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Structure and Function of Poly ADP-Ribose Glycohydrolases

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Structure and Function of Poly ADP-Ribose Glycohydrolases Structure and function of poly ADP-ribose glycohydrolases A thesis submitted to the University of Manchester for the degree of Ph.D in the Faculty of Life Sciences 2015 Amy Brassington Structure and function of poly ADP-ribose glycohydrolases CONTENTS TABLE FIGURE LIST 9 TABLE LIST 14 ABBREVIATIONS 15 ABSTRACT 17 DECLARATION 18 COPYRIGHT STATEMENT 18 ACKNOWLEDGEMENTS 19 CHAPTER ONE 20 Introduction 20 1.1 Introduction to DNA damage 20 1.2 Poly ADP-ribosylation and glycosylation 21 1.3 ‘Readers’, ‘writers’ and ‘erasers’ in PARylation 24 1.4 Poly ADP-ribose glycohydrolase (PARG) the ‘erasers’ of 28 PARylation 1.5 The structures of bacterial and canonical PARGs 30 1.6 Mono ADP-ribsoylation by macrodomains 35 1.7 Therapeutic implications of PARG 36 1.8 Recruitment of PARG and the relationship between PARG 38 and PARP 1.9 Parthanatos and the release of PAR chains 39 1.10 PAR in bacteria and endotoxins 40 1.11 Project objectives 41 2 Structure and function of poly ADP-ribose glycohydrolases CHAPTER TWO 43 Materials and methods 43 2.1 Materials 43 2.2 E. coli and P. pastoris molecular biology methods 43 2.2.1 Determination of DNA concentration 43 2.2.2 Polymerase chain reaction (PCR) 43 2.2.3 PCR product purification 44 2.2.4 Agarose gel electrophoresis 45 2.2.5 Restriction enzyme DNA digest of plasmid DNA 45 2.2.6 Purification of Plasmid DNA from E. coli 45 2.2.7 In Fusion Cloning reaction 46 2.2.8 Colony PCR 47 2.2.9 DNA sequencing reactions 48 2.2.10 E. coli strains. 49 2.2.11 E. coli growth conditions. 49 2.2.12 Transformation of competent E. coli cells. 49 2.2.13 Purification of plasmid from E. coli transformants 50 2.2.14 Glycerol stocks of E. coli strains 50 2.2.15 P. pastoris growth conditions 50 2.2.16 P. pastoris strains 52 2.2.17 Transformation of P. pastoris 52 2.3 Protein expression and purification 53 2.3.1 Determining protein concentration 53 2.3.2 SDS-PAGE electrophoresis 53 3 Structure and function of poly ADP-ribose glycohydrolases 2.3.3 Western Blot analysis 53 2.3.4 E. coli protein expression trials 55 2.3.5 P. pastoris protein expression trials 55 2.3.6 E. coli large-scale protein expression 56 2.3.7 Production of selenomethione-labelled proteins in E. coli 56 2.3.8 Large-scale protein expression in P. pastoris 57 2.3.9 E. coli cell disruption by sonication. 58 2.3.10 P. pastoris cell disruption 58 2.3.11 Protein buffer exchange using de-salting columns 58 2.3.12 Batch nickel affinity chromatography 59 2.3.13 Reverse-batch nickel affinity chromatography 59 2.3.14 Gel filtration chromatography 59 2.4 Enzymatic and biophysical methods 60 2.4.1 Poly ADP-ribose glycohydrolase activity assays 60 2.4.2 Circular dichroism (CD) spectroscopy 61 2.4.3 Isothermal titration calorimetry (ITC) 61 2.4.4. Nuclear magnetic resonance (NMR) 63 2.4.5 Thermal shift assays. 64 2.4.6 Liquid Chromatography-Mass Spectrometry of trypic digests 64 2.4.7 Multi-Angle Laser Light Scattering (MALLS) 65 2.5 Crystallographic methods 65 2.5.1 Crystallisation and crystal handling methods 65 2.5.2 X-ray data collection 68 2.5.3 Structure elucidation and refinement 68 4 Structure and function of poly ADP-ribose glycohydrolases CHAPTER THREE 70 Biophysical characterization of Thermospora curvata poly 70 ADP-ribose glycohydrolase 3.1 Background information. 70 3.2 Biophysical characterization of bacterial PARG variants 71 E114A & E114Q 3.2.1 Expression and purification of bPARG WT, E114A & E114Q 71 3.2.2 Spectral analysis of bPARG E114A & E114Q 72 3.2.3 Thermal shift binding assays of bPARG WT, E114A and 73 E114Q 3.2.4 ITC measurement of bPARG E114A FAD and ADP-ribose 75 binding 3.3 Structural characterization of bPARG E114A 77 3.3.1 Crystallization of bPARG E114A. 77 3.3.2 Structure determination of bPARG E114A Apo and plus ADP- 78 ribose 3.3.3 Crystallisation of bPARG E114A with FAD 83 3.4 Engineering of T. curvata bPARG and H. sapiens MACROD2 83 hybrid variants 3.4.1 Design of a bPARG-MACROD2 Loop hybrid 83 3.4.2 Cloning of bPARG-MD2 and MACROD2-bP hybrid genes 84 3.4.3 Protein expression of bPARG-MD2 and MACROD2-bP hybrid 85 proteins 3.4.4 Purification of MACROD2, bPARG-MD2 and MACROD2-bP 85 proteins. 3.4.5 PARG Activity assay of MACROD2-bP hybrid 87 3.4.6 Thermal shift assay of modified proteins 89 3.5 Summary and discussion 91 5 Structure and function of poly ADP-ribose glycohydrolases CHAPTER FOUR 94 Biophysical and structural characterization of Tetrahymena 94 thermophila PARG bound to the poly (ADP-ribose) substrate 4.1 Background information 94 4.2 Expression and purification of inactive Tetrahymena 95 thermophila PARG 4.2.1 Expression and purification of TTPARG WT, E256A, E256Q, 95 E255Q and E255A 4.2.2 UV-Vis Spectral analysis of TTPARG E255A and E255Q. 97 4.3 Biophysical characterization of purified TTPARG 98 4.3.1 Thermal shift assays of TTPARG E256Q and E256A 98 4.3.2 ITC Binding assays of TTPARG WT and E256Q, E256A, 100 E255A, E255Q 4.3.3 SPR ligand-binding assays of TTPARG WT and E256Q, E256A, 102 E255A, E255Q 4.4 Structural characterization of the inactive TTPARG-PAR 103 complex 4.4.1 Crystallisation of inactive E256Q TTPARG with poly-ADP- 103 ribose (PAR) fragments 4.4.2 Structure determination of inactive TTPARG with PAR 104 4.4.3 Crystal structure of inactive TTPARG with PAR 107 4.4.4 Crystal structure of TTPARG-PAR reveals exo-glycohydrolase 110 binding mode 4.4.5. The catalytic mechanism of canonical PARG 110 4.5 Discussion 113 6 Structure and function of poly ADP-ribose glycohydrolases CHAPTER FIVE 115 Expression, purification and biophysical characterization of 115 the mammalian PARG regulatory domain 5.1 Background information 115 5.2 Cloning, expression and purification of full length human 117 PARG and human PARG regulatory domain variants in E.Coli 5.2.1 Rational design of hPARG regulatory domain truncations. 117 5.2.2 Cloning of hPARG C-terminally truncated forms. 117 5.2.3 Expression trials of full length hPARG and hPARG fragments. 119 5.2.4 Large-scale expression and purification full-length hPARG 120 5.2.5 Large-scale expression and purification of various hPARG 123 fragments 5.3 Cloning, expression and purification of human PARG 128 regulatory domain in P. pastoris 5.3.1 hPARG expression in P. pastoris 128 5.3.2 Cloning of hPARG 1-388 into pPICZ 3.1 plasmid 129 5.3.3 Transformation into P. pastoris strains 130 5.3.4 Expression trials of hPARG 1-388 in P. pastoris 130 5.3.5 Large-scale expression and purification of hPARG 1-388 in 134 P. pastoris 5.4 Biophysical characterization of purified hPARG regulatory 135 domain variants 5.4.1 Multi angle light scattering (MALLS) 135 5.4.2 Circular dichroism (CD) of various PARG fragments 136 5.4.3 Nuclear magnetic resonance (NMR) of hPARG1-388. 138 5.4.4 Pull-down assays of hPARG regulatory domain (1-388) and 139 catalytic domain hPARG (448-976). 5.5 Crystallization of PARG regulatory domain fragments 141 7 Structure and function of poly ADP-ribose glycohydrolases 5.5.1 Crystallization and initial data collection of hPARG regulatory 141 domain fragments 5.5.2 Crystallization trials of other mammalian PARG regulatory 145 domains 5.6 Summary and discussion 146 CHAPTER SIX 150 Structure determination of luciferase-like mono-oxygenase 150 from a SirTM operon 6.1 Background information 152 6.2 Biophysical characterization of SAV0323 152 6.2.1 Expression and purification of SAV0323 153 6.2.2 Thermal shift ligand binding assay. 154 6.2.3 Multi angle light scattering (MALLS) 155 6.3 Elucidation of the SAV0323 protein structure 157 6.3.1 Crystallization and data collection of native SAV0323 159 6.3.2 Expression, purification and crystallization of 159 selenomethionine-labelled SAV0323 6.3.3 Structure elucidation of SAV0323 159 6.4 Discussion 161 CHAPTER SEVEN 165 Discussion 165 REFERENCES 173 APPENDIX 186 8 Structure and function of poly ADP-ribose glycohydrolases Figure list Figure 1. Schematic representation of proposed PAR mediated 21 single and double strand DNA repair mechanisms Figure 2. PAR synthesis/ degradation pathway and the role of 22 key enzymes Figure 3. DNA repair mechanism and chromatin 25 re-organisation by PARylation Figure 4. Structures of ADP-ribose binder domains 28 Figure 5. Phylogenetic distribution of PARG proteins with clear 30 division between canonical and bacterial PARGs Figure 6. Proposed catalytic mechanism for the bacterial PARG 31 Figure 7. Comparisons of the overall structures and active sites 34 for bacterial, eukaryotic and human PARGs Figure 8. Surface representation of TARG1 bound to 35 ADP-ribose, (PDB:4J5S) Figure 9. Schematic representation of genome arrangements 41 of the macrodomain- sirtuin linked operons Figure 10. Basic configuration of an isothermal titration 62 calorimetry (ITC) instrument Figure 11. Diagram of protein crystallization solubility curve 65 Figure 12. Diagram of sitting drop vapor diffusion method 67 Figure 13. SDS-PAGE analysis of nickel affinity 72 chromatography fractions of bPARG E114A, E114Q and WT respectively Figure 14. Absorbance spectrum of purified bPARG E114A 73 and E114Q Figure 15. Thermal shift assays of bPARG variants 74 Figure 16. ITC binding data for bPARG WT and E114A 76 with ADP-ribose and FAD Figure 17.
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