FREE ELECTRONIC LIBRARY - Abstract, dissertation, book

Pages:   || 2 | 3 | 4 | 5 |   ...   | 23 |

«     A University of Sussex DPhil thesis  Available online via Sussex Research Online:    ...»

-- [ Page 1 ] --



A University of Sussex DPhil thesis 

Available online via Sussex Research Online: 


This thesis is protected by copyright which belongs to the author.   

This thesis cannot be reproduced or quoted extensively from without first 

obtaining permission in writing from the Author   

The content must not be changed in any way or sold commercially in any 

format or medium without the formal permission of the Author    When referring to this work, full bibliographic details including the  author, title, awarding institution and date of the thesis must be given  Please visit Sussex Research Online for more information and further details    Cellular and biochemical analyses of TDP1 mediated chromosomal break repair A thesis submitted to the University of Sussex for the degree of Doctor of Philosophy By Owen Spencer Wells 2 Declaration I hereby declare that this thesis has not been and will not be, submitted in whole or in part to another University for the award of any other degree.

Signed ………………………………………………………………………… Owen S. Wells Acknowledgements First of all I would like to express my gratitude to Dr Sherif El-Khamisy for giving me the opportunity to undertake a PhD and for his continued guidance and support throughout. A special thank you to Jessica Hudson for looking after me, spending time listening to my half-baked ideas and sharing her expertise within the laboratory. I would like to thank Aaron, Mariella and Kyle for their encouragement and input in my work, and also to all the members in the El-Khamisy lab, Jude and Conny, my office and the GDSC for putting up with me for the last 3.5 years and making my time here enjoyable.

Finally, a special mention to Diana, Chris, Sophie, Carol and Marcel for helping me through this last year.

Outside of the centre I would like to thank my Mother for whom I am extremely grateful and proud of, for raising my brother and myself and helping us accomplish the goals in our life. My brother, Robert, who I look up too and whose drive and success in anything he puts his mind to has inspired me to do the same, or at least try. The rest of my family especially my Nan, Grandma and Grandad who have all had a pivotal role in my upbringing and enabled me to be the person I am today. Last but not least my uncle Mark whose modest persona has rubbed off on me and has alwayskept me grounded.

Of my friends back home I would like to thank Huw Wilkinson, Jonathan James, Jon Gouran, Gareth Sherrington, James Edwards, Richard Martin and Chris Jones for helping me maintain a social life, for always showing an interest in what I do and listening when I talk about my research. Everyone at Littlemore rugby club and my old house mates Bunk and Tim for helping take my mind off work.

–  –  –

Tyrosyl DNA phosphodiesterase 1 (TDP1) is an end-processing enzyme involved in the repair of abortive topoisomerase I (Top1) complexes. Although not essential for survival, a hypomorphic mutation in TDP1 is linked to the autosomal recessive ataxia, spinocerebellar ataxia with axonal neuropathy 1 (SCAN1).

SCAN1 is a rare human condition linked with neurodegeneration and ataxic gait and patients are usually wheel chair bound by their early teens. TDP1 primarily cleaves lesions at the 3’-end of DNA breaks and its most prominent substrate is stalled Top1 linked to the 3’-terminus of DNA. The enzymatic mechanism by which TDP1 functions are well understood and inhibitors are now being investigated for treatment of cancer. In contrast, the processes involved in TDP1 recruitment, localisation and regulation during the DNA damage response remain unclear. This thesis investigates how the evolutionarily driven N-terminus of TDP1, not conserved in lower Eukaryotes, is required for optimal cellular protection against genotoxic stress. I also characterise how post-translational modifications of TDP1 allow for efficient repair of transcriptionally associated, chromosomal single-strand breaks and uncover new protein interacting partners of TDP1 and their role in TDP1 mediated repair.

–  –  –

Table of Contents Chapter I


1.1 General introduction

1.2 Sources of DNA damage

1.2.1 Endogenous sources of DNA damage Reactive oxygen species Methylation Hydrolysis Mismatch of bases

1.2.2 Exogenous sources of DNA damage UV Ionising radiation

1.3 DNA repair

1.3.1 DNA double-strand break repair Homologous recombination Non-homologous end-joining

1.3.2 Nucleotide excision repair

1.3.3 Mismatch repair

1.3.4 Base excision repair

1.3.5 Single-strand break repair Detection: PARP End-processing Gap filling Ligation

1.3.6 Ligase family Ligase I Ligase III Ligase IV

1.3.7 Protein-linked DNA single-strand breaks

1.4 Tyrosyl DNA phosphodiesterase 1

1.5 Diseases associated with defects in single-strand break repair

1.5.1 Neural development

1.5.2 Autosomal recessive ataxias

1.5.3 Spinocerebellar ataxia with axonal neuropathy 1 (SCAN1)

1.5.4 Microcephaly, infantile-onset seizures, and developmental delay (MCSZ).. 31 1.5.5 X-linked mental retardation (XLMR)

1.5.6 Ataxia oculomotor apraxia 1 (AOA1)

1.5.7 Ataxia oculomotor apraxia 2 (AOA2)

1.6 Neurodegeneration: a consequence of repair defect in the nucleus, mitochondria or both?

1.7 Aims and objectives

Chapter II


Materials and Methods

2.1 Materials

2.2 Cloning and Molecular methods

2.2.1 PCR, restriction digests and ligations

2.2.2 Site-directed mutagenesis

2.2.3 Competent cells and transformations E.coli DH5α cells Transformations

2.3 Electrophoresis and western blot analysis

2.3.1 Electrophoresis of DNA

2.3.2 Electrophoresis of proteins Coomassie blue staining Silver staining

2.4 Western blots

2.5 Protein expression and purification

2.5.1 Protein expression

2.5.2 Protein purification Cell lysis Immobilised metal affinity chromatography (IMAC) purification......... 47

2.6 Biochemical assays

2.6.1 SUMOylation assay

2.6.2 SUMO and TDP1 non-covalent binding studies Recombinant TDP1 binding to SUMO immobilised resin

2.6.3 Casein Kinase 2 phosphorylation

2.7 Protein structure and folding

2.7.1 Circular dichroism Quantitative analysis of circular dichroism by Dichroweb

2.7.2 Protein denaturation curves

2.8 Mammalian cell culture

2.8.1 Maintenance of cell lines

2.8.2 Transfection Calcium phosphate transfection Transfection by electroporation

2.9 Cell harvest and lysis

2.10 Co-immunoprecipitation

2.11 Creating stable cell lines in DT40 Tdp1-/- cells

2.12 Viability assays

2.13 FACS analysis

2.14 DNA single-strand break assays

2.14.1 Alkaline comet assay Preparation

2.14.2 Gyrosyl assay Preparation of oligonucleotide substrates: TDP1 fluorescence assay:

2.15 Mass spectroscopy

Chapter III

Results 1: The N-terminal evolutionarily driven domain of TDP1 is required for optimal cellular protection against genotoxic stress

3.1 Introduction


3.1.1 TDP1; discovery, substrate and mechanism of action

3.1.2 TDP1’s role in SCAN1

3.1.3 Chapter objectives

3.2 Results

3.2.1 Full-length TDP1 and the catalytic domain show no difference in activity, structure or stability The C-terminal domain does not demonstrate a reduction in enzymatic activity in vitro TDP1 and TDP1151-608 have no difference in thermal stability, or structure

3.2.2 Full-length hTDP1 is required for optimal protection against DNA damaging agents in DT40 cells, whilst hTDP1151-608 gives partial protection Tdp1-/- DT40 cells, complemented with Myc, Myc-hTDP1 or MychTDP1151-608 maintain similar proliferation rates and cell cycle progression..... 77 TDP1151-608 does not exhibit reduced activity in cell extracts................. 77 Protection of cell lines treated with different genotoxic agents shows that the TDP1151-608 gives partial resistance when compared to the full-length protein

3.2.3 Accumulation of DNA single-strand breaks in hTDP1151-608 compared to hTDP1, is in part transcription dependent DT40 Tdp1-/- cells, and cells complemented with hTDP1151-608 accumulate more single-strand breaks than full-length hTDP1 DT40 Tdp1-/- cells complemented with hTDP1151-608 accumulate more single-strand breaks than full-length hTDP1, and these appear to be transcription dependent

3.3 Discussion

Chapter IV

Results 2: SUMOylation facilitates TDP1 driven chromosomal break repair........ 91

4.1 Introduction

4.1.2 Post-translational modification by SUMOylation

4.1.3 SUMO

4.1.4 SUMOylation

4.1.5 SUMO family

4.1.6 Phospho-dependent SUMO motif

4.1.7 SUMO interacting motifs – SIMS

4.1.8 Molecular consequences of SUMOylation and SUMO-SIM interactions..... 96 4.1.9 Chapter objectives

4.2 Results

4.2.1 TDP1 is SUMOylated at lysine 111 by SUMO1 in vitro and vivo.............. 100 TDP1 is SUMOylated in vitro TDP1 SUMOylation occurs within the N-terminus in vitro................. 104 TDP1 SUMOylation occurs within the N-terminus at K111 in vitro... 106 Mutation of lysine to arginine at 111 abrogates SUMOylation in vitro and in vivo

4.2.2 Mutation of lysine 111 to arginine does not alter protein structure, stability or catalytic activity TDP1 and TDP1K111R give similar melting temperatures and unfolding patterns

vi TDP1K111R does not result in a reduced activity compared to wild-type TDP1K111R and TDP1 exhibit no significant structural differences...... 111 4.2.3 SUMOylation-deficient mutant accrues more DNA single-strand breaks that are in part transcription dependent TDP1 K111R mutant shows reduced survival in the presence of CPT 114 TDP1 K111R accumulates more DNA single-strand breaks in the presence of CPT Accumulation of DNA single-strand breaks is in part transcription dependant

4.2.4 TDP1 interacts non-covalently with SUMO2/3 TDP1 interacts with SUMO2 in vitro TDP1 interacts with SUMO2 via the catalytic domain in vitro............ 119 Identification of SIM/s in TDP1 Mutation of the first two amino acids in potential SIM sites results in instability and co-expression with GroEL TDP1 fragments within the catalytic domain causes loss of expression in vivo

4.3 Discussion

Chapter V

Results 3: The role of DNA ligases and CK2 during TDP1 mediated repair........ 132

5.1 Introduction

5.1.2 Ligase family and TDP1

5.1.3 Casein kinase II

5.1.4 Chapter objectives

5.2 Results

5.2.1 Ligase I and not ligase IIIα is implicated in TDP1-mediated nuclear repair136 Ligase I deficient cells results in hypersensitivity to camptothecin...... 136 The conserved catalytic domain of TDP1 is sufficient to maintain interaction with Lig I

5.2.2 TDP1 is a novel substrate for Casein Kinase II TDP1 is phosphorylated in vivo Phosphorylation of TDP1 occurs in the N-terminal domain................. 141 5.2.3 Phosphorylation of TDP1 by CK2 inhibits SUMOylation, in vitro............. 144 5.2.4 TDP1 is phosphorylated by CK2 in whole cell extracts

5.3 Discussion

Chapter VI

Discussion: Cellular and biochemical analyses of TDP1 mediated chromosomal break repair

6.1 Discussion



–  –  –

A-T ataxia-telangiectasia ATM ataxia-telangiectasia mutated ATR ataxia-telangiectasia mutated and Rad3-related ATRIP ATR interacting protein BER base excision repair BSA bovine serum albumin CDK cyclin-dependent kinase CHX cycloheximide CK2 casein kinase 2 CS Chicken serum CPT camptothecin DAPI 4`6-diamino-2-phenylindole DNA-PK DNA-dependent protein kinase DSB double-strand DNA break DRB 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole FCS foetal calf serum HR homologous recombination hrs hours HU hydroxyurea IF immunofluorescence IR ionising radiation LCL lymphoblastoid cell line MCPH primary microcephaly MEF mouse embryonic fibroblast


MMS methyl methanesulfonate MRN Mre11-Rad50-Nbs1 NER nucleotide excision repair NHEJ non-homologous end-joining NBS Nijmegen Breakage syndrome PARP Poly (ADP-ribose) polymerase PARPi PARP inhibitor PBS phosphate buffered saline PIKK phosphatidylinositol 3-kinase like kinase RNAi RNA interference ROS reactive oxygen species RPA replication protein A SCAN1 spinocerebellar ataxia with axonal neuropathy 1 SD standard deviation S.E.M standard error of the mean SSB single-strand DNA break ssDNA single-stranded DNA TBS tris-buffered saline TBB 4,5,6,7-Tetrabromobenzotriazole TDP1 tyrosyl DNA phosphodiesterase 1 TOP1 topoisomerase I Ub ubiquitin UNT untreated UV ultra-violet WB western blot WCE whole cell extract WT wild-type XRCC1 X-ray repair cross-complementing protein 1

–  –  –

Figure 1.1 DNA strand break repair

8 Figure 1.2 Outcome of DNA strand breaks if left unrepaired.................

13 Figure 1.3 Enzymes involved in the repair of DNA strand breaks........

14 Figure 1.4 DNA structure and damage modifying 3’ and 5’ phosphate backbone of DNA

Pages:   || 2 | 3 | 4 | 5 |   ...   | 23 |

Similar works:

«Abschätzung zur Änderung der TreibhausgasEmissionen in den EU-Beitrittsstaaten zur Vorbereitung der Verhandlungen im Rahmen der Ausgestaltung des Kioto-Protokolls Im Auftrag des Bundesministerium für Umwelt, Naturschutz und Reaktorsicherheit G II 1 – 45134 – 18/6 Endbericht März 2001 Dr. Sebastian Oberthür Uwe Büsgen Dennis Tänzler Ecologic Institut für Internationale und Europäische Umweltpolitik Pfalzburger Str. 43/44, 10 717 Berlin, Tel: +49-30-86 880-118; Fax: +49-30-86 880-100...»

«Das Lande der Hobbits vom Weidental und Sonnental in Sonnmath und Mittjahr, den fünften Monat der Menschen Wetter: Der erste der beiden Sommermonate hat begonnen!! Vergessen ist das Unwetter des letzten Monats die Kälte vom Anfang des Jahres!! Es ist Sommer und die Hobbits schlagen sich vor Freude über das warme und trockene Wetter auf ihre runden Bäuchlein. Picknicks auf den blühenden Wiesen des Weidentals und des Sonnentals bestimmen das Leben der ruhigen und gemütlichen Hobbits....»

«PASTURES THE OF BEYOND Also by Dayton O. Hyde Fiction One Summer in Montana The Major, the Poacher, and the Wonderful One Trout River Nonfiction Sandy Yamsi The Last Free Man Raising Waterfowl in Captivity Don Coyote For Children The Brand of a Boy Cranes in My Corral Strange Companions Island of the Loons Thunder Down the Track The Bells of Lake Superior Mr. Beans PASTURES THE OF BEYOND AN OLD COWBOY LOOKS BACK AT THE OLD WEST Dayton O. Hyde Arcade Publishing • New York Copyright © 2005,...»

«1. Freshman Orientation (imdb aka Home of Phobia) A film directed by Ryan Shiraki RT: 91 min, 35mm color, aspect ratio 1.85, Dolby 5.1, 5 reels, Rated R for strong sexual content, language and some drug use Press site: www.regentreleasing.com/freshmanorientationpress Website: www.freshmanorientation-themovie.com Theatrical booking information: Materials: John A. Lambert 310-806-4278 Helene Nielsen Beal 310-806-4279 President, U.S. Theatrical Distribution Director, U.S. Theatrical Distribution...»

«Unterrichtsprinzip Erziehung zur Gleichstellung von Frauen und Männern Informationen und Anregungen zur Umsetzung in der Volksschule Unterrichtsprinzip „Erziehung zur Gleichstellung von Frauen und Männern“ Informationen und Anregungen zur Umsetzung in der Volksschule Wien, 2011 Impressum: Medieninhaber, Verleger und Herausgeber: Bundesministerium für Unterricht, Kunst und Kultur (Abteilung GM/Gender und Schule) A-1014 Wien, Minoritenplatz 5 Manuskripterstellung: Claudia Schneider, Renate...»

«INAUGURAL-DISSERTATION zur Erlangung der Doktorwürde der Naturwissenschaftlich-Mathematischen Gesamtfakultät der Ruprechts – Karl – Universität Heidelberg vorgelegt von Diplom-Biologin Elke Schultz aus: Pirmasens Tag der mündlichen Prüfung: 01. Juni 2006 Identifizierung und Charakterisierung einer Protein-ProteinInteraktion beim Morbus Parkinson Gutachter: Prof. Dr. Lutz Gissmann Gutachter: Priv.-Doz. Dr. Dieter Kübler An dieser Stelle möchte ich mich bei allen bedanken, die zum...»

«The New Yor Cit e w rk ty Elect Ve tric ehicle Rea ess Pl e adine lan: Unlock king Urban Dem Un mand           O T    w e l k n   r p o er 6  t d n g    e t c r   s a e o y s   m t e t  h e y m t     u w m e   y t m e u   y e s                 Academic Citation: The New York City Electric Vehicle Readiness Pla n: Unlocking Urban Demand, December 2012  e a d 0 by Ari Kahn and Ch–  –  –  ...»

«Menkes van den Briel Contact Menkes van den Briel Name National ICT Australia Company Researcher Position 223 Anzac Parade, Kensington 2033, NSW, Australia Address +61 (0)2 8306 0464 Phone +61 (0)2 8306 0405 Fax menkes@nicta.com.au Email http://www.menkes76.com Homepage Summary Menkes van den Briel is an operations research scientist with a consistent track record in developing practical solutions for complex real-world problems. His work is impactful both academically and in practice. His...»

«IN THE SUPREME COURT OF THE STATE OF FLORIDA CORPORATE SECURITIES GROUP, INC., Petitioner, v. Case No. SC-00-931 SHIRLEY LIND, Respondent. / AMICUS CURIAE BRIEF OF PUBLIC INVESTORS ARBITRATION BAR ASSOCIATION Stephen Krosschell, Esq. Goodman & Nekvasil, P.A. 14020 Roosevelt Blvd., Suite 808 P.O. Box 17709 Clearwater, FL 33762 Attorney for Amicus Curiae Public Investors Arbitration Bar Association TABLE OF CONTENTS TABLE OF CITATIONS..................................»

«GENERAL INFORMATION SUSTAINABLE DEVELOPMENT, GLOBAL CHANGE AND ECOSYSTEMS EUR 21935 Interested in European research? RTD info is our quarterly magazine keeping you in touch with main developments (results, programmes, events, etc.). It is available in English, French and German. A free sample copy or free subscription can be obtained from: European Commission Directorate-General for Research Information and Communication Unit B-1049 Brussels Fax (32-2) 29-58220 E-mail: research@cec.eu.int...»

«TECHNISCHE UNIVERSITÄT DORTMUND FAKULTÄT FÜR INFORMATIK Arthur Pyka Multikriterielle Exploration von Compileroptimierungen und Cacheparametern Diplomarbeit 21. Februar 2011 INTERNE BERICHTE INTERNAL REPORTS Lehrstuhl Informatik XII (Technische Informatik und Eingebettete Systeme) Gutachter: Prof. Dr. Peter Marwedel Dipl-Inform. Sascha Plazar GERMANY · D-44221 DORTMUND Ich danke allen voran meinem Betreuer Sascha Plazar, der mit viel Engagement und Geduld die Entstehung dieser Arbeit...»

«RFP CONTRACTOR City of Rialto Purchasing Division Purchasing Manager: William Jernigan Phone: (909) 820-2570 Fax: (909) 820-2600 Email: procurement@rialtoca.gov REQUEST FOR PROPOSAL #15-048 Auto Body Repair Services Closing Date: January 15, 2015, 4:00 P.M. RFP Number: 15-048 Due Date: January 15, 2015 Time: 4:00 P.M. Project: Auto Body Repair Services The prospective supplier shall submit a fully executed sealed proposal, to be received no later than 4:00 P.M., January 15, 2015. Sealed...»

<<  HOME   |    CONTACTS
2016 www.abstract.xlibx.info - Free e-library - Abstract, dissertation, book

Materials of this site are available for review, all rights belong to their respective owners.
If you do not agree with the fact that your material is placed on this site, please, email us, we will within 1-2 business days delete him.