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University of Dundee DOCTOR of PHILOSOPHY the Identification University of Dundee DOCTOR OF PHILOSOPHY The identification and characterisation of proteins interacting with SUMO-ubiquitin hybrid chains Anderson, Oliver Award date: 2015 Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 04. Oct. 2021 The identification and characterisation of proteins interacting with SUMO-ubiquitin hybrid chains A thesis submitted in the fulfillment of the requirements for the degree of Doctor of Philosophy by Oliver Fraser Anderson College of Life Sciences, University of Dundee, United Kingdom September 2015 Ph.D. Supervisor: Professor Ron Hay Declaration I hereby declare that the following thesis is based on results of investigations conducted by me, and that this thesis is of my own composition. Work other than my own is clearly indicated in the text by reference to the relevant researchers or their publications. All references cited here have been consulted by me. This thesis has not, in whole or part, been previously accepted for a higher degree. Oliver Anderson I certify that Oliver Anderson performed the work for which this thesis is a record. The conditions of the relevant Ordinance and Regulations have been fulfilled. Professor Ronald T. Hay 2 Acknowledgement Firstly, I would like to thank my supervisor Professor Ron Hay for allowing me to undertake the research for this thesis in his laboratory, and for all his help and guidance over the last 3 years. I would also like to thank all the members of the RTH lab that I have had the pleasure of working alongside. In particular, I would like to thank Ellis Jaffray for all his technical assistance, Michael Tatham for all the discussions and help with proteomics, Federico Pelisch for help with microscopy, and Anne Seifert and Yili Yin for all their great advice. I would also like to thank the Biotechnology and Biological Sciences Research Council for generously providing the funding for this research. My thanks must also go out to the all the people of the College of Life Sciences, University of Dundee for making it such a great place to work and learn. Finally, Thank you to all my friends and especially to my family Mum, Dad, Sarah, Megan, Bethany, Morgan and Michael for the endless support over the last 3 years, without which none of this would be possible. 3 Contents Table of Contents Declaration ..................................................................................................... 2 Acknowledgement ......................................................................................... 3 Contents ......................................................................................................... 4 List of figures ................................................................................................. 7 Abbreviations ................................................................................................. 9 Abstract ........................................................................................................ 13 1 Introduction ............................................................................................... 15 1.1 Ubiquitin .............................................................................................. 15 1.1.1 Ubiquitin and the ubiquitin-like modifiers ....................................... 15 1.1.2 The ubiquitin conjugation pathway and associated enzymes ........ 16 1.1.3 Ubiquitin chain types ...................................................................... 18 1.1.4 Recognition of ubiquitin chains types by ubiquitin binding domains ................................................................................................................ 20 1.1.5 Ubiquitin deconjugating enzymes .................................................. 23 1.2 SUMO ................................................................................................... 25 1.2.1 SUMO the small ubiquitin-like modifier .......................................... 25 1.2.2 SUMO conjugation pathway .......................................................... 26 1.2.3 Consensus SUMO modification motif ............................................ 28 1.2.4 SUMO interaction motifs ................................................................ 29 1.2.5 SUMO specific proteases .............................................................. 31 1.3 Aims and objectives ........................................................................... 34 2 Materials and Methods ............................................................................. 35 2.1 Materials .............................................................................................. 35 2.2 Methods ............................................................................................... 35 2.2.1 General Methods ........................................................................... 35 2.2.2 Protein Expression and purification protocols ................................ 40 2.2.3 Biochemical assays ....................................................................... 43 2.2.4 Pull-down experiments .................................................................. 45 4 2.2.5 Cell Culture .................................................................................... 46 2.2.6 Immunoprecipitation (IP) ................................................................ 49 2.2.7 Microscopy ..................................................................................... 50 2.2.8 Mass spectrometry ........................................................................ 52 3 Establishment and validation of approaches to identify proteins interacting with SUMO-ubiquitin hybrid chains ........................................ 58 3.1 Introduction ........................................................................................ 58 3.1.1 SUMO targeted ubiquitin E3 ligases .............................................. 58 3.1.2 SUMO-ubiquitin hybrid chain assembly ......................................... 61 3.2 Results ................................................................................................ 64 3.2.1 Generating SUMO-ubiquitin hybrid chains. ................................... 64 3.2.2 A specific elution to elucidate information on interaction characteristic of hybrid chain interacting proteins ................................... 68 3.2.3 Detection of SUMO-ubiquitin hybrid chain binding proteins via protein affinity chromatograph coupled with high resolution mass spectrometry ........................................................................................... 74 3.2.4 Verifying potential hybrid chain interacting proteins ...................... 80 3.2.5 A bioinformatic approach to identifying proteins with the potential to interact with SUMO-ubiquitin hybrid chains ............................................ 90 3.3 Discussion .......................................................................................... 96 3.3.1 Generating SUMO-ubiquitin hybrid chains .................................... 96 3.3.2 USP2 CaD/SENP1 specific elution ................................................ 97 3.3.3 An assessment of the approach to detect SUMO-ubiquitin hybrid chain interacting proteins via affinity baits coupled with high resolution mass spectrometry. ................................................................................ 98 3.3.4 An assessment of the approach to detect SUMO-ubiquitin hybrid chain interacting proteins via primary sequence searches ................... 101 3.3.5 Conclusions ................................................................................. 102 4 Exploring a role for SUMO-ubiquitin hybrid chains in the recruitment of RAP80 to the sites of DNA repair ............................................................. 103 4.1 Introduction ...................................................................................... 103 4.1.1 DNA damage response, RAP80 and the BRCA 1A complex ...... 103 4.1.2 SUMO, ubiquitin and the DNA damage response ....................... 105 5 4.1.3 SUMO-ubiquitin hybrid chains at the sites of DNA damage ........ 107 4.2 Results .............................................................................................. 110 4.2.1 BRCA 1A complex component RAP80 shows an association for SUMO and ubiquitin in a cellular context .............................................. 110 4.2.2 A proximity ligation assay identifies a close association by SUMO and ubiquitin in RAP80 positive foci after DNA damage ....................... 113 4.2.2 The role of STUbLs in the recruitment RAP80 to sites of DNA repair .............................................................................................................. 119 4.2.3 Exploring the effect of SUMO proteases on RAP80
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