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Characterisation of Novel Functions of the Anaphase Promoting Complex/Cyclosome and Its Regulation Through Post-Translational Modification

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Characterisation of Novel Functions of the Anaphase Promoting Complex/Cyclosome and Its Regulation Through Post-Translational Modification CHARACTERISATION OF NOVEL FUNCTIONS OF THE ANAPHASE PROMOTING COMPLEX/CYCLOSOME AND ITS REGULATION THROUGH POST-TRANSLATIONAL MODIFICATION by PAUL EDWARD MINSHALL A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Cancer Sciences College of Medical and Dental Sciences University of Birmingham October 2014 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT The Anaphase Promoting Complex/Cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that regulates mitotic progression through targeting substrates for degradation by the 26S proteasome. In order to assess APC/C post-translational modification status, and identify novel APC/C substrates and regulators, a comprehensive analysis of the APC/C and APC/C-interacting proteins by mass spectrometry was undertaken. RNA polymerase I was identified as an APC/C-interacting complex, and the interaction was validated by reciprocal co-immunoprecipitation, GST pull-down and immunofluorescent confocal microscopy. Both RPA194 protein levels and RNA Polymerase I transcription were shown to be dependent upon APC/C activity. Ablation of APC/C function by RNAi increased RPA194 protein levels, and elevated RNA polymerase I activity significantly, as quantified by 5’-Fluorouridine incorporation into nascent pre-rRNA, and the increase in absolute levels of 45S, 28S and 18S rRNA transcripts, relative to non- silencing controls. A number of other potential APC/C substrates and regulators were identified by mass spectrometry. Many of these interacting proteins contained APC/C consensus degron motifs. The APC/C was also shown to be a major substrate for acetylation; a number of APC/C subunits were identified as being acetylated in vivo. In this regard, APC3 was shown to be a substrate for both CBP and p300 acetyltransferases. ACKNOWLEDGEMENTS I would like to take this opportunity to thank my supervisors, Andy Turnell and Roger Grand, for all their help over the last few years. I am extremely grateful for all their assistance in the lab and guidance with my research, as well as their advice on the content of this thesis. I would also like to thank all members of our group, past and present, particularly Nat and Rakesh, for teaching me how to do everything and for helping me to settle in. I am also extremely grateful to Cancer Research UK for funding this research. I would also like to thank Ashley Martin and Neil Shimwell for all things mass spec-related. Without them, a lot of the research within this thesis would not have happened. I am also extremely grateful to Joe Teodoro and the McGill University for hosting me for three months, to Joe’s student Thomas Kucharski for his help whilst there, and to the College of Medical and Dental Sciences, University of Birmingham for providing the travel grant that allowed me this opportunity. Finally I would like to thank Kathryn for all her support and understanding, and for driving over a hundred miles to see me on weekends, only for me to spend most of it in the lab. LIST OF CONTENTS 1. INTRODUCTION ................................................................................................................................................... 1 1.1. The Anaphase Promoting Complex/Cyclosome ................................................................................ 1 1.2. The Ubiquitin-Proteasome Pathway ...................................................................................................... 2 1.3. Form and Function of the APC/C ............................................................................................................. 6 1.3.1. Structure and Subunits of the APC/C ..................................................................................................... 6 1.3.2. Substrate Recognition ............................................................................................................................... 10 1.3.3. Mechanism of APC/C E3 ubiquitylation ............................................................................................. 16 1.4. The APC/C as a cell cycle regulator ...................................................................................................... 19 1.4.1. The APC/C is inhibited by the Spindle Assembly Checkpoint .................................................. 19 1.4.2. SAC reactivates the APC/C ....................................................................................................................... 26 1.4.3. The APC/C promotes sister chromatid separation ....................................................................... 27 1.4.4. The APC/C coordinates mitotic exit..................................................................................................... 30 1.4.5. The APC/C prevents early S phase entry ........................................................................................... 32 1.4.6. APC/C-Cdh1 is inhibited to promote S phase progression ........................................................ 33 1.4.7. The APC/C and replication licencing ................................................................................................... 36 1.4.8. The role of the APC/C in differentiation and G0 ............................................................................. 40 1.4.9. The APC/C is reactivated following DNA damage ......................................................................... 43 1.5. Regulation of APC/C-mediated ubiquitylation ................................................................................ 49 1.5.1. Regulation of co-activators and APC/C subunits ........................................................................... 49 1.5.2. Regulation of APC/C activity by modulation of substrates ........................................................ 52 1.6. Transcriptional Regulation by the APC/C ......................................................................................... 54 1.7. The APC/C and Cancer .............................................................................................................................. 56 2.1. THE NUCLEOLUS AND RNA POLYMERASE I ........................................................................................ 59 2.2. Nucleolar architecture and mitotic regulation ................................................................................ 59 2.3. RNA Polymerase I ........................................................................................................................................ 61 2.4. Ribosomal Assembly .................................................................................................................................. 62 2.4.1. Pol I transcription ....................................................................................................................................... 62 2.4.2. Pre-rRNA processing and ribosomal assembly ............................................................................... 66 2.5. The nucleolus and stress .......................................................................................................................... 70 3. Aims .................................................................................................................................................................. 73 4. MATERIALS AND METHODS ....................................................................................................................... 74 4.1. Cell Culture, Drug Treatments and Irradiation .......................................................................... 74 4.2. siRNA-mediated knockdown ............................................................................................................. 75 4.3. Generation of dox-inducible APC3 shRNA cell line................................................................... 76 4.4. Targeted recombination of DNA into HeLa-FRT cell lines .................................................... 77 4.5. Adenoviral infection .............................................................................................................................. 77 4.6. Preparation of Whole-Cell Extracts ................................................................................................. 77 4.7. Protein Concentration Determination ........................................................................................... 78 4.8. Nuclear Fractionation ........................................................................................................................... 78 4.9. FPLC (Fast Protein Liquid Chromatography) ............................................................................. 79 4.10. Immunoprecipitation (IP) ................................................................................................................... 79 4.11. SDS-PAGE ..................................................................................................................................................
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