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Stortz, Jennifer Ann (2017) Identification and Characterisation of Novel Trypanosoma Brucei Protein Kinases Involved in Repair of Cellular Damage

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Stortz, Jennifer Ann (2017) Identification and Characterisation of Novel Trypanosoma Brucei Protein Kinases Involved in Repair of Cellular Damage Stortz, Jennifer Ann (2017) Identification and characterisation of novel Trypanosoma brucei protein kinases involved in repair of cellular damage. PhD thesis. http://theses.gla.ac.uk/8055/ 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 http://theses.gla.ac.uk/ [email protected] Identification and characterisation of novel Trypanosoma brucei protein kinases involved in repair of cellular damage Jennifer Ann Stortz BSc (Hons) Submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy Wellcome Trust Centre for Molecular Parasitology Institute of Infection, Immunity and Inflammation College of Medical Veterinary and Life Sciences University of Glasgow April 2017 2 Abstract Under genotoxic stress conditions, the genome of any organism may become compromised thus undermining cellular functions and the high fidelity transmission of the genome. Should integrity become compromised, cells have evolved a plethora of pathways to monitor, assess and direct the removal or bypass of genomic lesions. Collectively, this response is known as the DNA damage response (DDR). At the forefront of the DDR are specialised enzymes known as protein kinases (PKs), which act to co-ordinate many aspects of this response. In the kinetoplastid parasite Trypanosoma brucei, the role of PKs in other processes, such as the control of the cell cycle and during differentiation between the mammalian and insect lifecycle stages, have already been investigated using mutant cells lines, RNA interference (RNAi) and genome wide and kinome focused screens. However, virtually no work has examined the role of PKs in the context of genome repair. To this end, two RNAi targeted screens (RITseqs), one to examine the genome as a whole and the other to focus directly on the kinome compliment, were performed by others in the mammalian infection stage of T. brucei, searching for genes whose loss sensitises the cells to the presence of the DNA alkylating agent Methyl methanesulfonate (MMS). To validate both screens here, putative DNA damage associated PKs were examined by RNA interference (RNAi). Across both screens, a total of eleven PKs, whose loss sensitised cells to MMS, were validated in this study by monitoring their proliferation. Amongst these novel PKs were a pseudokinase (Tb6560) and an aurora kinase (TbAUK2), whose functions were investigated using disruption mutants generated in BSF T. brucei cells combined with immunolocalisaton. Analysis of the Tb6560 mutants revealed the pseudokinase to be non-essential in vitro and uncovered a potential role for Tb6560 during endocytosis or internal trafficking. Analysis of the TbAUK2 mutants also revealed this PK to be non-essential in vitro. However, when exposed to a variety of genotoxic agents, the growth of the mutants was significantly reduced suggesting TbAUK2 is required for parasite survival under DNA damage conditions. Further phenotypic analysis revealed a potential role for TbAUK2 in the maintenance of the parasite’s nuclear genome. 3 Additionally, the roles of two PKs central to the DDR in other organisms were examined by RNAi. The atypical PKs Ataxia Telangiectasia Mutated (ATM) and the related PK ATM and Rad3 related (ATR) are activated in response to double stranded break (DSB) lesions or replicative lesions, respectively, in other eukaryotes. RNAi of TbATR was associated with perturbation of proliferation, extensive nuclear defects and sensitivity to genotoxic agents. Further analysis also revealed a potential role for this kinase in maintaining transcriptional silencing of bloodstream VSG expression sites (BESs). Conversely, RNAi of TbATM did not disrupt cellular proliferation in vitro and nor were TbATM deficient cells sensitised to alkylating or replication blocking agents, suggesting TbATM is not required for T. brucei survival or for maintenance of growth in these conditions. Collectively, these data reveal a diverse array of PKs required for genome maintainance, in BSF parasites, under genotoxic stress conditions. An indepth characterisation of three DNA damage associated PKs uncovered a variety of putative roles ranging from nuclear and chromosomal segregation to cell cycle regulation, proliferation and endocytosis. These data in particular highlight the complexity underpinning a cell’s response when threatened with genomic instability. Furthermore, depletion of one kinase, TbATR, was also associated with loss of transcriptional silencing within BESs suggesting that PK activity could operate during antigenic variation, a process vital for the persistence of this parasite within a host. 4 Table of Contents Abstract ...................................................................................... 2 List of Tables ............................................................................... 13 List of Figures .............................................................................. 14 List of Accompanying Material ........................................................... 18 Acknowledgements ........................................................................ 19 Author’s Declaration ...................................................................... 21 List of Abbreviations and definitions ................................................... 22 1 Introduction ........................................................................ 27 1.1 The Kinetoplastida .............................................................. 28 1.1.1 HAT is a debilitating disease .............................................. 29 1.1.1.1 Current HAT treatment options ............................................................................ 31 1.2 The lifecycle of T. brucei; a dixenous parasite ............................. 32 1.3 T. brucei: molecular biology ................................................... 35 1.3.1 The cell body and cell cycle .............................................. 35 1.4 Avoiding host clearance ........................................................ 39 1.4.1.1 The VSG Coat ......................................................................................................... 40 1.4.1.2 T. brucei genome is unusually arranged ............................................................... 41 1.4.1.2.1 BESs ................................................................................................................. 43 1.4.1.3 VSG switching ........................................................................................................ 44 1.4.1.3.1 Monoallelic expression and transcriptional switching ................................... 44 1.4.1.3.2 Recombinational switching ............................................................................. 46 1.4.1.4 Endocytosis as a method of immune evasion in T. brucei .................................... 51 1.5 Kinases and phosphorylation: Co-ordination of cellular communication 52 1.5.1 The human kinome; eukaryotic kinases and atypical kinases ........ 52 1.5.1.1 Eukaryotic Protein Kinases (ePKs) ......................................................................... 52 1.5.1.2 Atypical Protein kinases (aPKs) and Protein Kinase Like (PKL) kinases ................. 55 1.5.1.3 Pseudokinases: just a dead kinase? ...................................................................... 55 1.5.1.4 Pseudokinases in parasitic organisms ................................................................... 56 1.5.2 The T. brucei kinome ...................................................... 56 1.5.2.1 Pseudokinases in T. brucei .................................................................................... 58 1.6 DNA Repair and Damage ........................................................ 58 1.6.1.1 Replicative stress................................................................................................... 60 1.6.2 DNA Repair in T. brucei .................................................... 61 1.7 Project Objectives .............................................................. 62 2 Materials and Methods ............................................................ 64 5 2.1 General Bioinformatics ......................................................... 65 2.1.1 Sequence retrieval and examination .................................... 65 2.1.2 Homology searching, protein domain analysis and structure prediction ............................................................................. 65 2.1.3 General Statistics ........................................................... 65 2.2 Molecular Techniques ........................................................... 66 2.2.1 Genomic DNA extraction and preparation .............................. 66 2.2.2 Agarose Gel .................................................................. 66 2.2.3 General Primer design ..................................................... 66 2.2.4 Polymerase Chain Reaction (PCR) ........................................ 66 2.2.5 DNA fragment purification ...............................................
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