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PloS One 2019C9orf82 protein, or conserved anti-apoptotic protein 1 or caspase activity and apoptosis inhibitor 1 (CAAP1) has been implicated as a negative regulator of the intrinsic...
C9orf82 protein, or conserved anti-apoptotic protein 1 or caspase activity and apoptosis inhibitor 1 (CAAP1) has been implicated as a negative regulator of the intrinsic apoptosis pathway by modulating caspase expression and activity. In contrast, an independent genome wide screen for factors capable of driving drug resistance to the topoisomerase II (Topo II) poisons doxorubicin and etoposide, implicated a role for the nuclear protein C9orf82 in delaying DSBs repair downstream of Topo II, hereby sensitizing cells to DSB induced apoptosis. To determine its function in a genetically defined setting in vivo and ex vivo, we here employed CRISPR/Cas9 technology in zygotes to generate a C9orf82 knockout mouse model. C9orf82ko/ko mice were born at a Mendelian ratio and did not display any overt macroscopic or histological abnormalities. DSBs repair dependent processes like lymphocyte development and class switch recombination (CSR) appeared normal, arguing against a link between the C9orf82 encoded protein and V(D)J recombination or CSR. Most relevant, primary pre-B cell cultures and Tp53 transformed mouse embryo fibroblasts (MEFs) derived from C9orf82ko/ko E14.5 and wild type embryos displayed comparable sensitivity to a number of DNA lesions, including DSBs breaks induced by the topoisomerase II inhibitors, etoposide and doxorubicin. Likewise, the kinetics of γH2AX formation and resolution in response to etoposide of C9orf82 protein proficient, deficient and overexpressing MEFs were indistinguishable. These data argue against a direct role of C9orf82 protein in delaying repair of Topo II generated DSBs and regulating apoptosis. The genetically defined systems generated in this study will be of value to determine the actual function of C9orf82 protein.
Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; B-Lymphocytes; CRISPR-Cas Systems; Caspase 3; Cell Proliferation; Cells, Cultured; DNA Breaks, Double-Stranded; DNA Damage; DNA Repair; DNA Topoisomerases, Type II; Immunoglobulin Class Switching; Mice; Mice, Inbred C57BL; Mice, Knockout; T-Lymphocytes
PubMed: 30629682
DOI: 10.1371/journal.pone.0210526 -
Science (New York, N.Y.) Jun 2023Cyanotriazole compounds "poison" topoisomerase II of pathogenic trypanosomatids.
Cyanotriazole compounds "poison" topoisomerase II of pathogenic trypanosomatids.
Topics: Antiparasitic Agents; DNA Topoisomerases, Type II; Triazoles; Topoisomerase II Inhibitors; Trypanosomatina; Animals; Humans
PubMed: 37384682
DOI: 10.1126/science.adi5925 -
Cell Cycle (Georgetown, Tex.) Dec 2016
Topics: Aurora Kinase B; Centromere; Chromosome Segregation; DNA Topoisomerases, Type II; Sumoylation
PubMed: 27484981
DOI: 10.1080/15384101.2016.1216928 -
Developmental Cell Jan 2020GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan (DVC-1) and Wss1 resolve DNA-protein...
GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan (DVC-1) and Wss1 resolve DNA-protein crosslinks (DPCs), including Topoisomerase-DNA adducts, during DNA replication. Here, we show that GCNA mutants in mouse and C. elegans display defects in genome maintenance including DNA damage, aberrant chromosome condensation, and crossover defects in mouse spermatocytes and spontaneous genomic rearrangements in C. elegans. We show that GCNA and topoisomerase II (TOP2) physically interact in both mice and worms and colocalize on condensed chromosomes during mitosis in C. elegans embryos. Moreover, C. elegans gcna-1 mutants are hypersensitive to TOP2 poison. Together, our findings support a model in which GCNA provides genome maintenance functions in the germline and may do so, in part, by promoting the resolution of TOP2 DPCs.
Topics: Animals; Caenorhabditis elegans; DNA Damage; DNA Repair; DNA Replication; DNA Topoisomerases, Type II; DNA-Binding Proteins; Genome; Genomic Instability; Germ Cells; Male; Mice; Mice, Inbred C57BL; Mitosis; Mutation; Nuclear Proteins; Spermatocytes; Spermatogenesis
PubMed: 31839538
DOI: 10.1016/j.devcel.2019.11.006 -
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 -
International Journal of Molecular... Jun 2018Type IIA topoisomerases allow DNA double helical strands to pass through each other by generating transient DNA double strand breaks βDSBs), and in so doing, resolve... (Review)
Review
Type IIA topoisomerases allow DNA double helical strands to pass through each other by generating transient DNA double strand breaks βDSBs), and in so doing, resolve torsional strain that accumulates during transcription, DNA replication, chromosome condensation, chromosome segregation and recombination. Whereas most eukaryotes possess a single type IIA enzyme, vertebrates possess two distinct type IIA topoisomerases, Topo IIα and Topo IIβ. Although the roles of Topo IIα, especially in the context of chromosome condensation and segregation, have been well-studied, the roles of Topo IIβ are only beginning to be illuminated. This review begins with a summary of the initial studies surrounding the discovery and characterization of Topo IIβ and then focuses on the insights gained from more recent studies that have elaborated important functions for Topo IIβ in transcriptional regulation.
Topics: Animals; Chromosome Segregation; DNA Breaks, Double-Stranded; DNA Replication; DNA Topoisomerases, Type II; Humans; Transcription, Genetic
PubMed: 29966298
DOI: 10.3390/ijms19071917 -
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