DOCSLIB.ORG

915.Full.Pdf

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
915.Full.Pdf A Potently Neutralizing Antibody Protects Mice against SARS-CoV-2 Infection Wafaa B. Alsoussi, Jackson S. Turner, James B. Case, Haiyan Zhao, Aaron J. Schmitz, Julian Q. Zhou, Rita E. This information is current as Chen, Tingting Lei, Amena A. Rizk, Katherine M. McIntire, of September 27, 2021. Emma S. Winkler, Julie M. Fox, Natasha M. Kafai, Larissa B. Thackray, Ahmed O. Hassan, Fatima Amanat, Florian Krammer, Corey T. Watson, Steven H. Kleinstein, Daved H. Fremont, Michael S. Diamond and Ali H. Ellebedy Downloaded from J Immunol 2020; 205:915-922; Prepublished online 26 June 2020; doi: 10.4049/jimmunol.2000583 http://www.jimmunol.org/content/205/4/915 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2020/06/23/jimmunol.200058 Material 3.DCSupplemental References This article cites 51 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/205/4/915.full#ref-list-1 by guest on September 27, 2021 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology A Potently Neutralizing Antibody Protects Mice against SARS-CoV-2 Infection Wafaa B. Alsoussi,*,1 Jackson S. Turner,*,1 James B. Case,†,1 Haiyan Zhao,*,1 Aaron J. Schmitz,*,1 Julian Q. Zhou,‡,1 Rita E. Chen,† Tingting Lei,* Amena A. Rizk,* Katherine M. McIntire,* Emma S. Winkler,*,† Julie M. Fox,† Natasha M. Kafai,*,† Larissa B. Thackray,† Ahmed O. Hassan,† Fatima Amanat,x,{ Florian Krammer,x Corey T. Watson,‖ Steven H. Kleinstein,‡,#,** Daved H. Fremont,*,††,‡‡ Michael S. Diamond,*,†,††,xx and Ali H. Ellebedy*,††,xx Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for millions of infections and hundreds of thousands of deaths globally. There are no widely available licensed therapeutics against SARS-CoV-2, highlighting an urgent need for effective Downloaded from interventions. The virus enters host cells through binding of a receptor-binding domain within its trimeric spike glycoprotein to human angiotensin-converting enzyme 2. In this article, we describe the generation and characterization of a panel of murine mAbs directed against the receptor-binding domain. One mAb, 2B04, neutralized wild-type SARS-CoV-2 in vitro with remarkable potency (half-maximal inhibitory concentration of <2 ng/ml). In a murine model of SARS-CoV-2 infection, 2B04 protected challenged animals from weight loss, reduced lung viral load, and blocked systemic dissemination. Thus, 2B04 is a promising candidate for an effective antiviral that can be used to prevent SARS-CoV-2 infection. The Journal of Immunology, 2020, 205: 915–922. http://www.jimmunol.org/ ost members of the Coronaviridae family infect the (2–4). In December 2019, a third highly pathogenic human respiratory tract of mammals, causing mild respira- coronavirus, SARS-CoV 2 (SARS-CoV-2) emerged in Wuhan, M tory disease (1). In the past two decades, however, two Hubei province of China (5–7). Compared with SARS-CoV and highly pathogenic coronaviruses, severe acute respiratory syn- MERS-CoV, SARS-CoV-2 is more readily transmitted among drome coronavirus (SARS-CoV) and Middle East respiratory humans, spreading to multiple continents and leading to the World syndrome coronavirus (MERS-CoV), crossed the species barrier Health Organization’s declaration of a coronavirus disease 2019 and led to epidemics with high morbidity and mortality in humans (COVID-19) pandemic (8, 9). As of June 16, 2020, SARS-CoV-2 by guest on September 27, 2021 *Department of Pathology and Immunology, Washington University School of Med- of Genetics at Washington University School of Medicine]), the National Center for icine, St. Louis, MO 63110; †Department of Medicine, Washington University Research Resources (an Institute for Clinical and Translational Science/Clinical and School of Medicine, St. Louis, MO 63110; ‡Interdepartmental Program in Compu- Translational Sciences Award, Grant UL1 TR000448, to the Genome Technology tational Biology and Bioinformatics, Yale School of Medicine, Yale University, New Access Center in the Department of Genetics at Washington University School of Haven, CT 06511; xDepartment of Microbiology, Icahn School of Medicine at Mount Medicine), and a Helen Hay Whitney postdoctoral fellowship (to J.B.C.). The Sinai, New York, NY 10024; {Graduate School of Biomedical Sciences, Icahn contents of this publication are solely the responsibility of the authors and do not School of Medicine at Mount Sinai, New York, NY 10024; ‖Department of Biochem- necessarily represent the official views of the NIAID or NIH. istry and Molecular Genetics, University of Louisville School of Medicine, Louis- W.B.A., A.J.S., and J.S.T. isolated the Abs. W.B.A., J.B.C., K.M.M., H.Z., R.E.C., ville, KY 40292; #Department of Pathology, Yale School of Medicine, New Haven, A.O.H., J.M.F., E.S.W., L.B.T., and N.M.K. functionally characterized the mAbs. A.A.R., CT 06511; **Department of Immunobiology, Yale School of Medicine, New Haven, T.L., and F.A. expressed and purified the mAbs and recombinant viral proteins. J.Q.Z., CT 06511; ††Department of Molecular Microbiology, Washington University School C.T.W., and S.H.K. analyzed the scRNA-seq and BCR data. F.K. provided critical of Medicine, St. Louis, MO 63110; ‡‡Department of Biochemistry and Molecular reagents. W.B.A., J.S.T., D.H.F., M.S.D., and A.H.E. conceptualized the study and Biophysics, Washington University School of Medicine, St. Louis, MO 63110; and xx wrote the manuscript. The Andrew M. and Jane M. Bursky Center for Human Immunology and Immu- notherapy Programs, Washington University School of Medicine, St. Louis, MO Raw fastq files, associated RNA-sequencing, and processed gene expression data 63110 presented in this article have been submitted to the National Center for Biotechnol- ogy Information Gene Expression Omnibus/Sequence Read Archive database 1W.B.A., J.S.T., J.B.C., H.Z., A.J.S., and J.Q.Z. contributed equally to this work. (https://www.ncbi.nlm.nih.gov/geo/, https://www.ncbi.nlm.nih.gov/sra) under acces- ORCIDs: 0000-0002-4396-6265 (J.S.T.); 0000-0001-7331-5511 (J.B.C.); 0000-0001- sion numbers SRP256045 and GSE149036. Ab sequences presented in this article 5123-5537 (H.Z.); 0000-0002-8077-6751 (A.J.S.); 0000-0001-9602-2092 (J.Q.Z.); have been submitted to the GenBank/European Molecular Biology Laboratory/DNA 0000-0003-0567-738X (J.M.F.); 0000-0002-3115-3400 (N.M.K.); 0000-0002-9638- Data Base in Japan (https://www.ncbi.nlm.nih.gov/genbank/) under accession num- 1260 (A.O.H.); 0000-0003-4121-776X (F.K.); 0000-0001-7248-8787 (C.T.W.); 0000- bers MT341590–MT341641). 0003-4957-1544 (S.H.K.); 0000-0002-8544-2689 (D.H.F.). Address correspondence and reprint requests to Dr. Ali Ellebedy, Washington Uni- Received for publication May 19, 2020. Accepted for publication June 8, 2020. versity in St. Louis, St. Louis, MO. E-mail address: [email protected] This work was supported or partially supported by the National Institute of Allergy The online version of this article contains supplemental material. and Infectious Diseases (NIAID) (R21 AI139813 and U01 AI141990, to the Ellebedy Abbreviations used in this article: COVID-19, coronavirus disease 2019; FRNT, laboratory; HHSN272201700060C, to the Fremont laboratory; 75N93019C00062, focus reduction neutralization test; hACE2, human angiotensin-converting enzyme to the Fremont and Diamond laboratories; 5T32CA009547, to J.S.T.; and 2; HVG, highly variable gene; IMGT, ImMunoGeneTics information system; i.n., R01AI104739, to the Kleinstein laboratory), the NIAID Centers of Excellence for intranasal(ly); MERS-CoV, Middle East respiratory syndrome coronavirus; MGI, Influenza Research and Surveillance (CEIRS) (Contract HHSN272201400008C, to Mouse Genome Informatics; PB, plasmablast; PFA, paraformaldehyde; RBD, the Ellebedy and Krammer laboratories), the National Institutes of Health (NIH) receptor-binding domain; RSS, recombination signal sequence; S, spike; SARS- (Grant R01 AI127828, to the Diamond laboratory), the Defense Advanced Research CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2, SARS-CoV 2; Projects Agency (HR001117S0019, to the Diamond laboratory), the Collaborative scRNA-seq, single-cell RNA-sequencing; UMI, unique molecular identifier. Influenza Vaccine Innovation Centers (Contract 75N93019C00051, to the Krammer laboratory), the National Cancer Institute Cancer Center (Grant P30 CA91842, to the Ó Siteman Cancer Center [The Genome Technology Access Center in the Department Copyright 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000583 916 A PROTECTIVE, POTENTLY NEUTRALIZING SARS-COV-2 Ab caused .8 million confirmed cases globally, leading to at least after transfection. The soluble S proteins were recovered using 2 ml of 440,000 deaths (10). Currently, there are no widely available li- cobalt-charged resin (G-Biosciences). Mammalian SARS-CoV-2 RBD 2 censed therapeutics to prevent or treat COVID-19. This underlines (residues 331 524) was cloned into vector pFM1.2 with N-terminal m-phosphatase signal peptide and C-terminal 6XHis Tag. The protein the need for immediate development of prophylactic and thera- was expressed as S protein and recovered by nickel agarose beads peutic reagents to combat SARS-CoV-2 virus infection.
Load more

Recommended publications
  • Killed Whole-Genome Reduced-Bacteria Surface-Expressed Coronavirus Fusion Peptide Vaccines Protect Against Disease in a Porcine Model
    Killed whole-genome reduced-bacteria surface-expressed coronavirus fusion peptide vaccines protect against disease in a porcine model Denicar Lina Nascimento Fabris Maedaa,b,c,1, Debin Tiand,e,1, Hanna Yua,b,c, Nakul Dara,b,c, Vignesh Rajasekarana,b,c, Sarah Menga,b,c, Hassan M. Mahsoubd,e, Harini Sooryanaraind,e, Bo Wangd,e, C. Lynn Heffrond,e, Anna Hassebroekd,e, Tanya LeRoithd,e, Xiang-Jin Mengd,e,2, and Steven L. Zeichnera,b,c,f,2 aDepartment of Pediatrics, University of Virginia, Charlottesville, VA 22908-0386; bPendleton Pediatric Infectious Disease Laboratory, University of Virginia, Charlottesville, VA 22908-0386; cChild Health Research Center, University of Virginia, Charlottesville, VA 22908-0386; dDepartment of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0913; eCenter for Emerging, Zoonotic, and Arthropod-borne Pathogens, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0913; and fDepartment of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA 22908-0386 Contributed by Xiang-Jin Meng, February 14, 2021 (sent for review December 13, 2020; reviewed by Diego Diel and Qiuhong Wang) As the coronavirus disease 2019 (COVID-19) pandemic rages on, it Vaccination specifically with conserved E. coli antigens did not is important to explore new evolution-resistant vaccine antigens alter the gastrointestinal (GI) microbiome (6, 9). In human stud- and new vaccine platforms that can produce readily scalable, in- ies, volunteers were immunized orally with a KWCV against expensive vaccines with easier storage and transport. We report ETEC (10) with no adverse effects. An ETEC oral KWCV along here a synthetic biology-based vaccine platform that employs an with a cholera B toxin subunit adjuvant was studied in children expression vector with an inducible gram-negative autotransporter and found to be safe (11).
    [Show full text]
  • Genomic Sequencing of SARS-Cov-2: a Guide to Implementation for Maximum Impact on Public Health
    Genomic sequencing of SARS-CoV-2 A guide to implementation for maximum impact on public health 8 January 2021 Genomic sequencing of SARS-CoV-2 A guide to implementation for maximum impact on public health 8 January 2021 Genomic sequencing of SARS-CoV-2: a guide to implementation for maximum impact on public health ISBN 978-92-4-001844-0 (electronic version) ISBN 978-92-4-001845-7 (print version) © World Health Organization 2021 Some rights reserved. This work is available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 IGO licence (CC BY-NC-SA 3.0 IGO; https://creativecommons.org/licenses/by-nc-sa/3.0/igo). Under the terms of this licence, you may copy, redistribute and adapt the work for non-commercial purposes, provided the work is appropriately cited, as indicated below. In any use of this work, there should be no suggestion that WHO endorses any specific organization, products or services. The use of the WHO logo is not permitted. If you adapt the work, then you must license your work under the same or equivalent Creative Commons licence. If you create a translation of this work, you should add the following disclaimer along with the suggested citation: “This translation was not created by the World Health Organization (WHO). WHO is not responsible for the content or accuracy of this translation. The original English edition shall be the binding and authentic edition”. Any mediation relating to disputes arising under the licence shall be conducted in accordance with the mediation rules of the World Intellectual Property Organization (http://www.wipo.int/amc/en/mediation/rules/).
    [Show full text]
  • DNA Topoisomerase 1 and 2A Function As Oncogenes in Liver Cancer and May Be Direct Targets of Nitidine Chloride
    INTERNATIONAL JOURNAL OF ONCOLOGY 53: 1897-1912, 2018 DNA topoisomerase 1 and 2A function as oncogenes in liver cancer and may be direct targets of nitidine chloride LI-MIN LIU1*, DAN-DAN XIONG2*, PENG LIN3, HONG YANG3, YI-WU DANG2 and GANG CHEN2 1Department of Toxicology, College of Pharmacy, Guangxi Medical University; 2Department of Pathology; 3Ultrasonics Division, Radiology Department, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530021, P.R. China Received March 16, 2018; Accepted July 31, 2018 DOI: 10.3892/ijo.2018.4531 Abstract. The aim of the present study was to determine the role of patients with LC and for identification of high-risk cases, of topoisomerase 1 (TOP1) and topoisomerase 2A (TOP2A) thereby optimizing individual treatment management. More in liver cancer (LC), and to investigate the inhibitory effect importantly, the findings support TOP1 and TOP2A as poten- of nitidine chloride (NC) on these two topoisomerases. tial drug targets of NC for the treatment of LC. Immunohistochemistry (IHC) staining and microarray or RNA sequencing data mining showed markedly higher expression Introduction of TOP1 and TOP2A at the protein and mRNA levels in LC tissues compared with that in control non-tumor tissues. The Liver cancer (LC) is the second main cause of cancer-associated prognostic values of TOP1 and TOP2A expression were also mortality threatening global public health, with hepatocellular estimated based on data from The Cancer Genome Atlas. The carcinoma (HCC) being the main histopathological subtype (1,2). elevated expression levels of TOP1 and TOP2A were closely In the United States, the American Cancer Society projects that associated with poorer overall survival and disease-free >30,000 patients will succumb to LC in 2018 (3).
    [Show full text]
  • The Role of Sumoylation of DNA Topoisomerase Iiα C-Terminal Domain in the Regulation of Mitotic Kinases In
    SUMOylation at the centromere: The role of SUMOylation of DNA topoisomerase IIα C-terminal domain in the regulation of mitotic kinases in cell cycle progression. By Makoto Michael Yoshida Submitted to the graduate degree program in the Department of Molecular Biosciences and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ________________________________________ Chairperson: Yoshiaki Azuma, Ph.D. ________________________________________ Roberto De Guzman, Ph.D. ________________________________________ Kristi Neufeld, Ph.D. _________________________________________ Berl Oakley, Ph.D. _________________________________________ Blake Peterson, Ph.D. Date Defended: July 12, 2016 The Dissertation Committee for Makoto Michael Yoshida certifies that this is the approved version of the following dissertation: SUMOylation at the centromere: The role of SUMOylation of DNA topoisomerase IIα C-terminal domain in the regulation of mitotic kinases in cell cycle progression. ________________________________________ Chairperson: Yoshiaki Azuma, Ph.D. Date approved: July 12, 2016 ii ABSTRACT In many model systems, SUMOylation is required for proper mitosis; in particular, chromosome segregation during anaphase. It was previously shown that interruption of SUMOylation through the addition of the dominant negative E2 SUMO conjugating enzyme Ubc9 in mitosis causes abnormal chromosome segregation in Xenopus laevis egg extract (XEE) cell-free assays, and DNA topoisomerase IIα (TOP2A) was identified as a substrate for SUMOylation at the mitotic centromeres. TOP2A is SUMOylated at K660 and multiple sites in the C-terminal domain (CTD). We sought to understand the role of TOP2A SUMOylation at the mitotic centromeres by identifying specific binding proteins for SUMOylated TOP2A CTD. Through affinity isolation, we have identified Haspin, a histone H3 threonine 3 (H3T3) kinase, as a SUMOylated TOP2A CTD binding protein.
    [Show full text]
  • Sumoylation of DNA Topoisomerase Iiα Regulates Histone H3 Kinase Haspin and H3 Phosphorylation in Mitosis
    JCB: Article SUMOylation of DNA topoisomerase IIα regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis Makoto M. Yoshida,1 Lily Ting,2 Steven P. Gygi,2 and Yoshiaki Azuma1 1Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 2Department of Cell Biology, Harvard Medical School, Boston, MA 02115 DNA topoisomerase II (TOP2) plays a pivotal role in faithful chromosome separation through its strand-passaging ac- tivity that resolves tangled genomic DNA during mitosis. Additionally, TOP2 controls progression of mitosis by activating cell cycle checkpoints. Recent work showed that the enzymatically inert C-terminal domain (CTD) of TOP2 and its post- translational modification are critical to this checkpoint regulation. However, the molecular mechanism has not yet been determined. By using Xenopus laevis egg extract, we found that SUMOylation of DNA topoisomerase IIα (TOP2A) CTD Downloaded from regulates the localization of the histone H3 kinase Haspin and phosphorylation of histone H3 at threonine 3 at the cen- tromere, two steps known to be involved in the recruitment of the chromosomal passenger complex (CPC) to kinetochores in mitosis. Robust centromeric Haspin localization requires SUMOylated TOP2A CTD binding activity through SUMO- interaction motifs and the phosphorylation of Haspin. We propose a novel mechanism through which the TOP2 CTD regulates the CPC via direct interaction with Haspin at mitotic centromeres. jcb.rupress.org Introduction Cell stage–specific kinases are important for the progression of the centromere through the activity of histone H3 kinase Haspin, on September 20, 2017 mitosis. These kinases play a role in specific pathways to en- which phosphorylates histone H3 at threonine 3 (H3T3) for its sure that chromosomes segregate properly to daughter cells to direct interaction with the BIR domain of Survivin (Kelly et prevent aneuploidy.
    [Show full text]
  • Direct Interaction Between Hnrnp-M and CDC5L/PLRG1 Proteins Affects Alternative Splice Site Choice
    Direct interaction between hnRNP-M and CDC5L/PLRG1 proteins affects alternative splice site choice David Llères, Marco Denegri, Marco Biggiogera, Paul Ajuh, Angus Lamond To cite this version: David Llères, Marco Denegri, Marco Biggiogera, Paul Ajuh, Angus Lamond. Direct interaction be- tween hnRNP-M and CDC5L/PLRG1 proteins affects alternative splice site choice. EMBO Reports, EMBO Press, 2010, 11 (6), pp.445 - 451. 10.1038/embor.2010.64. hal-03027049 HAL Id: hal-03027049 https://hal.archives-ouvertes.fr/hal-03027049 Submitted on 26 Nov 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. scientificscientificreport report Direct interaction between hnRNP-M and CDC5L/ PLRG1 proteins affects alternative splice site choice David Lle`res1*, Marco Denegri1*w,MarcoBiggiogera2,PaulAjuh1z & Angus I. Lamond1+ 1Wellcome Trust Centre for Gene Regulation & Expression, College of Life Sciences, University of Dundee, Dundee, UK, and 2LaboratoriodiBiologiaCellulareandCentrodiStudioperl’IstochimicadelCNR,DipartimentodiBiologiaAnimale, Universita’ di Pavia, Pavia, Italy Heterogeneous nuclear ribonucleoprotein-M (hnRNP-M) is an and affect the fate of heterogeneous nuclear RNAs by influencing their abundant nuclear protein that binds to pre-mRNA and is a structure and/or by facilitating or hindering the interaction of their component of the spliceosome complex.
    [Show full text]
  • Modification of Topoisomerase Genes Copy Number in Newly Diagnosed
    Leukemia (2003) 17, 532–540 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu Modification of topoisomerase genes copy number in newly diagnosed childhood acute lymphoblastic leukemia E Gue´rin1,2, N Entz-Werle´3, D Eyer3, E Pencreac’h1, A Schneider1, A Falkenrodt4, F Uettwiller3, A Babin3, A-C Voegeli1,5, M Lessard4, M-P Gaub1,2, P Lutz3 and P Oudet1,5 1Laboratoire de Biochimie et de Biologie Mole´culaire Hoˆpital de Hautepierre, Strasbourg, France; 2INSERM U381, Strasbourg, France; 3Service d’Onco-He´matologie Pe´diatrique Hoˆpital de Hautepierre, Strasbourg, France; 4Laboratoire Hospitalier d’He´matologie Biologique Hoˆpital de Hautepierre, Strasbourg, France; and 5INSERM U184, Illkirch, France Topoisomerase genes were analyzed at both DNA and RNA segregation.4 These enzymes act by promoting transient DNA levels in 25 cases of newly diagnosed childhood acute breakage in order to allow strand-passage events and DNA lymphoblastic leukemia (ALL). The results of molecular analy- 5 sis were compared to risk group classification of children in relaxation to occur before rejoining the broken DNA ends. order to identify molecular characteristics associated with Two types of DNA topoisomerases have been described. Type response to therapy. At diagnosis, allelic imbalance at topo- I enzymes, encoded in humans by the TOP1, TOP3A and isomerase IIa (TOP2A) gene locus was found in 75% of TOP3B genes,6–8 cleave only one strand of the DNA helix informative cases whereas topoisomerase I and IIb gene loci whereas type II enzymes, encoded by the human TOP2A and are altered in none or only one case, respectively.
    [Show full text]
  • TOPBP1 Recruits TOP2A to Ultra-Fine Anaphase Bridges to Aid In
    ARTICLE Received 3 Dec 2014 | Accepted 9 Feb 2015 | Published 12 Mar 2015 DOI: 10.1038/ncomms7572 TOPBP1 recruits TOP2A to ultra-fine anaphase bridges to aid in their resolution Ronan Broderick1, Jadwiga Nieminuszczy1,2, Andrew N. Blackford1,w, Alicja Winczura1,w & Wojciech Niedzwiedz1 During mitosis, sister chromatids must be faithfully segregated to ensure that daughter cells receive one copy of each chromosome. However, following replication they often remain entangled. Topoisomerase IIa (TOP2A) has been proposed to resolve such entanglements, but the mechanisms governing TOP2A recruitment to these structures remain poorly understood. Here, we identify TOPBP1 as a novel interactor of TOP2A, and reveal that it is required for TOP2A recruitment to ultra-fine anaphase bridges (UFBs) in mitosis. The C-terminal region of TOPBP1 interacts with TOP2A, and TOPBP1 recruitment to UFBs requires its BRCT domain 5. Depletion of TOPBP1 leads to accumulation of UFBs, the majority of which arise from centromeric loci. Accordingly, expression of a TOPBP1 mutant that is defective in TOP2A binding phenocopies TOP2A depletion. These findings provide new mechanistic insights into how TOP2A promotes resolution of UFBs during mitosis, and highlights a pivotal role for TOPBP1 in this process. 1 Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. 2 Institute of Biochemistry and Biophysics, PAS, 02-106 Warsaw, Poland. w Present addresses: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK (A.N.B.); Division of Biomedical Cell Biology, Warwick Medical School, The University of Warwick, Coventry CV4 7AL, UK (A.W.).
    [Show full text]
  • Original Article Over-Expression of TOP2A As a Prognostic Biomarker in Patients with Glioma
    Int J Clin Exp Pathol 2018;11(3):1228-1237 www.ijcep.com /ISSN:1936-2625/IJCEP0070536 Original Article Over-expression of TOP2A as a prognostic biomarker in patients with glioma Tianmin Zhou1, Yan Wang2, Dongmeng Qian1, Qing Liang1, Bin Wang1 1Key Laboratory of Medicine and Biotechnology of Qingdao, Department of Microbiology, Medical College of Qingdao University, Qingdao, Shandong, P. R. China; 2Department of Pathology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, P. R. China Received December 7, 2017; Accepted January 19, 2018; Epub March 1, 2018; Published March 15, 2018 Abstract: Topoisomerase (DNA) II alpha (TOP2A), an enzyme that controls and alters the topologic states of DNA during transcription, is aberrantly expressed in many cancers. However, few studies have investigated expression of TOP2A and its clinical significance in glioma. We retrieved six independent investigations from the Oncomine da- tabase and found that TOP2A is highly expressed in glioma tissues compared with corresponding normal controls. Similar results were also found in clinical specimens at the protein level. Immunohistochemical analysis indicated that TOP2A over expression was highly correlated with grade stage, KI67 positive percentage, IDH1 mutation, and age, but other clinical parameters such as sex distribution and tumor size were barely associated with high TOP2A gene expression. Meanwhile we used Prognos can to assess the prognostic value of TOP2A expression in glioma patients, and found that high expression was associated with poor prognosis of patients with glioma. Furthermore, we used the Gene-Cloud of Biotechnology Information (GCBI) bioinformatics platform predict the role of TOP2A in glioma. It was not only involved in DNA replication, chromosome condensation, and responses to DNA damage stimuli, but also promoted cancer cell mitotic cell cycle and apoptosis, and phosphatidylinositol-mediated signaling by regulating gene expression.
    [Show full text]
  • An In-Depth Analysis of the HER2 Amplicon and Chromosome 17
    Rondón-Lagos et al. BMC Cancer 2014, 14:922 http://www.biomedcentral.com/1471-2407/14/922 RESEARCH ARTICLE Open Access Unraveling the chromosome 17 patterns of FISH in interphase nuclei: an in-depth analysis of the HER2 amplicon and chromosome 17 centromere by karyotyping, FISH and M-FISH in breast cancer cells Milena Rondón-Lagos1,4, Ludovica Verdun Di Cantogno2, Nelson Rangel1,4, Teresa Mele3, Sandra R Ramírez-Clavijo4, Giorgio Scagliotti3, Caterina Marchiò1,2*† and Anna Sapino1,2*† Abstract Background: In diagnostic pathology, HER2 status is determined in interphase nuclei by fluorescence in situ hybridization (FISH) with probes for the HER2 gene and for the chromosome 17 centromere (CEP17). The latter probe is used as a surrogate for chromosome 17 copies, however chromosome 17 (Chr17) is frequently rearranged. The frequency and type of specific structural Chr17 alterations in breast cancer have been studied by using comparative genomic hybridization and spectral karyotyping, but not fully detailed. Actually, balanced chromosome rearrangements (e.g. translocations or inversions) and low frequency mosaicisms are assessable on metaphases using G-banding karyotype and multicolor FISH (M-FISH) only. Methods: We sought to elucidate the CEP17 and HER2 FISH patterns of interphase nuclei by evaluating Chr17 rearrangements in metaphases of 9 breast cancer cell lines and a primary culture from a triple negative breast carcinoma by using G-banding, FISH and M-FISH. Results: Thirty-nine rearranged chromosomes containing a portion of Chr17 were observed. Chromosomes 8 and 11 were the most frequent partners of Chr17 translocations. The lowest frequency of Chr17 abnormalities was observed in the HER2-negative cell lines, while the highest was observed in the HER2-positive SKBR3 cells.
    [Show full text]
  • Genome-Wide TOP2A DNA Cleavage Is Biased Toward Translocated and Highly Transcribed Loci
    Downloaded from genome.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Method Genome-wide TOP2A DNA cleavage is biased toward translocated and highly transcribed loci Xiang Yu,1,8 James W. Davenport,2,8 Karen A. Urtishak,2 Marie L. Carillo,2,9 Sager J. Gosai,1,10 Christos P. Kolaris,2,11 Jo Ann W. Byl,3 Eric F. Rappaport,4 Neil Osheroff,3,5,6 Brian D. Gregory,1 and Carolyn A. Felix2,7 1Biology Department, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA; 2Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA; 3Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232, USA; 4NAPCore, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA; 5Department of Medicine (Hematology/Oncology), Vanderbilt University, Nashville, Tennessee 37232, USA; 6VA Tennessee Valley Healthcare System, Nashville, Tennessee 37212, USA; 7Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA Type II topoisomerases orchestrate proper DNA topology, and they are the targets of anti-cancer drugs that cause treat- ment-related leukemias with balanced translocations. Here, we develop a high-throughput sequencing technology to define TOP2 cleavage sites at single-base precision, and use the technology to characterize TOP2A cleavage genome-wide in the human K562 leukemia cell line. We find that TOP2A cleavage has functionally conserved local sequence preferences, occurs in cleavage cluster regions (CCRs), and is enriched in introns and lincRNA loci. TOP2A CCRs are biased toward the distal regions of gene bodies, and TOP2 poisons cause a proximal shift in their distribution.
    [Show full text]
  • Type II DNA Topoisomerases Cause Spontaneous Double-Strand Breaks in Genomic DNA
    G C A T T A C G G C A T genes Review Type II DNA Topoisomerases Cause Spontaneous Double-Strand Breaks in Genomic DNA Suguru Morimoto 1, Masataka Tsuda 1, Heeyoun Bunch 2 , Hiroyuki Sasanuma 1, Caroline Austin 3 and Shunichi Takeda 1,* 1 Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan; [email protected] (S.M.); [email protected] (M.T.); [email protected] (H.S.) 2 Department of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 41566, Korea; [email protected] 3 The Institute for Cell and Molecular Biosciences, the Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK; [email protected] * Correspondence: [email protected]; Tel.: +81-(075)-753-4411 Received: 30 September 2019; Accepted: 26 October 2019; Published: 30 October 2019 Abstract: Type II DNA topoisomerase enzymes (TOP2) catalyze topological changes by strand passage reactions. They involve passing one intact double stranded DNA duplex through a transient enzyme-bridged break in another (gated helix) followed by ligation of the break by TOP2. A TOP2 poison, etoposide blocks TOP2 catalysis at the ligation step of the enzyme-bridged break, increasing the number of stable TOP2 cleavage complexes (TOP2ccs). Remarkably, such pathological TOP2ccs are formed during the normal cell cycle as well as in postmitotic cells. Thus, this ‘abortive catalysis’ can be a major source of spontaneously arising DNA double-strand breaks (DSBs).
    [Show full text]