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Open Biology Oct 2021RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIβ (TOP2B)-mediated DNA strand break and the activation of DNA...
RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIβ (TOP2B)-mediated DNA strand break and the activation of DNA damage response signalling in humans. Here, we report a novel function of the breast cancer 1 (BRCA1)-BRCA1-associated ring domain 1 (BARD1) complex in this process. We found that BRCA1 is phosphorylated at S1524 by the kinases ataxia-telangiectasia mutated and ATR during gene activation, and that this event is important for productive transcription. Our biochemical and genomic analyses showed that the BRCA1-BARD1 complex interacts with TOP2B in the transcription start site and in a large number of protein-coding genes. Intriguingly, the BRCA1-BARD1 complex ubiquitinates TOP2B, which stabilizes TOP2B binding to DNA while BRCA1 phosphorylation at S1524 controls the TOP2B ubiquitination by the complex. Together, these findings suggest the novel function of the BRCA1-BARD1 complex in the regulation of TOP2B and Pol II-mediated gene expression.
Topics: Ataxia Telangiectasia Mutated Proteins; BRCA1 Protein; DNA Topoisomerases, Type II; Early Growth Response Protein 1; Gene Expression Regulation; HEK293 Cells; HeLa Cells; Humans; Immediate-Early Proteins; Mutation; Phosphorylation; Poly-ADP-Ribose Binding Proteins; Transcription Initiation Site; Transcription, Genetic; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 34610268
DOI: 10.1098/rsob.210221 -
ELife Dec 2022Spatial organization of chromatin plays a critical role in genome regulation. Previously, various types of affinity mediators and enzymes have been attributed to...
Spatial organization of chromatin plays a critical role in genome regulation. Previously, various types of affinity mediators and enzymes have been attributed to regulate spatial organization of chromatin from a thermodynamics perspective. However, at the mechanistic level, enzymes act in their unique ways and perturb the chromatin. Here, we construct a polymer physics model following the mechanistic scheme of Topoisomerase-II, an enzyme resolving topological constraints of chromatin, and investigate how it affects interphase chromatin organization. Our computer simulations demonstrate Topoisomerase-II's ability to phase separate chromatin into eu- and heterochromatic regions with a characteristic wall-like organization of the euchromatic regions. We realized that the ability of the euchromatic regions to cross each other due to enzymatic activity of Topoisomerase-II induces this phase separation. This realization is based on the physical fact that partial absence of self-avoiding interaction can induce phase separation of a system into its self-avoiding and non-self-avoiding parts, which we reveal using a mean-field argument. Furthermore, motivated from recent experimental observations, we extend our model to a bidisperse setting and show that the characteristic features of the enzymatic activity-driven phase separation survive there. The existence of these robust characteristic features, even under the non-localized action of the enzyme, highlights the critical role of enzymatic activity in chromatin organization.
Topics: Chromatin; Interphase; Genome; DNA Topoisomerases, Type II; Polymers
PubMed: 36472500
DOI: 10.7554/eLife.79901 -
Journal of Medicinal Chemistry Feb 2020Human DNA topoisomerase II is an important target in anticancer therapy. Despite the clinical success of drugs that target topoisomerase II, the development of resistant... (Review)
Review
Human DNA topoisomerase II is an important target in anticancer therapy. Despite the clinical success of drugs that target topoisomerase II, the development of resistant cancer cells can limit their clinical efficacy. To maximize the therapeutic potential of anticancer drugs, combination therapies and multitarget drugs have been suggested in many studies, where the use of multitarget drugs is advantageous from a pharmacokinetic point of view. There are various different options for the preparation of dual-target or multiple-target inhibitors, as topoisomerase II is both structurally (e.g., topoisomerase I, Hsp90, and kinases) and functionally (e.g., histone deacetylases and proteasome) connected to many validated anticancer targets. In this Perspective, we discuss the scientific background behind targeting topoisomerase II together with a number of other targets important in cancer therapy, review the present status, and discuss further options in the field.
Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; DNA Topoisomerases, Type II; Drug Design; HSP90 Heat-Shock Proteins; Humans; Neoplasms; Topoisomerase II Inhibitors; Tubulin Modulators
PubMed: 31592646
DOI: 10.1021/acs.jmedchem.9b00726 -
BMC Molecular and Cell Biology Jul 2019The processes of DNA supercoiling and transcription are interdependent because the movement of a transcription elongation complex simultaneously induces under- and... (Review)
Review
BACKGROUND
The processes of DNA supercoiling and transcription are interdependent because the movement of a transcription elongation complex simultaneously induces under- and overwinding of the DNA duplex and because the initiation, elongation and termination steps of transcription are all sensitive to the topological state of the DNA.
RESULTS
Policing of the local and global supercoiling of DNA by topoisomerases helps to sustain the major DNA-based transactions by eliminating barriers to the movement of transcription complexes and replisomes. Recent data from whole-genome and single-molecule studies have provided new insights into how interactions between transcription and the supercoiling of DNA influence the architecture of the chromosome and how they create cell-to-cell diversity at the level of gene expression through transcription bursting.
CONCLUSIONS
These insights into fundamental molecular processes reveal mechanisms by which bacteria can prevail in unpredictable and often hostile environments by becoming unpredictable themselves.
Topics: Binding Sites; Chromosomes, Bacterial; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; DNA, Bacterial; DNA, Single-Stranded; DNA, Superhelical; Escherichia coli; Mycobacterium tuberculosis; Promoter Regions, Genetic; Protein Binding; Transcription, Genetic; Transcriptional Activation
PubMed: 31319794
DOI: 10.1186/s12860-019-0211-6 -
International Journal of Molecular... Sep 2022Topoisomerases are essential enzymes that recognize and modify the topology of DNA to allow DNA replication and transcription to take place. Topoisomerases are divided... (Review)
Review
Topoisomerases are essential enzymes that recognize and modify the topology of DNA to allow DNA replication and transcription to take place. Topoisomerases are divided into type I topoisomerases, that cleave one DNA strand to modify DNA topology, and type II, that cleave both DNA strands. Topoisomerases normally rapidly religate cleaved-DNA once the topology has been modified. Topoisomerases do not recognize specific DNA sequences, but actively cleave positively supercoiled DNA ahead of transcription bubbles or replication forks, and negative supercoils (or precatenanes) behind, thus allowing the unwinding of the DNA-helix to proceed (during both transcription and replication). Drugs that stabilize DNA-cleavage complexes with topoisomerases produce cytotoxic DNA damage and kill fast-dividing cells; they are widely used in cancer chemotherapy. Oligonucleotide-recognizing topoisomerase inhibitors (OTIs) have given drugs that stabilize DNA-cleavage complexes specificity by linking them to either: (i) DNA duplex recognizing triplex forming oligonucleotide (TFO-OTIs) or DNA duplex recognizing pyrrole-imidazole-polyamides (PIP-OTIs) (ii) or by conventional Watson-Crick base pairing (WC-OTIs). This converts compounds from indiscriminate DNA-damaging drugs to highly specific targeted DNA-cleaving OTIs. Herein we propose simple strategies to enable DNA-duplex strand invasion of WC-OTIs giving strand-invading SI-OTIs. This will make SI-OTIs similar to the guide RNAs of CRISPR/Cas9 nuclease bacterial immune systems. However, an important difference between OTIs and CRISPR/Cas9, is that OTIs do not require the introduction of foreign proteins into cells. Recent successful oligonucleotide therapeutics for neurodegenerative diseases suggest that OTIs can be developed to be highly specific gene editing agents for DNA lesions that cause neurodegenerative diseases.
Topics: DNA; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; DNA, Superhelical; Humans; Imidazoles; Neurodegenerative Diseases; Nylons; Oligonucleotides; Pyrroles; Topoisomerase I Inhibitors; Topoisomerase II Inhibitors; Topoisomerase Inhibitors
PubMed: 36232843
DOI: 10.3390/ijms231911541 -
Journal of Molecular Biology Aug 2019Type II topoisomerases regulate DNA topology by making a double-stranded break in one DNA duplex, transporting another DNA segment through this break and then resealing... (Review)
Review
Type II topoisomerases regulate DNA topology by making a double-stranded break in one DNA duplex, transporting another DNA segment through this break and then resealing it. Bacterial type IIA topoisomerase inhibitors, such as fluoroquinolones and novel bacterial topoisomerase inhibitors, can trap DNA cleavage complexes with double- or single-stranded cleaved DNA. To study the mode of action of such compounds, 21 crystal structures of a "gyrase" fusion truncate of Staphyloccocus aureus DNA gyrase complexed with DNA and diverse inhibitors have been published, as well as 4 structures lacking inhibitors. These structures have the DNA in various cleavage states and appear to track trajectories along the catalytic paths of the DNA cleavage/religation steps. The various conformations sampled by these multiple "gyrase" structures show rigid body movements of the catalytic GyrA WHD and GyrB TOPRIM domains across the dimer interface. Conformational changes common to all compound-bound structures suggest common mechanisms for DNA cleavage-stabilizing compounds. The structures suggest that S. aureus gyrase uses a single moving-metal ion for cleavage and that the central four base pairs need to be stretched between the two catalytic sites, in order for a scissile phosphate to attract a metal ion to the A-site to catalyze cleavage, after which it is "stored" in another coordination configuration (B-site) in the vicinity. We present a simplified model for the catalytic cycle in which capture of the transported DNA segment causes conformational changes in the ATPase domain that push the DNA gate open, resulting in stretching and cleaving the gate-DNA in two steps.
Topics: Anti-Bacterial Agents; Catalytic Domain; DNA; DNA Cleavage; DNA Gyrase; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; Fluoroquinolones; Metals; Models, Molecular; Protein Conformation; Quinolones; Staphylococcus aureus; Topoisomerase II Inhibitors; Topoisomerase Inhibitors
PubMed: 31301408
DOI: 10.1016/j.jmb.2019.07.008 -
International Journal of Molecular... Feb 2021The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by... (Comparative Study)
Comparative Study
The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.
Topics: Chromosomes, Plant; DNA Topoisomerases, Type II; Fluorescent Dyes; Hordeum; Indoles; Metaphase; Microscopy, Confocal; Microscopy, Fluorescence; Reproducibility of Results; Single Molecule Imaging
PubMed: 33672992
DOI: 10.3390/ijms22041903 -
The Journal of Cell Biology Jan 2020Topoisomerase II (Topo II) is essential for mitosis since it resolves sister chromatid catenations. Topo II dysfunction promotes aneuploidy and drives cancer. To protect...
Topoisomerase II (Topo II) is essential for mitosis since it resolves sister chromatid catenations. Topo II dysfunction promotes aneuploidy and drives cancer. To protect from aneuploidy, cells possess mechanisms to delay anaphase onset when Topo II is perturbed, providing additional time for decatenation. Molecular insight into this checkpoint is lacking. Here we present evidence that catalytic inhibition of Topo II, which activates the checkpoint, leads to SUMOylation of the Topo II C-terminal domain (CTD). This modification triggers mobilization of Aurora B kinase from inner centromeres to kinetochore proximal centromeres and the core of chromosome arms. Aurora B recruitment accompanies histone H3 threonine-3 phosphorylation and requires Haspin kinase. Strikingly, activation of the checkpoint depends both on Haspin and Aurora B. Moreover, mutation of the conserved CTD SUMOylation sites perturbs Aurora B recruitment and checkpoint activation. The data indicate that SUMOylated Topo II recruits Aurora B to ectopic sites, constituting the molecular trigger of the metaphase checkpoint when Topo II is catalytically inhibited.
Topics: Aurora Kinase B; Chromosome Segregation; Chromosomes, Human; DNA Topoisomerases, Type II; Diketopiperazines; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Kinetochores; Metaphase; Mitosis; Phosphorylation; Piperazines; Protein Serine-Threonine Kinases; Sumoylation; Topoisomerase II Inhibitors
PubMed: 31712254
DOI: 10.1083/jcb.201807189 -
Frontiers in Bioscience (Landmark... Apr 2023Cholangiocarcinoma (CCA) is a malignant tumor with an increasing incidence worldwide. Although radiation therapy has improved the therapeutic efficiency of CCA...
BACKGROUND
Cholangiocarcinoma (CCA) is a malignant tumor with an increasing incidence worldwide. Although radiation therapy has improved the therapeutic efficiency of CCA treatment, differential expression of genes among cholangiocarcinoma subtypes has been revealed through precise sequencing. However, no specific molecular therapeutic targets or biomarkers have been figured out for use in precision medicine, and the exact mechanism by which antitumorigenic effects occur is still unclear. Therefore, it is necessary to conduct further studies on the development and mechanisms associated with CCA.
METHODS
We examined the clinical data and pathological features of patients with cholangiocarcinomas. We investigated the associations between DNA Topoisomerase II Alpha (TOP2A) expression and patient outcomes, such as metastasis-free survival (MFS) and disease-specific survival (DSS), as well as clinical characteristics and pathological results.
RESULTS
expression was shown to be upregulated in CCA tissue sections by immunohistochemistry staining and data mining. Moreover, we observed that the expression correlated with clinical features, such as the primary tumor stage, histological variants, and patients with hepatitis. Furthermore, high expression of was associated with worse survival outcomes in terms of the overall survival ( < 0.0001), disease-specific survival ( < 0.0001), and metastasis-free survival ( < 0.0001) compared with patients in the low expression group. This indicates that a high level of expression is related to an unfavorable prognosis.
CONCLUSIONS
Our results show that is highly expressed in CCA tissues, and its upregulation is correlated with the primary disease stage and poor prognosis significantly. Consequently, is a prognostic biomarker and a novel therapeutic target for the treatment of CCA.
Topics: Humans; Prognosis; DNA Topoisomerases, Type II; Cholangiocarcinoma; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Biomarkers, Tumor
PubMed: 37114547
DOI: 10.31083/j.fbl2804075 -
Molecules (Basel, Switzerland) Nov 2022Fluoroquinolones (FQs) are synthetic broad-spectrum antimicrobial agents that have been recently repurposed to anticancer candidates. Designing new derivatives of FQs...
Fluoroquinolones (FQs) are synthetic broad-spectrum antimicrobial agents that have been recently repurposed to anticancer candidates. Designing new derivatives of FQs with different moieties to target DNA topoisomerases could improve their anticancer efficacy. The present study aimed to synthesize a novel ciprofloxacin derivative, examine its anticancer activity against HepG2 and A549 cancer cells, and investigate the possible molecular mechanism underlying this activity by examining its ability to inhibit the topo I/II activity and to induce the apoptotic and necro-apoptotic pathways. Molecular docking, cell viability, cell migration, colony formation, cell cycle, Annexin V, lactate dehydrogenase (LDH) release, ELISA, and western blotting assays were utilized. Molecular docking results showed that this novel ciprofloxacin derivative exerted dual topo I and topo II binding and inhibition. It significantly inhibited the proliferation of A549 and HepG2 cancer cells and decreased their cell migration and colony formation abilities. In addition, it significantly increased the % of apoptotic cells, caused cell cycle arrest at G2/M phase, and elevated the LDH release levels in both cancer cells. Furthermore, it increased the expression of cleaved caspase 3, RIPK1, RIPK3, and MLKL proteins. This novel ciprofloxacin derivative exerted substantial dual inhibition of topo I/II enzyme activities, showed antiproliferative activity, suppressed the cell migration and colony formation abilities for A549 and HepG2 cancer cells and activated the apoptotic pathway. In addition, it initiated another backup deadly pathway, necro-apoptosis, through the activation of the RIPK1/RIPK3/MLKL pathway.
Topics: Apoptosis; Ciprofloxacin; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; Molecular Docking Simulation; Neoplasms; Protein Kinases
PubMed: 36432094
DOI: 10.3390/molecules27227993