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3.3.2 Telomere Dynamics of DNA Ligase I Deficient Fibroblasts D e p a r t m e n t o f P a t h o l o g y S c h o o l o f M e d ic in e C a r d if f U n iv e r s it y Ca r d if f UNIVERSITY PRIFYSGOL CAERDYg> Investigating the mechanistic basis of telomeric fusions A thesis submitted to the School of Medicine, Cardiff University in partial fulfilment for the degree of Doctor of Philosophy BY N ico le H. H eppel 2011 UMI Number: U567010 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U567010 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Declaration This work has not previously been accepted in substance for any degree and is not concurrently submittedjn candidature for any degree. Signed .T. (candidate) Date .5 ^ . .*. STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of ....$}{& (insert MCh, MD, MPhil, PhD etc, as appropriate) Signed^Sr^ferTr*.. (candidate) D a t e *0 STATEMENT 2 This thesis is the resuit of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. Signed (candidate) Date STATEMENTS I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed (candidate) Date MS*#*. STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loans after expiry of a bar on access previously approved by the Graduate Development Committee. Signed ......................................................... (candidate) Date I Acknowledgements I would like to thank my supervisor Dr Duncan Baird, for his continuous support and guidance through out the project. First and foremost I would like to thank him for giving me the opportunity to undertake this PhD in the first place. Furthermore,-1 would like to thank all the other members of the STELA group: Jan Rowson, Dr. Maira Tankinmanova, Dr. Bethan Britt-Compton, Dr Laureline Roger, Dr Thet Thet Lin, Dr Rhiannon Robinson and Biotelo Letsolo for all their advice and encouragement in the last four years. I also would like to thank all the technical staff in the Pathology department for their help. A special thank you goes to Rebecca Capper, Jane Bond and Amy Brook, which have been a tremendous help in tissue culture. I especially would like to thank my family and friends who have been a continual support and encouragement during the course of this PhD. A huge thank you goes to my dad and my brother who have been supporting me both financially and mentally over the course of the last 7 years. They have been home in everyway possible even if they were hundreds of kilometres away. Most importantly I have to thank my friend Laureline, without her strength and support this PhD thesis would not have been possible. Finally, I would like to thanks Tennessee Williams and Marlon Brando for perfectly summarising my feelings about single telomere length analysis (STELA). Stanley Kowalski:Hey, STELLA. Tennessee Williams A Streetcar named Desire II Summary Telomeres are essential nucleoprotein structures located at the ends of chromosomes that provide both end protection and avoid recognition through the DNA damage response. In humans, telomeres consists of arrays of TTAGGG repeats which associate with, and are protected by, shelterin, a complex of telomere binding proteins. Dysfunctional telomeres arise by either gradual telomere erosion as a result of the end replication problem to critical lengths, or by the removal of telomere binding proteins. It is widely accepted that short dysfunctional telomeres are recognised as DNA double strand breaks. The aberrant repair of telomeric double strand breaks and the subsequent formation of dicentric chromosomes, can initiate cycles of anaphase bridging, breakage and fusion that can drive genomic instability. In eukaryotes, double strand breaks are repaired by either error-free homologous recombination or error-prone non-homologous end joining. In this thesis, single molecule approaches were used to observe the telomere dynamics of DNA repair deficient fibroblast cultures and to characterise possible fusion events in cells undergoing crisis following die forced expression of HPV16 E6E7 oncoproteins. All the DNA repair deficient human fibroblast strains observed in this thesis exhibited a similar limited replicative capacity. In contrast to previous reports, the data presented here supports the assumption that DNA repair deficiency does not result in accelerated telomere erosion and therefore the reduced replicative lifespan of these fibroblast cultures appears to be telomere independent. The main aspect of this diesis was to examine the role that components of the DNA repair mechanisms might play in the fusion of short dysfunctional telomeres. The results presented here indicate that the fusion of short dysfunctional telomeres is largely independent of DNA Ligase I and DNA Ligase IV. These observations lead to the assumption that DNA Ligase III might be the predominant ligase involved in the fusion of short dysfunctional telomeres. Furthermore, NBS1 deficiency results in a paucity of sporadic telomere deletion events which is consistent with the hypothesis that NBS1 is involved in the resolution of Holliday junctions within T-loops creating telomeric deletion events. In contrast, DNA Ligase IV deficiency and PARP1 inhibition caused distinct short telomere length distributions. Both these phenotypes might be related to problems with telomere replication. Ill Table of Contents DECLARATION— ------------------- 1 ACKNOWLEDGEMENTS...................................................... II SUMMARY_____________________________________________________________________ III TABLE OF CONTENTS___________________________________________________________ IV ABBREVIATIONS----------------------------------------------------------------------------------------------VIII CHAPTER 1: INTRODUCTION--------------------------------------------------------------------------------- 1 l .l History of Telomeres .....................................................................................................................................l l .2 Structure of Telom eres ........................................ 2 1.2.1 Protozoa ........................................................................................................................................................ 4 1.2.2 Fungi...................................................................................................................................................................... 4 1.2.3 Plants........................................................................................................................................... 5 1.2.4 Mammalian..................... 6 l .3 Telomere A ssociated Proteins ..................................................................................................................6 1.3.1 The Shelterin complex................................................ 8 1.3.1.1 Telomere Repeat Binding Factors 1 and 2 (TRF1 and TRF2) .............................................. 8 1.3.1.2 TIN2............................................. '.......................................................................................................9 1.3.1.3 TPP1....................................................................................................................................................10 1.3.1.4 POT1................................... %..............................................................................................................10 1.3.1.5 Rapl....................................................... 11 1.3.2 Transient Associated Telomeres Proteins.............................................................................................. 11 1.3.2.1 PinXl...................................................................................................................................................12 1.3.2.2 Tankyrasel and Tankyrase2 .............................................................................................................. 12 1.4 Telomere Repeat Containing RNA (TERRA).........................................................................................13 1.5 Telomeric Function ......................................................................................................................................... 14 1.5.1 Capping the Chromosome termini...................................................................................... 14 1.5.2 Protection from DNA Damage Response (DDR) ........................................................................................ 15 1.5.3 Telomeres in mitosis and meiosis..................................................................................................................... 16 1.6 TELOMERE DYNAMICS IN SOMATIC CELLS ...................................................................................................17
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