DOCSLIB.ORG

Analysis of the Multivalent Binding Properties of the Novel H2A.Z Interactor PWWP2A

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Analysis of the Multivalent Binding Properties of the Novel H2A.Z Interactor PWWP2A Aus dem Adolf-Butenandt-Institut, Lehrstuhl: Molekularbiologie im Biomedizinischen Centrum München (BMC) der LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN Vorstand: Prof. Dr. Peter B. Becker und der JUSTUS-LIEBIG-UNIVERSITÄT GIESSEN Analysis of the multivalent binding properties of the novel H2A.Z interactor PWWP2A Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München Ramona Spitzer aus Günzburg 2018 Mit Genehmigung der Medizinischen Fakultät der Ludwig-Maximilians-Universität München Betreuerin: Prof. Dr. Sandra B. Hake Zweitgutachter: Prof. Dr. Andreas Ladurner Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 14.09.2018 Eidesstattliche Versicherung Ich, Ramona Spitzer, erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Titel: “Analysis of the multivalent binding properties of the novel H2A.Z interactor PWWP2A” selbständig verfasst, mich außer der angegebenen keiner weiteren Hilfsmittel bedient und alle Erkenntnisse, die aus dem Schrifttum ganz oder annähernd übernommen sind, als solche kenntlich gemacht und nach ihrer Herkunft unter Bezeichnung der Fundstelle einzeln nachgewiesen habe. Ich erkläre des Weiteren, dass die hier vorgelegte Dissertation nicht in gleicher oder in ähnlicher Form bei einer anderen Stelle zur Erlangung eines akademischen Grades eingereicht wurde. München, den ……14.09.2018……. …………………………................ (Ramona Spitzer) Acknowledgements First, I would like to thank my supervisor Prof. Dr. Sandra B. Hake for the opportunity to work as a PhD student in her lab. Sandra, you always pushed my thesis with constant input and ideas and it was always fun to discuss my project or to defend my point of view. You gave me the opportunity to improve myself in giving talks, discussing and defending scientific issues and strengthening my personality. On top, you always had a sympathetic ear for me, no matter if it was about scientific questions or personal problems. Thank you so much for letting me be a part of the amazing “HAKE- GIRLS”. Next, I want to thank Dr. Catherine Regnard (“PWWP”-Team) and Dr. Felix Müller-Planitz for being members of my thesis advisory committee. There was always an “open door” to discuss questions and to get helpful advice for my PhD project. I am happy for the opportunity to work in the Biomedical Center (BMC) and being part of a great “chromatin-network-community” and the IRTG1064 (thanks Elizabeth for being such a great coordinator). There was no question, method or reagent which could not be answered, learned or “borrowed”. I want to thank all former members of the Hake-Lab and of course our former master student Lucia (sushi & rice-balls-4-ever). It was a pleasure to work together with you and I really enjoyed your company. Furthermore, I want to thank Stephi Link (the crazy cat lady) for being the other 50% of the Hake-Pair (LMU-Crew). It was always fun to work together with you and discuss all the gossip, which was floating around. I think with you, it is not possible to miss anything new or secret in the lab community. I was also lucky to share the lab with the former and current FMP´s (especially Sabrina, Ashish and Petra), where I got constantly input for “in vitro” questions like purification (Äkta), assemblies and cloning strategies or just having a little chit-chat. In addition, I am grateful that I could experience collaborative work with outstanding scientists here in Munich and all over the world: Stephanie Link, Moritz Völker-Albert, Axel Imhof, Masahiko Harata, Thomas Burgold, Brian Hendrich, Mariam Sana, Mario Torrado del Rey and Joel Mackay. Last but not least I want to thank my mum Elisabeth and my dad Anton for always standing behind me, my brothers Jonathan and Fabian and my whole family and friends for their constant support and motivating words. A special thanks goes to my better half Tobi who was always at my side, motivating me when things weren´t running well and pushing me to go on. Thank you! In silent remembrance of Karl Bader who always believed in science and technical progress and Agnes Müller, my beloved grandmother, who is greatly missed here. TABLE OF CONTENTS Summary .................................................................................................................................... 1 Zusammenfassung ...................................................................................................................... 3 1 Introduction ........................................................................................................................ 5 1.1 The structure of chromatin ......................................................................................... 5 1.2 Alteration of chromatin structure ............................................................................... 8 1.2.1 Posttranslational modifications of histones ....................................................... 11 1.2.2 Histone variants .................................................................................................. 13 1.2.3 H2A variants ....................................................................................................... 15 1.3 The histone variant H2A.Z ......................................................................................... 17 1.4 H2A.Z binding proteins .............................................................................................. 21 1.5 PWWP domain-containing proteins .......................................................................... 25 1.6 Objectives .................................................................................................................. 27 2 Material and Methods ...................................................................................................... 28 2.1 Materials .................................................................................................................... 28 2.1.1 Technical devices ................................................................................................ 28 2.1.2 Consumables ...................................................................................................... 30 2.1.3 Chemicals ........................................................................................................... 31 2.1.4 Kits, enzymes and markers ................................................................................. 33 2.1.5 Antibodies .......................................................................................................... 34 2.1.6 Plasmids .............................................................................................................. 35 2.1.7 Oligonucleotides ................................................................................................. 36 2.1.8 Bacterial strains and cell lines ............................................................................ 38 2.1.9 Software ............................................................................................................. 38 2.1.10 Buffers and solutions .......................................................................................... 39 2.2 Cell biological methods .............................................................................................. 41 2.2.1 Mammalian cell cultivation ................................................................................ 41 2.2.2 Transient transfection of mammalian cells ........................................................ 42 2.3 Molecular biological methods ................................................................................... 42 2.3.1 Agarose gel electrophoresis ............................................................................... 42 2.3.2 Cloning of PWWP2A truncations ........................................................................ 42 2.3.3 Site-directed mutagenesis .................................................................................. 43 I 2.4 Biochemical methods ................................................................................................ 44 2.4.1 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ........................................ 44 2.4.2 Coomassie staining of SDS-PAGE gels ................................................................ 44 2.4.3 Immunoblotting ................................................................................................. 44 2.4.4 Mononucleosome preparation of HeLa Kyoto cells ........................................... 45 2.4.5 Purification of MNase digested DNA .................................................................. 46 2.4.6 Purification of recombinant GST-tagged PWWP2A and PWWP2A truncations 47 2.4.7 Identification of histone modifications by Mass-Spectrometry (MS) ................ 50 2.4.8 Reconstitution of recombinant mononucleosomes .......................................... 52 2.4.9 In vitro binding assays with recombinant PWWP2A constructs ........................ 58 2.4.10 NMR spectroscopy ............................................................................................. 62 2.5 Bioinformatics ............................................................................................................ 62 2.5.1 MS Data analysis................................................................................................. 62 3 Results ..............................................................................................................................
Load more

Recommended publications
  • The Functions of DNA Damage Factor RNF8 in the Pathogenesis And
    Int. J. Biol. Sci. 2019, Vol. 15 909 Ivyspring International Publisher International Journal of Biological Sciences 2019; 15(5): 909-918. doi: 10.7150/ijbs.31972 Review The Functions of DNA Damage Factor RNF8 in the Pathogenesis and Progression of Cancer Tingting Zhou 1, Fei Yi 1, Zhuo Wang 1, Qiqiang Guo 1, Jingwei Liu 1, Ning Bai 1, Xiaoman Li 1, Xiang Dong 1, Ling Ren 2, Liu Cao 1, Xiaoyu Song 1 1. Institute of Translational Medicine, China Medical University; Key Laboratory of Medical Cell Biology, Ministry of Education; Liaoning Province Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, Shenyang, Liaoning Province, China 2. Department of Anus and Intestine Surgery, First Affiliated Hospital of China Medical University, Shenyang, Liaoning Province, China Corresponding authors: Xiaoyu Song, e-mail: [email protected] and Liu Cao, e-mail: [email protected]. Key Laboratory of Medical Cell Biology, Ministry of Education; Institute of Translational Medicine, China Medical University; Collaborative Innovation Center of Aging Related Disease Diagnosis and Treatment and Prevention, Shenyang, Liaoning Province, 110122, China. Tel: +86 24 31939636, Fax: +86 24 31939636. © Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2018.12.03; Accepted: 2019.02.08; Published: 2019.03.09 Abstract The really interesting new gene (RING) finger protein 8 (RNF8) is a central factor in DNA double strand break (DSB) signal transduction.
    [Show full text]
  • Functional Roles of Bromodomain Proteins in Cancer
    cancers Review Functional Roles of Bromodomain Proteins in Cancer Samuel P. Boyson 1,2, Cong Gao 3, Kathleen Quinn 2,3, Joseph Boyd 3, Hana Paculova 3 , Seth Frietze 3,4,* and Karen C. Glass 1,2,4,* 1 Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Colchester, VT 05446, USA; [email protected] 2 Department of Pharmacology, Larner College of Medicine, University of Vermont, Burlington, VT 05405, USA; [email protected] 3 Department of Biomedical and Health Sciences, University of Vermont, Burlington, VT 05405, USA; [email protected] (C.G.); [email protected] (J.B.); [email protected] (H.P.) 4 University of Vermont Cancer Center, Burlington, VT 05405, USA * Correspondence: [email protected] (S.F.); [email protected] (K.C.G.) Simple Summary: This review provides an in depth analysis of the role of bromodomain-containing proteins in cancer development. As readers of acetylated lysine on nucleosomal histones, bromod- omain proteins are poised to activate gene expression, and often promote cancer progression. We examined changes in gene expression patterns that are observed in bromodomain-containing proteins and associated with specific cancer types. We also mapped the protein–protein interaction network for the human bromodomain-containing proteins, discuss the cellular roles of these epigenetic regu- lators as part of nine different functional groups, and identify bromodomain-specific mechanisms in cancer development. Lastly, we summarize emerging strategies to target bromodomain proteins in cancer therapy, including those that may be essential for overcoming resistance. Overall, this review provides a timely discussion of the different mechanisms of bromodomain-containing pro- Citation: Boyson, S.P.; Gao, C.; teins in cancer, and an updated assessment of their utility as a therapeutic target for a variety of Quinn, K.; Boyd, J.; Paculova, H.; cancer subtypes.
    [Show full text]
  • Mediator of DNA Damage Checkpoint Protein 1 Facilitates V(D)J Recombination in Cells Lacking DNA Repair Factor XLF
    biomolecules Article Mediator of DNA Damage Checkpoint Protein 1 Facilitates V(D)J Recombination in Cells Lacking DNA Repair Factor XLF Carole Beck 1,2, Sergio Castañeda-Zegarra 1,2 , Camilla Huse 1,2, Mengtan Xing 1,2 and Valentyn Oksenych 1,2,3,* 1 Department of Clinical and Molecular Medicine (IKOM), Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway; [email protected] (C.B.); [email protected] (S.C.-Z.); [email protected] (C.H.); [email protected] (M.X.) 2 St. Olavs Hospital, Trondheim University Hospital, Clinic of Medicine, Postboks 3250 Sluppen, 7006 Trondheim, Norway 3 Department of Biosciences and Nutrition (BioNuT), Karolinska Institutet, 14183 Huddinge, Sweden * Correspondence: [email protected] Received: 27 November 2019; Accepted: 24 December 2019; Published: 30 December 2019 Abstract: DNA double-strand breaks (DSBs) trigger the Ataxia telangiectasia mutated (ATM)-dependent DNA damage response (DDR), which consists of histone H2AX, MDC1, RNF168, 53BP1, PTIP, RIF1, Rev7, and Shieldin. Early stages of B and T lymphocyte development are dependent on recombination activating gene (RAG)-induced DSBs that form the basis for further V(D)J recombination. Non-homologous end joining (NHEJ) pathway factors recognize, process, and ligate DSBs. Based on numerous loss-of-function studies, DDR factors were thought to be dispensable for the V(D)J recombination. In particular, mice lacking Mediator of DNA Damage Checkpoint Protein 1 (MDC1) possessed nearly wild-type levels of mature B and T lymphocytes in the spleen, thymus, and bone marrow. NHEJ factor XRCC4-like factor (XLF)/Cernunnos is functionally redundant with ATM, histone H2AX, and p53-binding protein 1 (53BP1) during the lymphocyte development in mice.
    [Show full text]
  • Clinical Report of a Neonate Carrying a Large Deletion in the 10P15.3P13
    Shao et al. Mol Cytogenet (2021) 14:29 https://doi.org/10.1186/s13039-021-00546-1 CASE REPORT Open Access Clinical report of a neonate carrying a large deletion in the 10p15.3p13 region and review of the literature Qiao‑Yan Shao, Pei‑Lin Wu, Bi‑Yun Lin, Sen‑Jing Chen, Jian Liu and Su‑Qing Chen* Abstract Background: Terminal deletion of chromosome 10p is a rare chromosomal abnormality. We report a neonatal case with a large deletion of 10p15.3p13 diagnosed early because of severe clinical manifestations. Case presentation: Our patient presented with specifc facial features, hypoparathyroidism, sen sorineural deafness, renal abnormalities, and developmental retardation, and carried a 12.6 Mb deletion in the 10p15.3 p13 region. The terminal 10p deletion involved in our patient is the second largest reported terminal deletion reported to date, and includes the ZMYND11 and GATA3 genes and a partial critical region of the DiGeorge syndrome 2 gene (DGS2). Conclusion: On the basis of a literature review, this terminal 10p deletion in the present case is responsible for a spe‑ cifc contiguous gene syndrome. This rare case may help the understanding of the genotype–phenotype spectrum of terminal deletion of chromosome 10p. Keywords: 10p15.3 microdeletion syndrome, HDR syndrome, DiGeorge critical region 2, ZMYND11, GATA3 Background Here, we report a Chinese infant showing specifc Terminal deletion of chromosome 10p is a rare chromo- facial features, congenital hypoparathyroidism, sen- somal disorder. Te haploinsufciency in the distal region sorineural hearing loss, absence of the right kidney, a of 10p15.3 is responsible for 10p15.3 microdeletion syn- sacrococcygeal mass, a right frontal cyst, and psycho- drome (OMIM 608,668), characterized by specifc facial motor retardation.
    [Show full text]
  • Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model
    Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 T + is online at: average * The Journal of Immunology , 34 of which you can access for free at: 2016; 197:1477-1488; Prepublished online 1 July from submission to initial decision 4 weeks from acceptance to publication 2016; doi: 10.4049/jimmunol.1600589 http://www.jimmunol.org/content/197/4/1477 Molecular Profile of Tumor-Specific CD8 Cell Hypofunction in a Transplantable Murine Cancer Model Katherine A. Waugh, Sonia M. Leach, Brandon L. Moore, Tullia C. Bruno, Jonathan D. Buhrman and Jill E. Slansky J Immunol cites 95 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html http://www.jimmunol.org/content/suppl/2016/07/01/jimmunol.160058 9.DCSupplemental This article http://www.jimmunol.org/content/197/4/1477.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 25, 2021. The Journal of Immunology Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model Katherine A.
    [Show full text]
  • Identification of Cyclophilin Gene Family in Soybean and Characterization of Gmcyp1
    Western University Scholarship@Western Electronic Thesis and Dissertation Repository 7-4-2013 12:00 AM Identification of Cyclophilin gene family in soybean and characterization of GmCYP1 Hemanta Raj Mainali The University of Western Ontario Supervisor Dr. Sangeeta Dhaubhadel The University of Western Ontario Graduate Program in Biology A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Hemanta Raj Mainali 2013 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Agricultural Science Commons, Biochemistry Commons, Bioinformatics Commons, Biology Commons, Biotechnology Commons, Molecular Biology Commons, Plant Biology Commons, and the Plant Breeding and Genetics Commons Recommended Citation Mainali, Hemanta Raj, "Identification of Cyclophilin gene family in soybean and characterization of GmCYP1" (2013). Electronic Thesis and Dissertation Repository. 1362. https://ir.lib.uwo.ca/etd/1362 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Identification of Cyclophilin gene family in soybean and characterization of GmCYP1 (Thesis format: Monograph) by Hemanta Raj Mainali Graduate Program in Biology A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science The School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada © Hemanta Raj Mainali 2013 Abstract I identified members of the Cyclophilin (CYP) gene family in soybean (Glycine max) and characterized the GmCYP1, one of the members of soybean CYP.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • 4-6 Weeks Old Female C57BL/6 Mice Obtained from Jackson Labs Were Used for Cell Isolation
    Methods Mice: 4-6 weeks old female C57BL/6 mice obtained from Jackson labs were used for cell isolation. Female Foxp3-IRES-GFP reporter mice (1), backcrossed to B6/C57 background for 10 generations, were used for the isolation of naïve CD4 and naïve CD8 cells for the RNAseq experiments. The mice were housed in pathogen-free animal facility in the La Jolla Institute for Allergy and Immunology and were used according to protocols approved by the Institutional Animal Care and use Committee. Preparation of cells: Subsets of thymocytes were isolated by cell sorting as previously described (2), after cell surface staining using CD4 (GK1.5), CD8 (53-6.7), CD3ε (145- 2C11), CD24 (M1/69) (all from Biolegend). DP cells: CD4+CD8 int/hi; CD4 SP cells: CD4CD3 hi, CD24 int/lo; CD8 SP cells: CD8 int/hi CD4 CD3 hi, CD24 int/lo (Fig S2). Peripheral subsets were isolated after pooling spleen and lymph nodes. T cells were enriched by negative isolation using Dynabeads (Dynabeads untouched mouse T cells, 11413D, Invitrogen). After surface staining for CD4 (GK1.5), CD8 (53-6.7), CD62L (MEL-14), CD25 (PC61) and CD44 (IM7), naïve CD4+CD62L hiCD25-CD44lo and naïve CD8+CD62L hiCD25-CD44lo were obtained by sorting (BD FACS Aria). Additionally, for the RNAseq experiments, CD4 and CD8 naïve cells were isolated by sorting T cells from the Foxp3- IRES-GFP mice: CD4+CD62LhiCD25–CD44lo GFP(FOXP3)– and CD8+CD62LhiCD25– CD44lo GFP(FOXP3)– (antibodies were from Biolegend). In some cases, naïve CD4 cells were cultured in vitro under Th1 or Th2 polarizing conditions (3, 4).
    [Show full text]
  • Further Insights Into the Regulation of the Fanconi Anemia FANCD2 Protein
    University of Rhode Island DigitalCommons@URI Open Access Dissertations 2015 Further Insights Into the Regulation of the Fanconi Anemia FANCD2 Protein Rebecca Anne Boisvert University of Rhode Island, [email protected] Follow this and additional works at: https://digitalcommons.uri.edu/oa_diss Recommended Citation Boisvert, Rebecca Anne, "Further Insights Into the Regulation of the Fanconi Anemia FANCD2 Protein" (2015). Open Access Dissertations. Paper 397. https://digitalcommons.uri.edu/oa_diss/397 This Dissertation is brought to you for free and open access by DigitalCommons@URI. It has been accepted for inclusion in Open Access Dissertations by an authorized administrator of DigitalCommons@URI. For more information, please contact [email protected]. FURTHER INSIGHTS INTO THE REGULATION OF THE FANCONI ANEMIA FANCD2 PROTEIN BY REBECCA ANNE BOISVERT A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CELL AND MOLECULAR BIOLOGY UNIVERSITY OF RHODE ISLAND 2015 DOCTOR OF PHILOSOPHY DISSERTATION OF REBECCA ANNE BOISVERT APPROVED: Dissertation Committee: Major Professor Niall Howlett Paul Cohen Becky Sartini Nasser H. Zawia DEAN OF THE GRADUATE SCHOOL UNIVERSITY OF RHODE ISLAND 2015 ABSTRACT Fanconi anemia (FA) is a rare autosomal and X-linked recessive disorder, characterized by congenital abnormalities, pediatric bone marrow failure and cancer susceptibility. FA is caused by biallelic mutations in any one of 16 genes. The FA proteins function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). The monoubiquitination of FANCD2 and FANCI is a central step in the activation of the FA-BRCA pathway and is required for targeting these proteins to chromatin.
    [Show full text]
  • Polo-Like Kinase 1 in Genomic Instability
    Polo-like Kinase 1 in genomic instability Xiaoqi Liu Department of Toxicology and Cancer Biology University of Kentucky The fate of a single parental chromosome throughout the eukaryotic cell cycle Cytokinesis Activation of cell division Preparation for mitosis The stages of mitosis and cytokinesis in an animal cell MT MT - + kinetochore Centrosome Spindle pole + (MTOC) Spindle pole Centrosome Spindle Nucleus G2 Prophase Prometaphase Metaphase Central spindle Contractile ring Midbody Anaphase Telophase Cytokinesis Control of mitotic entry and exit by Cdc2/cyclin B and APC Anaphase promoting complex (APC) Cdc2 Cyclin B Cdc2 Anaphase inhibitor Cyclin B Cdc2 G2 Prophase Metaphase Anaphase Telophase G1 (Liu & Erikson, PNAS 2002; Liu & Erikson, PNAS 2003; Liu et al, MCB 2005; Liu et al, MCB 2006) The polo-like kinases have a C-terminal polo-box domain M-phase kinase domain polo-box Plk1 603 Plx1 (Xl) 80% 598 polo (Dm) 65% 576 Plo1 (Sp) 50% 683 Cdc5 (Sc) 51% 705 Early phase of cell cycle Plk2 52% 683 Plk3 52% 631 Plk1 localizes to mitotic structures G2/pro Prometa Spindle poles Centrosomes kinetochores Centrosomes Centrosomes kinetochores Meta Telo Spindle poles Midbodies kinetochores Spindle poles Central spindles kinetochores Plk1 in cancer ---Plk1 overexpression is correlated with cell proliferation and carcinogenesis. ---Plk1 is a new diagnostic marker for cancer. ---Plk1 inhibitors are in various clinical trails. ---However, how Plk1 contributes to carcinogenesis is elusive. Does Plk1 elevation contribute to cancer progression? Approach:
    [Show full text]
  • Insights Into Regulation of Human RAD51 Nucleoprotein Filament Activity During
    Insights into Regulation of Human RAD51 Nucleoprotein Filament Activity During Homologous Recombination Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ravindra Bandara Amunugama, B.S. Biophysics Graduate Program The Ohio State University 2011 Dissertation Committee: Richard Fishel PhD, Advisor Jeffrey Parvin MD PhD Charles Bell PhD Michael Poirier PhD Copyright by Ravindra Bandara Amunugama 2011 ABSTRACT Homologous recombination (HR) is a mechanistically conserved pathway that occurs during meiosis and following the formation of DNA double strand breaks (DSBs) induced by exogenous stresses such as ionization radiation. HR is also involved in restoring replication when replication forks have stalled or collapsed. Defective recombination machinery leads to chromosomal instability and predisposition to tumorigenesis. However, unregulated HR repair system also leads to similar outcomes. Fortunately, eukaryotes have evolved elegant HR repair machinery with multiple mediators and regulatory inputs that largely ensures an appropriate outcome. A fundamental step in HR is the homology search and strand exchange catalyzed by the RAD51 recombinase. This process requires the formation of a nucleoprotein filament (NPF) on single-strand DNA (ssDNA). In Chapter 2 of this dissertation I describe work on identification of two residues of human RAD51 (HsRAD51) subunit interface, F129 in the Walker A box and H294 of the L2 ssDNA binding region that are essential residues for salt-induced recombinase activity. Mutation of F129 or H294 leads to loss or reduced DNA induced ATPase activity and formation of a non-functional NPF that eliminates recombinase activity. DNA binding studies indicate that these residues may be essential for sensing the ATP nucleotide for a functional NPF formation.
    [Show full text]
  • Identification of Novel Chemotherapeutic Strategies For
    www.nature.com/scientificreports OPEN Identification of novel chemotherapeutic strategies for metastatic uveal melanoma Received: 17 November 2016 Paolo Fagone1, Rosario Caltabiano2, Andrea Russo3, Gabriella Lupo1, Accepted: 09 February 2017 Carmelina Daniela Anfuso1, Maria Sofia Basile1, Antonio Longo3, Ferdinando Nicoletti1, Published: 17 March 2017 Rocco De Pasquale4, Massimo Libra1 & Michele Reibaldi3 Melanoma of the uveal tract accounts for approximately 5% of all melanomas and represents the most common primary intraocular malignancy. Despite improvements in diagnosis and more effective local therapies for primary cancer, the rate of metastatic death has not changed in the past forty years. In the present study, we made use of bioinformatics to analyze the data obtained from three public available microarray datasets on uveal melanoma in an attempt to identify novel putative chemotherapeutic options for the liver metastatic disease. We have first carried out a meta-analysis of publicly available whole-genome datasets, that included data from 132 patients, comparing metastatic vs. non metastatic uveal melanomas, in order to identify the most relevant genes characterizing the spreading of tumor to the liver. Subsequently, the L1000CDS2 web-based utility was used to predict small molecules and drugs targeting the metastatic uveal melanoma gene signature. The most promising drugs were found to be Cinnarizine, an anti-histaminic drug used for motion sickness, Digitoxigenin, a precursor of cardiac glycosides, and Clofazimine, a fat-soluble iminophenazine used in leprosy. In vitro and in vivo validation studies will be needed to confirm the efficacy of these molecules for the prevention and treatment of metastatic uveal melanoma. Uveal melanoma is the most common primary intraocular cancer, and after the skin, the uveal tract is the second most common location for melanoma1.
    [Show full text]