A University of Sussex PhD thesis Available online via Sussex Research Online: http://sro.sussex.ac.uk/ 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 Investigation into the role of the SMC5/6 Complex in human cells. A thesis submitted to the University of Sussex for the degree of Doctor of Philosophy By Grant Alexander McGregor. i ii 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…………………………………………………………………… iii Acknowledgements Firstly, I would like to express my utmost gratitude to Dr Jo Murray for the opportunity to undertake a PhD and for her continued guidance and support through it all. A great deal of thanks must also go to all the members of the Murray and Carr labs past and present with special thanks to Owen Wells and Hung Quang Dang for all their assistance. I would also like to thank all the members of the GDSC including the Caldecott lab, especially Stuart, and the Sweet lab for sharing many reagents and an office with me, the O’Driscoll lab, the Downs lab and the Hochegger lab for their hints, tips and tricks and not to forget reagents. It’s also important to thank the prep room and tissue culture staff without whom research would grind to a halt. I’d also like to give thanks to Velibor Savic for his many discussions and help in processing data and of course to Antony Oliver for his help, discussion and the sharing of an occasional gin. The thanks I owe to my mother is without limit, I am extremely grateful to her for all her love and help, without her I would not have made it this far. Together with my Nana and Di, she raised me to try hard, enjoy myself but also to remain upbeat when things got tough. My family have always been a large part of my life and the support and perspective provided by my Auntie Norma, Uncle Johnny, Uncle Keith, Auntie Julie, Cousins John, Jillian, Greig, Keith and Molly have helped me immeasurably. I also have to thank my friends, without whom I’d never had made it this far, Kevin, Abu, Tomisin, Ross, Rachel, Owen, Matt and Sam, you all played your part. If I haven’t mentioned you by name please note it’s not because I’ve forgotten you, you all know who you are. Thank you to everyone who has helped and supported me over these last 4 years, without you, I’d never have made it. iv University of Sussex Grant Alexander McGregor Doctor of Philosophy Biochemistry Investigation into the role of the SMC5/6 Complex in human cells. Summary The Structural Maintenance of Chromosome (SMC) family of proteins are required to regulate almost all aspects of chromosome biology and are critical for genomic stability. The SMC5/6 complex, a member of this family, is composed of two SMC heterodimers and six additional Non-SMC Elements 1- 6. The components of SMC5/6 possess activities including ATPases, ubiquitin and SUMO ligases. SMC5/6 is required in homologous recombination and for accurate chromosome segregation. Loss of SMC5/6 is lethal in yeasts, embryonic lethal in mice and mutations in NSMCE2 leads to primordial dwarfism and insulin resistance. This thesis focuses on a mutation in NSMCE3, found in American and Dutch families, that results in a novel chromosomal breakage syndrome characterized by fatal pulmonary disease. Another focus is the development, execution and validation of a microscopy based synthetic sick/lethal screen using cells with knockdown of NSMCE4a. Studies of SMC5/6 in yeasts predict that compromising SMC5/6 function would lead to a dependence on other DNA repair pathways. The results combined with patient data confirm that SMC5/6 is important in the absence of repair by non-homologous end joining and is particularly important under conditions of replication stress. v List of Abbreviations. ALT Alternate Lengthening of Telomeres Alt-NHEJ Alternate-NHEJ AT Ataxia-telangiectasia ATM Ataxia-telangiectasia mutated ATP Adenosine Triphosphate ATR Ataxia-telangiectasia mutated and Rad3-related ATRIP ATR interacting protein BER Base excision repair BrdU 5'-bromo-2'-deoxyuridine BSA Bovine Serum Albumin CDK Cyclin-dependent kinase CPD Cyclobutane pyrminidine dimer CPT Camptothecin (k)Da (kilo)Dalton DAPI 4' 6-diamino-2-phenylindole DNA Deoxyribonucleic acid DSB Double strand break dsDNA Double-strand DNA EdU 5'-ethynyl-2'-deoxyuridine FA Fanconi Anaemia FACS Fluorescence Activated Cell Sorting FCS Foetal calf serum GFP Green Fluorescent Protein HR Homologous Recombination hrs Hours HU Hydroxyurea IF Immunofluorescence IR Ionizing Radiation LB Lysogeny Broth mDNA Mitochondrial DNA MMC Mitomycin C MMR Mismatch Repair MMS Methyl Methanesulphonate vi MRN MRE11-RAD50-NBS1 nDNA Nuclear DNA NEBD Nuclear Envelope Breakdown NER Nucleotide Excision Repair NHEJ Non-Homologous End-Joining NSE Non-Smc Element NSMCE Non-SMC Element OD Optical density PARP Poly (ADP-Ribose) Polymerase PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction rDNA ribosomal DNA RFP Red Fluorescent Protein RISC RNA-Induced Silencing Complex RNA Ribonucleic Ccid RNAi RNA interference ROS Reactive Oxygen Species RPA Replication Protein A SAM S Adenosyl Methionine SC Synaptonemal Complex SCE Sister Chromatid Exchange SD Standard Deviation SEM Standard Error of the Mean shRNA short hairpin RNA siRNA small interfering RNA SMC Structural Maintenance of Chromosome SSB Single-Strand Break ssDNA single-strand DNA SUMO Small Ubiquitin-like Modifier TERT Telomerase Reverse Transcriptase TLS Translesion Synthesis UV Ultra-Violet WT Wild-type XP Xeroderma Pigmentosum vii Contents Table of Contents Chapter 1.0 – Introduction 1 1.1 – Introduction to DNA repair 2 1.1.1 – Sources of DNA damage 2 1.1.1.1 – Endogenous sources of DNA damage 2 1.1.1.1.1 – DNA replication 2 1.1.1.1.2 – Reactive oxygen species 3 1.1.1.1.3 – DNA methylation 4 1.1.1.1.4 – Hydrolysis of DNA 4 1.1.1.2 – Exogenous sources of DNA damage 5 1.1.1.2.1 – UV light damage 5 1.1.1.2.2. – Ionizing radiation 5 1.1.1.3 – Chemical sources of DNA damage 6 1.1.1.3.1 – Methylmethane sulphonate (MMS) 6 1.1.1.3.2 – Mitomycin C (MMC) 6 1.1.1.3.3 – Camptothecin (CPT) 7 1.1.1.3.4 – Hydroxyurea (HU) 7 1.1.2 – Mechanisms of DNA repair 8 1.1.2.1 – DNA-damage signalling 8 1.1.2.2 – DNA Double strand breaks 8 1.1.2.2.1 – Homologous recombination 9 1.1.2.2.2 – Non-homologous end-joining 12 1.1.2.3 – Repair of Altered Bases 13 1.1.2.3.1 – Nucleotide Excision Repair 13 1.1.2.3.2 – Mismatch Repair 14 1.1.2.3.3 – Base Excision Repair 15 1.1.2.3.4 – Single-Strand Break Repair 16 1.1.2.3.5 – Translesion Synthesis 17 1.1.2.3.6 – The Global Response to DNA Damage 17 1.2 – Structural Maintenance of Chromosome Family of Complexes18 viii 1.2.1 – Cohesin 20 1.2.1.1 – The Scc2-Scc4 Cohesin Loader Complex 22 1.2.1.2 – Cohesin complex and developmental disorders22 1.2.1.3 – Cohesin complex and cancer 23 1.2.2 – Condensin 24 1.2.2.1 – Cell cycle regulators of Condensins 25 1.2.3 – The SMC5/6 Complex 25 1.2.3.1 – Discovery of SMC5/6 25 1.2.3.2 – Composition of SMC5/6 26 1.2.3.2.1 – SMC6 28 1.2.3.2.2 – NSE2 28 1.2.3.2.3 – NSE1 29 1.2.3.2.4 – NSE3 30 1.2.3.2.5 – NSE4 30 1.2.3.2.6 – NSE5 and NSE6 30 1.2.3.3 – SMC5/6 localization on Chromatin 31 1.2.3.4 – SMC5/6 Complex promotes DNA DSB Repair 32 1.2.3.5 – SMC5/6 in Meiosis 34 1.2.3.6 – SMC5/6 in ALT pathway 36 1.3 – RNA interference 39 1.3.1 – Regulation of genes using RNAi 42 1.3.2 – Use of RNAi in research 42 1.4 – Screening 45 1.4.1 – Synthetic lethality screens. 46 1.4.2 – High-throughput and high content screens 49 1.5 – Aims and Objectives 52 Chapter 2.0 – Materials and Methods 54 2.1 – List of human cells used 55 2.2 – Strains of E. coli used 55 2.3 – Strains of Schizosaccharomyces pombe used 56 2.4 – Materials 56 ix 2.5 – Cloning and molecular methods 59 2.5.1 – PCR, restriction digests and ligations 59 2.5.2 – Site-directed mutagenesis and fusion PCR 59 2.5.3 – DNA plasmids created or used 60 2.5.4 – List of oligonucleotides used 61 2.5.5 – Competent cells and transformations 62 2.5.5.1 – Creating competent cells 62 2.5.5.2 – Transformations 62 2.5.6 – Electrophoresis of DNA and Western blot analysis 62 2.5.6.1 – Electrophoresis of DNA 62 2.5.6.2 – Western blotting 63 2.5.6.2.1 – List of antibodies used 64 2.6 – S.