Investigating the Role of CK2 in the DNA Damage Response By Edward Strenk Miller A thesis presented to the College of Medical and Dental Sciences, The University of Birmingham, for the degree of DOCTOR OF PHILOSOPHY School of Cancer Sciences College of Medical and Dental Sciences University of Birmingham June 2014 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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 Casein Kinase 2 (CK2) is a ubiquitous serine/threonine kinase. Due to its pleiotropic nature CK2 is involved in a multitude of cellular pathways including cell survival, proliferation and apoptosis. Therefore it has come as no surprise that a requirement for CK2 activity has been identified during the repair of DNA damage. Here we have described a novel role for CK2 in the response to DNA double-strand breaks. We have shown the mediator of DNA damage checkpoint 1 (MDC1) is constitutively phosphorylated by CK2, which is required for an interaction with the MRE11/RAD50/NBS1 (MRN) complex, via the FHA domain of NBS1. Moreover, disruption of this interaction resulted in loss of MRN foci following ionizing radiation and a partial G2/M checkpoint defect. Furthermore, the identification of three siblings presenting NBS/Seckel-like phenotypes with a unique mutation in the BRCT domain of NBS1, that phenocopied some of our observations, provided additional evidence for the importance of phospho-dependent interactions within the cell. Lastly, the identification of putative CK2 target residues in MRE11 and our preliminary data suggest that the kinase may play further roles in regulating the activity of the MRN complex that lie outside its activity in DNA double-strand break repair. This thesis is dedicated to my Mum and Dad for their unconditional support and encouragement and in loving memory of my Nan. You were right I did do it! Acknowledgements Firstly, I would like to thank my supervisors Dr Grant Stewart and Professor Malcolm Taylor for their continued support throughout my PhD, and for taking a chance on a Zoology graduate from Redditch. Extra gratitude must be given to Grant who has provided guidance, encouragement and confidence along the seemingly endless road of a part-time PhD. I would also like to thank Dr Roger Grand, who has always been there to offer advice both scientific and not, Dr Phil Byrd, my molecular biology mentor, Dr Manuel Stucki and members of his laboratory for their collaboration and Dr Xiaohua Wu and Dr Matthew Weitzman for generously providing reagents. Extended thanks go to members of the Stewart Laboratory, past and present Ellis, John, Helen and Rachael. In particular, both Dr Natasha Zlatanou and Dr Martin Higgs have provided valuable feedback in the writing of this thesis as well as sound scientific advice in the laboratory. Special thanks must go to Dr Kelly Endean whose support and friendship has been crucial throughout my PhD. I’m sorry you never did get to play your music in the laboratory. I would also like to thank Anoushka Thomas for both her friendship and constant optimism. I would also like to thank Dr Jim Last on so many levels. He has become a good friend, pillar of support, drinking companion and quiz buddy. I can take the tin hat off now right? Lastly my deepest gratitude goes to my family and friends, over the past 6 years or so they have kept me going in the harder times and without them I don’t think I would have made it to this point. I feel very lucky. Contents Chapter 1 General Introduction 1 1.1 Genomic instability and Cancer 1 1.2 The DNA damage response 3 1.2.1 ATM activation and the ATM-mediated DNA damage response 3 1.2.2 ATR activation and the ATR-mediated DNA damage reposnse 11 1.3 Cell cycle checkpoints in response to DNA damage 14 1.4 DNA double-strand break repair 16 1.4.1 Homologous recombination 17 1.4.1.1 Single-strand annealing and break-induced replication 22 1.4.2 Non-homologous end-joining 23 1.4.2.1 Alternative end-joining 26 1.4.3 Programmed double-strand breaks 27 1.5 Human DSB repair deficiency syndromes 31 1.5.1 Ataxia-Telangiectasia 31 1.5.2 Syndromes associated with mutations in the MRN complex 32 1.5.3 Syndromes associated with RNF168 mutations 38 1.5.4 Seckel syndrome 38 1.5.5 Ligase IV syndrome 41 1.5.6 RS-SCID (Associated with Artemis or XLF deficiency) 42 1.6 Mediators of DNA repair 43 1.6.1 Claspin 44 1.6.2 TopBP1 45 1.6.3 53BP1 46 1.6.4 BRCA1 48 1.6.5 MDC1 52 1.6.5.1 Structure 53 1.6.5.2 Function 56 1.6.5.2.1 DNA double-strand break repair 57 1.6.5.2.2 Cell cycle checkpoint control 61 1.6.5.2.3 Mitosis 62 1.6.5.3 Additional functions 63 1.6.6 The MRN complex 65 1.6.6.1 MRE11 66 1.6.6.2 RAD50 68 1.6.6.3 NBS1 69 1.6.6.4 Sensing DSBs and repair 71 1.6.6.5 MRN and cell cycle checkpoint control 74 1.6.6.6 Additional functions 76 Contents 1.7 Post-translational modifications and the DNA DSB damage response 78 1.7.1 Ubiquitylation in response to DNA DSBs 79 1.7.2 SUMOylation in response to DNA DSBs 83 1.7.3 Methylation and acetylation in response to DNA DSBs 85 1.7.4 Phosphorylation in response to DNA DSBs 88 1.8 Casein Kinase 2 (CK2) 90 1.8.1 Structure and activity 91 1.8.2 CK2 and cell cycle 93 1.8.3 CK2 and apoptosis 95 1.8.4 CK2 and the DNA damage response 95 1.9 Aims and objectives 99 Chapter 2 Materials and Methods 101 2.1 Molecular biology techniques 101 2.1.1 Bacterial strains 101 2.1.2 Media 102 2.1.3 Antibiotics 102 2.1.4 Primer design 102 2.1.5 Enzymes 102 2.1.6 Transformation of bacteria 103 2.1.7 DNA constructs 105 2.1.8 Small-scale preparation of DNA constructs 105 2.1.9 Large-scale preparation of DNA constructs 105 2.1.10 Measuring DNA concentrations 106 2.1.11 Cloning 106 2.1.11.1 PCR of a gene sequence from plasmid DNA constructs 106 2.1.11.2 Visualising PCR products via agrose gel electrophoresis 108 2.1.11.3 Gel extraction 108 2.1.11.4 Restriction digest and ligation 108 2.1.11.5 Screening of colonies via bacterial PCR 109 2.1.11.6 Sequencing 109 2.1.12 Mutagenesis 111 2.1.12.1 Primer design 111 2.1.12.2 PCR 113 2.1.12.3 Digestion and transformation 113 2.1.13 GST purification 113 2.1.14 Generation of cDNA for sequencing 115 2.1.14.1 RNA extraction and purification 115 2.1.14.2 Production of cDNA 115 2.2 Tissue culture techniques 116 2.2.1 Cell lines 116 2.2.2 Tissue culture media and solutions 116 Contents 2.2.3 Maintenance of cells 116 2.2.4 Cryopreservation of cell lines 118 2.2.5 Transient transfection of DNA constructs into cell lines 118 2.2.6 Generation of stable cell lines via retroviral transfection 119 2.2.7 Generation of stable Flp-In/T-Rex cell lines 120 2.2.8 Cell irradiation 121 2.2.9 Treatment of cells with DNA damaging agents 121 2.2.10 Transfection of cell lines with siRNA 121 2.2.11 Cell extraction and protein purification 122 2.2.12 G2/M checkpoint assay and cell cycle analysis 122 2.3 Protein biochemistry techniques 123 2.3.1 Protein dtermination 123 2.3.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 124 2.3.3 Visualisation of proteins 124 2.3.4 In-gel digestion of protein for analysis by mass spectrometry 124 2.3.5 In vitro kinase assay 125 2.3.6 GST pull-down 126 2.4 Immunochemistry techniques 126 2.4.1 Antibodies 126 2.4.2 Western blotting 126 2.4.3 Immunoprecipitation 127 2.4.4 Immunofluoresence 128 Chapter 3 Constitutive phosphorylation of MDC1 by CK2 is required for retention of the MRN complex at the sites of DNA double-strand breaks 132 3.1 Introduction 132 3.2 Results 134 3.2.1 Identification of potential phosphorylation motifs on MDC1 134 3.2.2 A N-terminal region of MDC1 is phosphorylated by CK2 in vitro and in vivo 137 3.2.3 Identification of MDC1 CK2 target sites by mass spectrometry analysis 142 3.2.4 MDC1 associates with CK2 in vivo in the presence and absence of damage 144 3.2.5 Loss of CK2 results in a persistent DNA damage response following IR 144 3.2.6 Loss of CK2 via siRNA or mutation of the CK2 target motifs does not affect MDC1 localisation 147 3.2.7 Loss of CK2 does not impair recruitment of other DNA damage factors following IR 149 3.2.8 siRNA-mediated reduction of CK2 results in loss of NBS1 foci following IR 151 Contents 3.2.9 The SDTD motif of MDC1 mediates its interaction with the MRN complex 153 3.2.10 The CK2 phosphorylated N-terminus of MDC1 interacts with NBS1 via its FHA domain 155 3.2.11 Abolishing the interaction between MDC1 and NBS1 results in a partial G2/M checkpoint defect 158 3.2.12 Identification of a Seckel-like family with a unique mutation in NBS1 162 3.2.13 ΔSer118 does not result in reduction of NBS1 protein levels or disrupt binding to MRE11 or RAD50 163 3.2.14 The ΔSer118 mutation disrupts NBS1 binding to MDC1 and ablates NBS1 foci following IR 171 3.2.15 ΔSer118 NBS1 mutation does not result in a G2/M checkpoint activation defect 173 3.3 Discussion 177 Chapter 4 Identification of putative CK2 motifs on MRE11 185 4.1 Introduction 185 4.2 Results 187 4.2.1 A C-terminal region of MRE11 is phosphorylated by CK2 in vitro 187 4.2.2 Generation of ATLD2 stable cell lines 193 4.2.3 Mutation of MRE11 putative CK2 sites does not lead to instability of the MRN