate sites were obtained by PCR from Mu-like prophage sequences present in Escherichia coli O157:H7 Sakai, Haemophilus influenzae Rd, Salmonella enterica serovar Typhi CT18, and two strains of Neisseria meningitidis. Each of the sites was used to replace the natural Mu SGS to form recombinant prophages, and the effects on Mu replication and host lysis were determined. The site from the E. coli prophage supported markedly enhanced replication and host lysis over that observed with a Mu derivative lacking the SGS, those from the N. meningitidis prophages allowed a small enhancement, and the sites from the Haemophilus and Salmonella prophages gave none. Each of the candidate sites was cleaved specifically by E. coli DNA gyrase both in vitro and in vivo. Supercoiling assays performed in vitro, with the five sites or the Mu SGS individually cloned into a pUC19 reporter plasmid, showed that the Mu SGS and the E. coli or N. meningitidis sequences allowed an enhancement of processive, gyrase-dependent supercoiling, whereas the H. influenzae or Salmonella serovar Typhi sequences did not. While consistent with a requirement for enhanced processivity of supercoiling for a site to function in Mu replication, these data suggest that other factors are also important. The relevance of these observations to an understanding of the function of the SGS is discussed.
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Aubry, Alexandra, Xiao-Su Pan, L. Mark Fisher, Vincent Jarlier i Emmanuelle Cambau. "Mycobacterium tuberculosis DNA Gyrase: Interaction with Quinolones and Correlation with Antimycobacterial Drug Activity". Antimicrobial Agents and Chemotherapy 48, nr 4 (kwiecień 2004): 1281–88. http://dx.doi.org/10.1128/aac.48.4.1281-1288.2004.
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ABSTRACT Genome studies suggest that DNA gyrase is the sole type II topoisomerase and likely the unique target of quinolones in Mycobacterium tuberculosis. Despite the emerging importance of quinolones in the treatment of mycobacterial disease, the slow growth and high pathogenicity of M. tuberculosis have precluded direct purification of its gyrase and detailed analysis of quinolone action. To address these issues, we separately overexpressed the M. tuberculosis DNA gyrase GyrA and GyrB subunits as His-tagged proteins in Escherichia coli from pET plasmids carrying gyrA and gyrB genes. The soluble 97-kDa GyrA and 72-kDa GyrB subunits were purified by nickel chelate chromatography and shown to reconstitute an ATP-dependent DNA supercoiling activity. The drug concentration that inhibited DNA supercoiling by 50% (IC50) was measured for 22 different quinolones, and values ranged from 2 to 3 μg/ml (sparfloxacin, sitafloxacin, clinafloxacin, and gatifloxacin) to >1,000 μg/ml (pipemidic acid and nalidixic acid). By comparison, MICs measured against M. tuberculosis ranged from 0.12 μg/ml (for gatifloxacin) to 128 μg/ml (both pipemidic acid and nalidixic acid) and correlated well with the gyrase IC50s (R 2 = 0.9). Quinolones promoted gyrase-mediated cleavage of plasmid pBR322 DNA due to stabilization of the cleavage complex, which is thought to be the lethal lesion. Surprisingly, the measured concentrations of drug inducing 50% plasmid linearization correlated less well with the MICs (R 2 = 0.7). These findings suggest that the DNA supercoiling inhibition assay may be a useful screening test in identifying quinolones with promising activity against M. tuberculosis. The quinolone structure-activity relationship demonstrated here shows that C-8, the C-7 ring, the C-6 fluorine, and the N-1 cyclopropyl substituents are desirable structural features in targeting M. tuberculosis gyrase.
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Cao, Nan, Kemin Tan, Xiaobing Zuo, Thirunavukkarasu Annamalai i Yuk-Ching Tse-Dinh. "Mechanistic insights from structure of Mycobacterium smegmatis topoisomerase I with ssDNA bound to both N- and C-terminal domains". Nucleic Acids Research 48, nr 8 (30.03.2020): 4448–62. http://dx.doi.org/10.1093/nar/gkaa201.
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Abstract Type IA topoisomerases interact with G-strand and T-strand ssDNA to regulate DNA topology. However, simultaneous binding of two ssDNA segments to a type IA topoisomerase has not been observed previously. We report here the crystal structure of a type IA topoisomerase with ssDNA segments bound in opposite polarity to the N- and C-terminal domains. Titration of small ssDNA oligonucleotides to Mycobacterium smegmatis topoisomerase I with progressive C-terminal deletions showed that the C-terminal region has higher affinity for ssDNA than the N-terminal active site. This allows the C-terminal domains to capture one strand of underwound negatively supercoiled DNA substrate first and position the N-terminal domains to bind and cleave the opposite strand in the relaxation reaction. Efficiency of negative supercoiling relaxation increases with the number of domains that bind ssDNA primarily with conserved aromatic residues and possibly with assistance from polar/basic residues. A comparison of bacterial topoisomerase I structures showed that a conserved transesterification unit (N-terminal toroid structure) for cutting and rejoining of a ssDNA strand can be combined with two different types of C-terminal ssDNA binding domains to form diverse bacterial topoisomerase I enzymes that are highly efficient in their physiological role of preventing excess negative supercoiling in the genome.
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Bhandari, Nirajan, Christine Rourke, Thomas Wilmoth, Alekya Bheemreddy, David Schulman, Dina Collins, Harold E. Smith, Andy Golden i Aimee Jaramillo-Lambert. "Identification of Suppressors of top-2 Embryonic Lethality in Caenorhabditis elegans". G3: Genes|Genomes|Genetics 10, nr 4 (21.02.2020): 1183–91. http://dx.doi.org/10.1534/g3.119.400927.
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Topoisomerase II is an enzyme with important roles in chromosome biology. This enzyme relieves supercoiling and DNA and RNA entanglements generated during mitosis. Recent studies have demonstrated that Topoisomerase II is also involved in the segregation of homologous chromosomes during the first meiotic division. However, the function and regulation of Topoisomerase II in meiosis has not been fully elucidated. Here, we conducted a genetic suppressor screen in Caenorhabditis elegans to identify putative genes that interact with topoisomerase II during meiosis. Using a temperature-sensitive allele of topoisomerase II, top-2(it7ts), we identified eleven suppressors of top-2-induced embryonic lethality. We used whole-genome sequencing and a combination of RNAi and CRISPR/Cas9 genome editing to identify and validate the responsible suppressor mutations. We found both recessive and dominant suppressing mutations that include one intragenic and 10 extragenic loci. The extragenic suppressors consist of a known Topoisomerase II-interacting protein and two novel interactors. We anticipate that further analysis of these suppressing mutations will provide new insights into the function of Topoisomerase II during meiosis.
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van Loenhout, M. T. J., M. V. de Grunt i C. Dekker. "Dynamics of DNA Supercoils". Science 338, nr 6103 (13.09.2012): 94–97. http://dx.doi.org/10.1126/science.1225810.
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DNA in cells exhibits a supercoiled state in which the double helix is additionally twisted to form extended intertwined loops called plectonemes. Although supercoiling is vital to many cellular processes, its dynamics remain elusive. In this work, we directly visualize the dynamics of individual plectonemes. We observe that multiple plectonemes can be present and that their number depends on applied stretching force and ionic strength. Plectonemes moved along DNA by diffusion or, unexpectedly, by a fast hopping process that facilitated very rapid (<20 milliseconds) long-range plectoneme displacement by nucleating a new plectoneme at a distant position. These observations directly reveal the dynamics of plectonemes and identify a mode of movement that allows long-distance reorganization of the conformation of the genome on a millisecond time scale.
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Dorman, Charles J. "Regulation of transcription by DNA supercoiling in Mycoplasma genitalium: global control in the smallest known self-replicating genome". Molecular Microbiology 81, nr 2 (16.06.2011): 302–4. http://dx.doi.org/10.1111/j.1365-2958.2011.07718.x.
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Gonzalez-Huici, V. "Genome wide, supercoiling-dependent in vivo binding of a viral protein involved in DNA replication and transcriptional control". Nucleic Acids Research 32, nr 8 (28.04.2004): 2306–14. http://dx.doi.org/10.1093/nar/gkh565.
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Menger, Katja E., Alejandro Rodríguez-Luis, James Chapman i Thomas J. Nicholls. "Controlling the topology of mammalian mitochondrial DNA". Open Biology 11, nr 9 (wrzesień 2021): 210168. http://dx.doi.org/10.1098/rsob.210168.
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The genome of mitochondria, called mtDNA, is a small circular DNA molecule present at thousands of copies per human cell. MtDNA is packaged into nucleoprotein complexes called nucleoids, and the density of mtDNA packaging affects mitochondrial gene expression. Genetic processes such as transcription, DNA replication and DNA packaging alter DNA topology, and these topological problems are solved by a family of enzymes called topoisomerases. Within mitochondria, topoisomerases are involved firstly in the regulation of mtDNA supercoiling and secondly in disentangling interlinked mtDNA molecules following mtDNA replication. The loss of mitochondrial topoisomerase activity leads to defects in mitochondrial function, and variants in the dual-localized type IA topoisomerase TOP3A have also been reported to cause human mitochondrial disease. We review the current knowledge on processes that alter mtDNA topology, how mtDNA topology is modulated by the action of topoisomerases, and the consequences of altered mtDNA topology for mitochondrial function and human health.
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Head, Nathan E., i Hongwei Yu. "Cross-Sectional Analysis of Clinical and Environmental Isolates of Pseudomonas aeruginosa: Biofilm Formation, Virulence, and Genome Diversity". Infection and Immunity 72, nr 1 (styczeń 2004): 133–44. http://dx.doi.org/10.1128/iai.72.1.133-144.2004.
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ABSTRACT Chronic lung infections with Pseudomonas aeruginosa biofilms are associated with refractory and fatal pneumonia in cystic fibrosis (CF). In this study, a group of genomically diverse P. aeruginosa isolates were compared with the reference strain PAO1 to assess the roles of motility, twitching, growth rate, and overproduction of a capsular polysaccharide (alginate) in biofilm formation. In an in vitro biofilm assay system, P. aeruginosa displayed strain-specific biofilm formation that was not solely dependent on these parameters. Compared with non-CF isolates, CF isolates expressed two opposing growth modes: reduced planktonic growth versus efficient biofilm formation. Planktonic cells of CF isolates showed elevated sensitivity to hydrogen peroxide, a reactive oxygen intermediate, and decreased lung colonization in an aerosol infection mouse model. Despite having identical genomic profiles, CF sequential isolates produced different amounts of biofilm. While P. aeruginosa isolates exhibited genomic diversity, the genome size of these isolates was estimated to be 0.4 to 19% (27 to 1,184 kb) larger than that of PAO1. To identify these extra genetic materials, random amplification of polymorphic DNA was coupled with PAO1-subtractive hybridization. Three loci were found within the genomes of two CF isolates encoding one novel homolog involved in retaining a Shigella virulence plasmid (mvpTA) and two divergent genes that function in removing negative supercoiling (topA) and biosynthesis of pyoverdine (PA2402). Together, P. aeruginosa biodiversity could provide one cause for the variation of morbidity and mortality in CF. P. aeruginosa may possess undefined biofilm adhesins that are important to the development of an antibiofilm therapeutic target.
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Ishii, Satoshi, Tetsuya Murakami i Kazuo Shishido. "A pSC101-parsequence-mediated study on the intracellular state of supercoiling of the pBR322 genome inEscherichia coliDNA topoisomerase I deletion mutant". FEMS Microbiology Letters 93, nr 2 (czerwiec 1992): 115–20. http://dx.doi.org/10.1111/j.1574-6968.1992.tb05076.x.
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del Val, Elsa, William Nasser, Hafid Abaibou i Sylvie Reverchon. "RecA and DNA recombination: a review of molecular mechanisms". Biochemical Society Transactions 47, nr 5 (18.10.2019): 1511–31. http://dx.doi.org/10.1042/bst20190558.
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Abstract Recombinases are responsible for homologous recombination and maintenance of genome integrity. In Escherichia coli, the recombinase RecA forms a nucleoprotein filament with the ssDNA present at a DNA break and searches for a homologous dsDNA to use as a template for break repair. During the first step of this process, the ssDNA is bound to RecA and stretched into a Watson–Crick base-paired triplet conformation. The RecA nucleoprotein filament also contains ATP and Mg2+, two cofactors required for RecA activity. Then, the complex starts a homology search by interacting with and stretching dsDNA. Thanks to supercoiling, intersegment sampling and RecA clustering, a genome-wide homology search takes place at a relevant metabolic timescale. When a region of homology 8–20 base pairs in length is found and stabilized, DNA strand exchange proceeds, forming a heteroduplex complex that is resolved through a combination of DNA synthesis, ligation and resolution. RecA activities can take place without ATP hydrolysis, but this latter activity is necessary to improve and accelerate the process. Protein flexibility and monomer–monomer interactions are fundamental for RecA activity, which functions cooperatively. A structure/function relationship analysis suggests that the recombinogenic activity can be improved and that recombinases have an inherently large recombination potential. Understanding this relationship is essential for designing RecA derivatives with enhanced activity for biotechnology applications. For example, this protein is a major actor in the recombinase polymerase isothermal amplification (RPA) used in point-of-care diagnostics.
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Aldag, Pierre, Fabian Welzel, Leonhard Jakob, Andreas Schmidbauer, Marius Rutkauskas, Fergus Fettes, Dina Grohmann i Ralf Seidel. "Probing the stability of the SpCas9–DNA complex after cleavage". Nucleic Acids Research 49, nr 21 (18.11.2021): 12411–21. http://dx.doi.org/10.1093/nar/gkab1072.
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Abstract CRISPR–Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After initial target strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability than the pre-cleavage state. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around Cas9 bound to the target strand. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress.
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Nam, Gi-Moon, i Gaurav Arya. "Torsional behavior of chromatin is modulated by rotational phasing of nucleosomes". Nucleic Acids Research 42, nr 15 (6.08.2014): 9691–99. http://dx.doi.org/10.1093/nar/gku694.
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Abstract Torsionally stressed DNA plays a critical role in genome organization and regulation. While the effects of torsional stresses on naked DNA have been well studied, little is known about how these stresses propagate within chromatin and affect its organization. Here we investigate the torsional behavior of nucleosome arrays by means of Brownian dynamics simulations of a coarse-grained model of chromatin. Our simulations reveal a strong dependence of the torsional response on the rotational phase angle Ψ0 between adjacent nucleosomes. Extreme values of Ψ0 lead to asymmetric, bell-shaped extension-rotation profiles with sharp maxima shifted toward positive or negative rotations, depending on the sign of Ψ0, and to fast, irregular propagation of DNA twist. In contrast, moderate Ψ0 yield more symmetric profiles with broad maxima and slow, uniform propagation of twist. The observed behavior is shown to arise from an interplay between nucleosomal transitions into states with crossed and open linker DNAs and global supercoiling of arrays into left- and right-handed coils, where Ψ0 serves to modulate the energy landscape of nucleosomal states. Our results also explain the torsional resilience of chromatin, reconcile differences between experimentally measured extension-rotation profiles, and suggest a role of torsional stresses in regulating chromatin assembly and organization.
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Eriksson, Peter R., Geetu Mendiratta, Neil B. McLaughlin, Tyra G. Wolfsberg, Leonardo Mariño-Ramírez, Tiffany A. Pompa, Mohendra Jainerin, David Landsman, Chang-Hui Shen i David J. Clark. "Global Regulation by the Yeast Spt10 Protein Is Mediated through Chromatin Structure and the Histone Upstream Activating Sequence Elements". Molecular and Cellular Biology 25, nr 20 (15.10.2005): 9127–37. http://dx.doi.org/10.1128/mcb.25.20.9127-9137.2005.
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ABSTRACT The yeast SPT10 gene encodes a putative histone acetyltransferase (HAT) implicated as a global transcription regulator acting through basal promoters. Here we address the mechanism of this global regulation. Although microarray analysis confirmed that Spt10p is a global regulator, Spt10p was not detected at any of the most strongly affected genes in vivo. In contrast, the presence of Spt10p at the core histone gene promoters in vivo was confirmed. Since Spt10p activates the core histone genes, a shortage of histones could occur in spt10Δ cells, resulting in defective chromatin structure and a consequent activation of basal promoters. Consistent with this hypothesis, the spt10Δ phenotype can be rescued by extra copies of the histone genes and chromatin is poorly assembled in spt10Δ cells, as shown by irregular nucleosome spacing and reduced negative supercoiling of the endogenous 2μm plasmid. Furthermore, Spt10p binds specifically and highly cooperatively to pairs of upstream activating sequence elements in the core histone promoters [consensus sequence, (G/A)TTCCN6TTCNC], consistent with a direct role in histone gene regulation. No other high-affinity sites are predicted in the yeast genome. Thus, Spt10p is a sequence-specific activator of the histone genes, possessing a DNA-binding domain fused to a likely HAT domain.
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Krassovsky, Kristina, Rajarshi P. Ghosh i Barbara J. Meyer. "Genome-wide profiling reveals functional interplay of DNA sequence composition, transcriptional activity, and nucleosome positioning in driving DNA supercoiling and helix destabilization in C. elegans". Genome Research 31, nr 7 (24.06.2021): 1187–202. http://dx.doi.org/10.1101/gr.270082.120.
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Barsoum, J., i P. Berg. "Simian virus 40 minichromosomes contain torsionally strained DNA molecules". Molecular and Cellular Biology 5, nr 11 (listopad 1985): 3048–57. http://dx.doi.org/10.1128/mcb.5.11.3048-3057.1985.
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Sundin and Varshavsky (J. Mol. Biol. 132:535-546, 1979) found that nearly two-thirds of simian virus 40 (SV40) minichromosomes obtained from nuclei of SV40-infected cells become singly nicked or cleaved across both strands after digestion with staphylococcal nuclease at 0 degrees C. The same treatment of SV40 DNA causes complete digestion rather than the limited cleavages produced in minichromosomal DNA. We have explored this novel behavior of the minichromosome and found that the nuclease sensitivity is dependent upon the topology of the DNA. Thus, if minichromosomes are pretreated with wheat germ DNA topoisomerase I, the minichromosomal DNA is completely resistant to subsequent digestion with staphylococcal nuclease at 0 degrees C. If the minichromosome-associated topoisomerase is removed, virtually all of the minichromosomes are cleaved to nicked or linear structures by the nuclease treatment. The cleavage sites are nonrandomly located; instead they occur at discrete loci throughout the SV40 genome. SV40 minichromosomal DNA is also cleaved to nicked circles and full-length linear fragments after treatment with the single strand-specific endonuclease S1; this cleavage is also inhibited by pretreatment with topoisomerase I. Thus, it may be that the nuclease sensitivity of minichromosomes is due to the transient or permanent unwinding of discrete regions of their DNA. Direct comparisons of the extent of negative supercoiling of native and topoisomerase-treated SV40 minichromosomes revealed that approximately two superhelical turns were removed by the topoisomerase treatment. The loss of these extra negative supercoils from the DNA probably accounts for the resistance of the topoisomerase-treated minichromosomes to the staphylococcal and S1 nucleases. These findings suggest that the DNA in SV40 intranuclear minichromosomes is torsionally strained. The functional significance of this finding is discussed.
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Barsoum, J., i P. Berg. "Simian virus 40 minichromosomes contain torsionally strained DNA molecules." Molecular and Cellular Biology 5, nr 11 (listopad 1985): 3048–57. http://dx.doi.org/10.1128/mcb.5.11.3048.
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Sundin and Varshavsky (J. Mol. Biol. 132:535-546, 1979) found that nearly two-thirds of simian virus 40 (SV40) minichromosomes obtained from nuclei of SV40-infected cells become singly nicked or cleaved across both strands after digestion with staphylococcal nuclease at 0 degrees C. The same treatment of SV40 DNA causes complete digestion rather than the limited cleavages produced in minichromosomal DNA. We have explored this novel behavior of the minichromosome and found that the nuclease sensitivity is dependent upon the topology of the DNA. Thus, if minichromosomes are pretreated with wheat germ DNA topoisomerase I, the minichromosomal DNA is completely resistant to subsequent digestion with staphylococcal nuclease at 0 degrees C. If the minichromosome-associated topoisomerase is removed, virtually all of the minichromosomes are cleaved to nicked or linear structures by the nuclease treatment. The cleavage sites are nonrandomly located; instead they occur at discrete loci throughout the SV40 genome. SV40 minichromosomal DNA is also cleaved to nicked circles and full-length linear fragments after treatment with the single strand-specific endonuclease S1; this cleavage is also inhibited by pretreatment with topoisomerase I. Thus, it may be that the nuclease sensitivity of minichromosomes is due to the transient or permanent unwinding of discrete regions of their DNA. Direct comparisons of the extent of negative supercoiling of native and topoisomerase-treated SV40 minichromosomes revealed that approximately two superhelical turns were removed by the topoisomerase treatment. The loss of these extra negative supercoils from the DNA probably accounts for the resistance of the topoisomerase-treated minichromosomes to the staphylococcal and S1 nucleases. These findings suggest that the DNA in SV40 intranuclear minichromosomes is torsionally strained. The functional significance of this finding is discussed.
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Brochu, Julien, Émilie Vlachos-Breton, Dina Irsenco i Marc Drolet. "Characterization of a pathway of genomic instability induced by R-loops and its regulation by topoisomerases in E. coli". PLOS Genetics 19, nr 5 (4.05.2023): e1010754. http://dx.doi.org/10.1371/journal.pgen.1010754.
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The prototype enzymes of the ubiquitous type IA topoisomerases (topos) family are Escherichia coli topo I (topA) and topo III (topB). Topo I shows preference for relaxation of negative supercoiling and topo III for decatenation. However, as they could act as backups for each other or even share functions, strains lacking both enzymes must be used to reveal the roles of type IA enzymes in genome maintenance. Recently, marker frequency analysis (MFA) of genomic DNA from topA topB null mutants revealed a major RNase HI-sensitive DNA peak bordered by Ter/Tus barriers, sites of replication fork fusion and termination in the chromosome terminus region (Ter). Here, flow cytometry for R-loop-dependent replication (RLDR), MFA, R-loop detection with S9.6 antibodies, and microscopy were used to further characterize the mechanism and consequences of over-replication in Ter. It is shown that the Ter peak is not due to the presence of a strong origin for RLDR in Ter region; instead RLDR, which is partly inhibited by the backtracking-resistant rpoB*35 mutation, appears to contribute indirectly to Ter over-replication. The data suggest that RLDR from multiple sites on the chromosome increases the number of replication forks trapped at Ter/Tus barriers which leads to RecA-dependent DNA amplification in Ter and to a chromosome segregation defect. Overproducing topo IV, the main cellular decatenase, does not inhibit RLDR or Ter over-replication but corrects the chromosome segregation defect. Furthermore, our data suggest that the inhibition of RLDR by topo I does not require its C-terminal-mediated interaction with RNA polymerase. Overall, our data reveal a pathway of genomic instability triggered by R-loops and its regulation by various topos activities at different steps.
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Corless, Samuel, i Nick Gilbert. "Investigating DNA supercoiling in eukaryotic genomes". Briefings in Functional Genomics 16, nr 6 (24.04.2017): 379–89. http://dx.doi.org/10.1093/bfgp/elx007.
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Thomas, Shelia, Francine Rezzoug i Donald Max Miller. "A Family Of G-Rich Genomic Sequences Iinteract Specifically With The Pu27 Silencer Element Of The c-Myc Promoter". Blood 122, nr 21 (15.11.2013): 1264. http://dx.doi.org/10.1182/blood.v122.21.1264.1264.
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Abstract Introduction A substantial proportion (∼80%) of c-Myc expression is regulated by the NHEIII1 element in the c-myc P1 promoter. The NHEIII1 element contains a G/C rich negative regulatory sequence (Pu27), which can form an intramolecular DNA quadruplex structure which results in c-myc silencing. Quadruplex DNA is a four-stranded structure which is quite stable and can form either “intramolecular” (one DNA strand) quadruplex, or “intermolecular” (two or four DNA strands) quadruplex. These quadruplex-forming sequences are very commonly present in the promoters of eukaryotic genes and generally are negative regulators of transcription. The equilibrium between quadruplex (transcriptionally inactive) and duplex (transcriptionally active) DNA structure is influenced by protein binding, salt concentration and supercoiling torsion (as well as other factors). Constitutive overexpression of the c-myc P0-noncoding transcript, containing the Pu27 sequence, inhibits tumorigenesis and growth of tumor cells. Oligonucleotides encoding the Pu27 sequence bind specifically to the Pu27 sequence in the c-myc promoter, inhibit c-myc expression and result in decreased cellular proliferation and cell death. This most likely reflects the formation of intermolecular quadruplex between the oligonucleotide and target sequence. In addition to the Pu27 sequence in the c-myc promoter, we have recently identified a family of 13 nearly identical Pu27 sequences in the noncoding human genome. Methods Pu27 family members were identified by a BLAT search from the UCSC Genome Browser. Circular dichroism was used to document quadruplex formation by each of the 14 Pu27 family members. Specific interaction of each Pu27 “family member” with the “parent” Pu27 sequence was documented by electrophoretic mobility shift (EMS) studies. Growth inhibition was measured for the U937, Raji, K562 and HL60 leukemic cell lines. C-Myc expression was measured by RT-PCR and Western blot analysis. Results We have identified fourteen nearly identical copies of the Pu27 sequence in the noncoding genome. They are located on Chromosomes 8 (c-Myc promoter), 1, 3 (2), 5, 7, 9, 10 (2), 11, 16, 17, 20, and X. Other than the c-Myc Pu27 sequence, these sequences do not appear to be located in promoter regions. Using gel shift analysis, we demonstrated that each of the Pu27 family member sequences binds in a sequence specific manner to the “parent” sequence in the c-myc promoter. Other genomic quadruplex-forming sequences (from the K-ras, Bcl2 and VEGF promoters) do not bind to the c-myc Pu27 promoter sequence. We have previously shown that treatment of leukemic cell lines with an oligonucleotide containing the Pu27 sequence results in downregulation of c-myc transcription and inhibition of cell proliferation with subsequent cell death. The Pu27 oligonucleotide does not affect the growth of nontransformed cell lines. Each of the Pu27 family members also inhibits the growth of all of the leukemic cell lines tested, with varying efficacy. This occurs with an IC50 as low as <200 nM in all of tumor cell lines tested. Analyzing data from the Human Cancer Genome project we have shown that one of the Pu27 family members (located on chromosome 5) is frequently deleted in leukemic cells of patients with AML (data from Human Cancer Genome Project). Conclusions This novel family of genomic sequences interacts specifically with the Pu27 c-Myc silencer sequence and down regulates c-myc expression and cellular proliferation. This suggests that they likely play a role in regulating c-Myc expression either through direct DNA-DNA interaction or sequence-specific interaction with transcripts of one or more of the Pu27 sequences. We hypothesize that DNA-DNA interactions between these sequences may also stabilize quadruplex formation by the c-myc promoter sequence, locking it in the “off” (transcriptionally inactive) position. Disruption of this interaction may play an important role in leukemia pathogenesis. Disclosures: No relevant conflicts of interest to declare.
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Herzel, Hanspeter, Olaf Weiss i Edward N. Trifonov. "Sequence Periodicity in Complete Genomes of Archaea Suggests Positive Supercoiling". Journal of Biomolecular Structure and Dynamics 16, nr 2 (październik 1998): 341–45. http://dx.doi.org/10.1080/07391102.1998.10508251.
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Yan, Yan, Wenxuan Xu, Sandip Kumar, Alexander Zhang, Fenfei Leng, David Dunlap i Laura Finzi. "Negative DNA supercoiling makes protein-mediated looping deterministic and ergodic within the bacterial doubling time". Nucleic Acids Research 49, nr 20 (1.11.2021): 11550–59. http://dx.doi.org/10.1093/nar/gkab946.
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Abstract Protein-mediated DNA looping is fundamental to gene regulation and such loops occur stochastically in purified systems. Additional proteins increase the probability of looping, but these probabilities maintain a broad distribution. For example, the probability of lac repressor-mediated looping in individual molecules ranged 0–100%, and individual molecules exhibited representative behavior only in observations lasting an hour or more. Titrating with HU protein progressively compacted the DNA without narrowing the 0–100% distribution. Increased negative supercoiling produced an ensemble of molecules in which all individual molecules more closely resembled the average. Furthermore, in only 12 min of observation, well within the doubling time of the bacterium, most molecules exhibited the looping probability of the ensemble. DNA supercoiling, an inherent feature of all genomes, appears to impose time-constrained, emergent behavior on otherwise random molecular activity.
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Champion, Keith, i N. Patrick Higgins. "Growth Rate Toxicity Phenotypes and Homeostatic Supercoil Control Differentiate Escherichia coli from Salmonella enterica Serovar Typhimurium". Journal of Bacteriology 189, nr 16 (30.03.2007): 5839–49. http://dx.doi.org/10.1128/jb.00083-07.
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ABSTRACT Escherichia coli and Salmonella enterica serovar Typhimurium share high degrees of DNA and amino acid identity for 65% of the homologous genes shared by the two genomes. Yet, there are different phenotypes for null mutants in several genes that contribute to DNA condensation and nucleoid formation. The mutant R436-S form of the GyrB protein has a temperature-sensitive phenotype in Salmonella, showing disruption of supercoiling near the terminus and replicon failure at 42°C. But this mutation in E. coli is lethal at the permissive temperature. A unifying hypothesis for why the same mutation in highly conserved homologous genes of different species leads to different physiologies focuses on homeotic supercoil control. During rapid growth in mid-log phase, E. coli generates 15% more negative supercoils in pBR322 DNA than Salmonella. Differences in compaction and torsional strain on chromosomal DNA explain a complex set of single-gene phenotypes and provide insight into how supercoiling may modulate epigenetic effects on chromosome structure and function and on prophage behavior in vivo.
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Jolivet-Gougeon, Anne, Sandrine David-Jobert, Zohreh Tamanai-Shacoori, Christian M�nard i Michel Cormier. "Osmotic Stress-Induced Genetic Rearrangements inEscherichia coli H10407 Detected by Randomly Amplified Polymorphic DNA Analysis". Applied and Environmental Microbiology 66, nr 12 (1.12.2000): 5484–87. http://dx.doi.org/10.1128/aem.66.12.5484-5487.2000.
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ABSTRACT Randomly amplified polymorphic DNA (RAPD) analysis is a DNA polymorphism assay commonly used for fingerprinting genomes. After optimizing the reaction conditions, samples of Escherichia coli H10407 DNA were assayed to determine the influence of osmotic and/or oligotrophic stress on variations in RAPD banding patterns. Genetic rearrangements or DNA topology variations could be detected as changes in agarose gel electrophoresis banding profiles. A new amplicon generated using DNA extracted from bacteria prestarved by an osmotic stress and resuscitated in rich medium was observed. Enrichment improved recovery of mutator cells and allowed them to be detected in samples, suggesting that DNA modifications, such as stress-induced alterations and supercoiling phenomena, should be taken into consideration before beginning RAPD analyses.
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Kravatskaya, G. I., Y. V. Kravatsky, V. R. Chechetkin i V. G. Tumanyan. "Coexistence of different base periodicities in prokaryotic genomes as related to DNA curvature, supercoiling, and transcription". Genomics 98, nr 3 (wrzesień 2011): 223–31. http://dx.doi.org/10.1016/j.ygeno.2011.06.006.
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Bowater, Richard P., Natália Bohálová i Václav Brázda. "Interaction of Proteins with Inverted Repeats and Cruciform Structures in Nucleic Acids". International Journal of Molecular Sciences 23, nr 11 (31.05.2022): 6171. http://dx.doi.org/10.3390/ijms23116171.
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Cruciforms occur when inverted repeat sequences in double-stranded DNA adopt intra-strand hairpins on opposing strands. Biophysical and molecular studies of these structures confirm their characterization as four-way junctions and have demonstrated that several factors influence their stability, including overall chromatin structure and DNA supercoiling. Here, we review our understanding of processes that influence the formation and stability of cruciforms in genomes, covering the range of sequences shown to have biological significance. It is challenging to accurately sequence repetitive DNA sequences, but recent advances in sequencing methods have deepened understanding about the amounts of inverted repeats in genomes from all forms of life. We highlight that, in the majority of genomes, inverted repeats are present in higher numbers than is expected from a random occurrence. It is, therefore, becoming clear that inverted repeats play important roles in regulating many aspects of DNA metabolism, including replication, gene expression, and recombination. Cruciforms are targets for many architectural and regulatory proteins, including topoisomerases, p53, Rif1, and others. Notably, some of these proteins can induce the formation of cruciform structures when they bind to DNA. Inverted repeat sequences also influence the evolution of genomes, and growing evidence highlights their significance in several human diseases, suggesting that the inverted repeat sequences and/or DNA cruciforms could be useful therapeutic targets in some cases.
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Mrázek, Jan. "Comparative Analysis of Sequence Periodicity among Prokaryotic Genomes Points to Differences in Nucleoid Structure and a Relationship to Gene Expression". Journal of Bacteriology 192, nr 14 (21.05.2010): 3763–72. http://dx.doi.org/10.1128/jb.00149-10.
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ABSTRACT Regular spacing of short runs of A or T nucleotides in DNA sequences with a period close to the helical period of the DNA double helix has been associated with intrinsic DNA bending and nucleosome positioning in eukaryotes. Analogous periodic signals were also observed in prokaryotic genomes. While the exact role of this periodicity in prokaryotes is not known, it has been proposed to facilitate the DNA packaging in the prokaryotic nucleoid and/or to promote negative or positive supercoiling. We developed a methodology for assessments of intragenomic heterogeneity of these periodic patterns and applied it in analysis of 1,025 prokaryotic chromosomes. This technique allows more detailed analysis of sequence periodicity than previous methods where sequence periodicity was assessed in an integral form across the whole chromosome. We found that most genomes have the periodic signal confined to several chromosomal segments while most of the chromosome lacks a strong sequence periodicity. Moreover, there are significant differences among different prokaryotes in both the intensity and persistency of sequence periodicity related to DNA curvature. We proffer that the prokaryotic nucleoid consists of relatively rigid sections stabilized by short intrinsically bent DNA segments and characterized by locally strong periodic patterns alternating with regions featuring a weak periodic signal, which presumably permits higher structural flexibility. This model applies to most bacteria and archaea. In genomes with an exceptionally persistent periodic signal, highly expressed genes tend to concentrate in aperiodic sections, suggesting that structural heterogeneity of the nucleoid is related to local differences in transcriptional activity.
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Rastelli, Luca, Karen Robinson, Yanbo Xu i Sadhan Majumder. "Reconstitution of Enhancer Function in Paternal Pronuclei of One-Cell Mouse Embryos". Molecular and Cellular Biology 21, nr 16 (15.08.2001): 5531–40. http://dx.doi.org/10.1128/mcb.21.16.5531-5540.2001.
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ABSTRACT How chromatin-mediated transcription regulates the beginning of mammalian development is currently unknown. Factors responsible for promoter repression and enhancer-mediated relief of this repression are not present in the paternal pronuclei of one-cell mouse embryos but are present in the zygotic nuclei of two-cell embryos. Here we show that coinjection of purified histones and a plasmid-encoded reporter gene into the paternal pronuclei of one-cell embryos at a specific histone-DNA concentration could recreate the behavior observed in two-cell embryos: acquisition of promoter repression and subsequent relief of this repression either by functional enhancers or by histone deacetylase inhibitors. Furthermore, the extent of enhancer-mediated stimulation in one-cell embryos depended on the acetylation status of the injected histones, on the treatment of embryos with a histone deacetylase inhibitor, and on the developmentally regulated appearance of enhancer-specific coactivator activity. The coinjected plasmids in one-cell embryos also exhibited chromatin assembly, as determined by a supercoiling assay. Thus, injection of histones into one-cell embryos faithfully reproduced the chromatin-mediated transcription observed in two-cell embryos. These results suggest that the need for enhancers to stimulate promoters through relief of chromatin-mediated repression occurs once the parental genomes are organized into chromatin. Furthermore, we present a model mammalian system in which the role of individual histones, and particular domains within the histones that are targeted in enhancer function, can be examined using purified mutant histones.
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Grohens, Théotime, Sam Meyer i Guillaume Beslon. "A Genome-Wide Evolutionary Simulation of the Transcription-Supercoiling Coupling". Artificial Life, 5.08.2022, 1–18. http://dx.doi.org/10.1162/artl_a_00373.
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Abstract DNA supercoiling, the level of under- or overwinding of the DNA polymer around itself, is widely recognized as an ancestral regulation mechanism of gene expression in bacteria. Higher levels of negative supercoiling facilitate the opening of the DNA double helix at gene promoters and thereby increase gene transcription rates. Different levels of supercoiling have been measured in bacteria exposed to different environments, leading to the hypothesis that variations in supercoiling could be a response to changes in the environment. Moreover, DNA transcription has been shown to generate local variations in the supercoiling level and, therefore, to impact the transcription rate of neighboring genes. In this work, we study the coupled dynamics of DNA supercoiling and transcription at the genome scale. We implement a genome-wide model of gene expression based on the transcription-supercoiling coupling. We show that, in this model, a simple change in global DNA supercoiling is sufficient to trigger differentiated responses in gene expression levels via the transcription-supercoiling coupling. Then, studying our model in the light of evolution, we demonstrate that this non-linear response to different environments, mediated by the transcription-supercoiling coupling, can serve as the basis for the evolution of specialized phenotypes.
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Visser, Bryan J., Sonum Sharma, Po J. Chen, Anna B. McMullin, Maia L. Bates i David Bates. "Psoralen mapping reveals a bacterial genome supercoiling landscape dominated by transcription". Nucleic Acids Research, 14.04.2022. http://dx.doi.org/10.1093/nar/gkac244.
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Abstract DNA supercoiling is a key regulator of all DNA metabolic processes including replication, transcription, and recombination, yet a reliable genomic assay for supercoiling is lacking. Here, we present a robust and flexible method (Psora-seq) to measure whole-genome supercoiling at high resolution. Using this tool in Escherichia coli, we observe a supercoiling landscape that is well correlated to transcription. Supercoiling twin-domains generated by RNA polymerase complexes span 25 kb in each direction – an order of magnitude farther than previous measurements in any organism. Thus, ribosomal and many other highly expressed genes strongly affect the topology of about 40 neighboring genes each, creating highly integrated gene circuits. Genomic patterns of supercoiling revealed by Psora-seq could be aptly predicted from modeling based on gene expression levels alone, indicating that transcription is the major determinant of chromosome supercoiling. Large-scale supercoiling patterns were highly symmetrical between left and right chromosome arms (replichores), indicating that DNA replication also strongly influences supercoiling. Skew in the axis of symmetry from the natural ori-ter axis supports previous indications that the rightward replication fork is delayed several minutes after initiation. Implications of supercoiling on DNA replication and chromosome domain structure are discussed.
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Guo, Monica S., Ryo Kawamura, Megan L. Littlehale, John F. Marko i Michael T. Laub. "High-resolution, genome-wide mapping of positive supercoiling in chromosomes". eLife 10 (19.07.2021). http://dx.doi.org/10.7554/elife.67236.
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Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to Escherichia coli and Saccharomyces cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently oriented genes, consistent with the ‘twin-domain’ model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin-binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.
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Fujita, Hironobu, Ayane Osaku, Yuto Sakane, Koki Yoshida, Kayoko Yamada, Seia Nara, Takahito Mukai i Masayuki Su’etsugu. "Enzymatic Supercoiling of Bacterial Chromosomes Facilitates Genome Manipulation". ACS Synthetic Biology, 23.08.2022. http://dx.doi.org/10.1021/acssynbio.2c00353.
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