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Theses Digitisation: This Is a Digitised https://theses.gla.ac.uk/ Theses Digitisation: https://www.gla.ac.uk/myglasgow/research/enlighten/theses/digitisation/ This is a digitised version of the original print thesis. Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work 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 Enlighten: Theses https://theses.gla.ac.uk/ [email protected] Identification ofTopBPl Chromatin Modification Domains and Transcriptional Targets Roni H G Wright A thesis submitted to the University o f Glasgow, Faculty of Veterinary Medicine for the degree o f Doctor of Philosophy UNIVERSITY of GLASGOW i r % Division of Pathological Sciences Faculty of Veterinary Medicine September 2007 © Roni Wright :v & ProQuest Number: 10391063 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. uest ProQuest 10391063 Published by ProQuest LLO (2017). C o pyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 For my Mum and Dad Acknowledgements I would like to thank to my supei-visor Iain Morgan for all his support, guidance and patience over the last thiee year's. Thanks to everyone in the PV lab especially Mary Donaldson for their interest in the project and help with experimental protocols, but above all thank you for your friendship. A special thanks to Edward Dornan for all the laughs, and perhaps somewhat less scientific conversations over the years. And last but by no means least thanlcs to Allan for all his love and unwavermg support -This thesis is as much yours as it is mine. Table of Contents Acknowledgements......................................................................................... i Table of Contents...................................................................................................ii-x List of Figures....................................................................................................xi-xiv List of Tables.....................................................................................................xv-xvii Abbreviations...................................................................................................xviii-xx Abstract..............................................................................................................xxi-xxii Author’s Declaration..............................................................................................xxiii Chapter 1: Introduction................................................. 1-39 1.1. The DNA Damage Response.....................................................1-9 1.1.1. Sensors.............................................................................................4 1.1.2. Transducers.....................................................................................7 1.1.3. Effectors.......................................................................................... 9 1.2.DNA Damage Checkpoints.......................................................9-12 1.2.1. G1 Checkpoint................................................................................9 1.2.2. S Phase Checkpoint.......................................................................10 1.2.3. G2 Checkpoint................................................................................. 11 1.3.Repai r................................................................................................... 13-19 1.3.1. Nucleotide Excision Repah...........................................................15 1.3.2. Mismatch Repair............................................................................. 15 1.3.3. Base Excision Repair......................................................................16 1.3.4. Double Strand Break Repair, Homologous Repah and Non-Homologous End Joining...................................................... 17 1.4.Apoptosi s................................................................................. 20 1.5. Role of Chromatin in the DNA Damage Response 21-25 1.5.1. The Modification of Histones........................................................21 1.5.2. Modification of Chromatin by ATP-dependent Chromatin Remodellers.......................... 23 1.6. TopBPl...............................................................................................26-38 1.6.1. Structure of TopB Pl............................................. 27 1.6.2. Homologues of TopB Pl................................................................ 27 1,6.2. LFission Yeast Schizosaccaromyces Pombe Homologue Rad4/Cut5..................................................................................28 ui 1.6.2.2.Budding Yeast Saccharomyces Cerevisiae Homologue D p b ll....................................................................................... 29 1.6.2.3. Drosophila Melanogaster Homologue Mus 101..................29 1.6.2.4.Xenopus Laevi Homologue....................................................32 1.6.2.5.Caernorhabditis elegans Homologue F37D6.1.................... 33 1.6.3. TopBPl -A Possible Transcription Factor?................................. 34 1.6.4. TopBP 1 and Breast Cancer........................................................... 35 C hapter 2: M aterials and M ethods .........................................40-87 2.1. Materials .......................................................................................40-55 2.1.1. Antibodies.............................................. 40 2.1.2. Bacteriology.....................................................................................42 2.1.3. Cell lines........................................................................................... 43 2.1.4. Chemicals and Reagents................................................................. 44 2.1.5. Enzymes............................................................................................ 46 2.1.6. Kits.....................................................................................................47 2.1.7. Miscellaneous....................................................................................49 2.1.8. Molecular Weight Markers............................................................. 50 2.1.9. Plasmids............................................................................................. 51 2.1.10. Tissue Culture....................................................................................53 2.1.11. Yeast Materials..................................................................................54 IV 2.2. Methods ............................................................................................56-87 2.2.1. Molecular Biology..................................................................... 56-76 2.2.1.1. Agarose Gel Electrophoresis...............................................56 2.2.1.2. DNA/RNA concentration Determination...................... ....57 2.2.1.3. Restriction Enzyme Digests.................................................57 2.2.1.4. DNA Purification using Phenol Chloroform.................... 58 2.2.1.5. Ethanol Precipitation of DNA.............................................58 2.2.1.6. Phophatase Treatment.......................................................... 59 2.2.1.7. Ligation of DNA.................................................................... 59 2.2.1.8. Transformation of Chemically Competent Bacterial Cells........................................................................................ 59 2.2.1.9. Electroporation of E. coli......................................................60 2.2.1.10. Large scale Preparation of Plasmid DNA............................60 2.2.1.11. Small Scale Plasmid Purification..........................................62 2.2.1.12. Polymerase Chain Reaction.................................................. 63 2.2.1.13. PCR Purification.....................................................................66 2.2.1.13.1. QIAqickPCR clean up Protocol.................................66 2.2.1.13.2. Chargeswitch® PCR Clean-Up..................................67 2.2.1.14. Gel Extraction......................................................................... 67 2.2.1.15. DNA Sequencing....................................................................68 2.2.1.16. BCA/CUSO4 Protein Assay...................................................68 2.2.1.17. Western Blotting.....................................................................69 2.2.1.18. Stripping Membranes....................................... 71 2.2.1.19. Coomassie Staining................................................................ 71 2.2.1.20. Immunoprécipitation.............................................................
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