Provided by the author(s) and NUI Galway in accordance with publisher policies. Please cite the published version when available. DNA mediated chromatin pull-down: a novel method for the Title study of chromatin replication Author(s) Kliszczak, Anna Ewelina Publication 2012-04-27 Date Item record http://hdl.handle.net/10379/2965 Downloaded 2023-02-03T00:05:52Z Some rights reserved. For more information, please see the item record link above. DNA mediated chromatin pull-down: a novel method for the study of chromatin replication Anna E. Kliszczak The Centre for Chromosome Biology, National Centre for Biomedical Engineering Science, NUI Galway, Ireland A thesis submitted to the National University of Ireland Galway for the degree of Doctor of Philosophy April 2012 Supervisor: Prof. Corrado Santocanale Table of contents Table of contents LIST OF FIGURES ........................................................................................... 10 LIST OF TABLES ............................................................................................. 14 ABBREVIATIONS ............................................................................................ 15 ACKNOWLEDGEMENTS ............................................................................... 17 DEDICATION .................................................................................................... 18 ABSTRACT ........................................................................................................ 19 CHAPTER 1 INTRODUCTION ...................................................................... 21 1.1 Cell division and the cell cycle .......................................................... 21 1.2 Molecular mechanism of the cell cycle progression ....................... 22 1.2.1 Cyclin-dependent kinases and cyclins ................................................. 22 1.2.2 Regulation of cyclin-dependent kinases activity ................................. 23 1.3 DNA damage response and cell cycle checkpoints ......................... 24 1.3.1 Cell cycle checkpoints ......................................................................... 26 1.3.1.1 G phase checkpoint ..................................................................... 27 1 1.3.1.2 S phase checkpoint ....................................................................... 27 1.3.1.3 G /M checkpoint ........................................................................... 28 2 1.3.1.4 Spindle assembly checkpoint ........................................................ 29 1.4 DNA synthesis .................................................................................... 30 1.4.1 Overview ............................................................................................. 30 1.4.2 Discovery of DNA ............................................................................... 30 1.4.3 Features of DNA synthesis in prokaryotes and eukaryotes ................. 31 1.5 Prokaryotic DNA replication ............................................................ 32 1.5.1 Bacterial DNA synthesis ..................................................................... 32 1.5.2 Regulation of bacterial origin firing .................................................... 34 1.6 Initiation of eukaryotic DNA replication ........................................ 34 1.6.1 Proteins involved in formation of pre-replication complexes ............. 35 2 Table of contents 1.6.1.1 Origin specification ...................................................................... 35 1.6.1.1.1 DNA sequences ........................................................................ 35 1.6.1.1.2 The Origin Recognition Complex (ORC) ................................ 36 1.6.1.1.3 Origin selection in higher eukaryotes ...................................... 37 1.6.1.2 The Cdt1 protein ........................................................................... 39 1.6.1.3 The Cdc6 protein .......................................................................... 39 1.6.1.4 The minichromosome maintenance (Mcm2-7) proteins ............... 40 1.6.2 Regulation of licensing ........................................................................ 44 1.6.3 Proteins involved in activation of licensed origins .............................. 45 1.6.3.1 The Cdc7 and Cdk2 kinases ......................................................... 45 1.6.3.2 The Cdc45 protein ........................................................................ 47 1.6.3.3 The GINS complex ........................................................................ 47 1.6.3.4 The Mcm10 protein....................................................................... 48 1.7 The elongation reaction ..................................................................... 49 1.7.1 Proteins involved in elongation of DNA synthesis ............................. 50 1.7.1.1 Replication protein A .................................................................... 50 1.7.1.2 PCNA and RFC complex .............................................................. 51 1.7.1.3 DNA polymerases ......................................................................... 52 1.7.1.4 Flap structure-specific endonuclease 1 ........................................ 54 1.7.2 Regulation at stalled replication forks ................................................. 56 1.8 Termination of DNA replication ...................................................... 60 1.8.1 Termination by converging replication forks ...................................... 60 1.8.2 Termination at telomeres ..................................................................... 61 1.9 Temporal regulation of DNA replication ........................................ 61 1.10 Structure and organisation of the chromatin.................................. 65 1.10.1 Euchromatin and heterochromatin....................................................... 66 1.10.2 The nucleosome organisation .............................................................. 67 3 Table of contents 1.10.3 Higher-order DNA structures .............................................................. 68 1.10.4 Canonical histones ............................................................................... 70 1.10.5 Histone variants ................................................................................... 71 1.10.6 Histone post-translational modifications ............................................. 76 1.10.6.1 Acetylation .................................................................................... 76 1.10.6.2 Methylation ................................................................................... 78 1.10.6.3 Phosphorylation ........................................................................... 79 1.10.7 3D organisation of nuclear structure and functions ............................. 79 1.11 Chromatin assembly .......................................................................... 83 1.11.1 Dynamics of chromatin assembly in vivo ............................................ 83 1.11.2 Replication-dependent chromatin assembly and maturation ............... 83 1.11.2.1 Role of chaperones in nucleosome assembly................................ 87 1.11.3 Replication-independent chromatin assembly ..................................... 90 1.11.3.1 Chromatin remodelling factors .................................................... 92 1.12 Techniques widely used for the study of DNA replication ............ 95 1.12.1 Evaluating cell proliferation methods ................................................. 95 1.12.1.1 [3H] thymidine incorporation ....................................................... 95 1.12.1.2 Incorporation of halogenated nucleotides.................................... 95 1.12.1.3 Incorporation of 5-ethynyl-2’-deoxyuridine ................................. 96 1.12.2 Chromatin immunoprecipitation ......................................................... 97 1.13 Aims of this study ............................................................................ 100 CHAPTER 2 MATERIALS AND METHODS ............................................. 101 2.1 Materials ........................................................................................... 101 2.1.1 Chemical reagents.............................................................................. 101 2.1.2 Molecular biology reagents ............................................................... 104 2.1.3 Tissue culture cell line and reagents .................................................. 106 2.1.3.1 Cell line ...................................................................................... 106 4 Table of contents 2.1.3.2 Cell culture reagents .................................................................. 107 2.1.4 Computer programmes ...................................................................... 107 2.2 Nucleic acid methods ....................................................................... 108 2.2.1 Preparation of genomic DNA ............................................................ 108 2.2.2 Agarose gel electrophoresis ............................................................... 108 2.2.3 DNA transfer and detection ............................................................... 108 2.2.4 Polymerase Chain Reaction (PCR) ................................................... 109 2.3 Protein methods ............................................................................... 110 2.3.1 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) .................. 110 2.3.2 Protein sample preparation ................................................................ 110 2.3.3 Methods to determine protein concentration ..................................... 111 2.3.3.1 Bradford Protein assay............................................................... 111 2.3.3.2 BCA Protein assay ...................................................................... 111 2.3.4 SDS-PAGE staining methods ............................................................ 112 2.3.4.1 Coomassie Blue staining ............................................................ 112 2.3.4.2 GelCode Blue staining ................................................................ 112 2.3.4.3 Silver staining ............................................................................. 112 2.3.5 Western blotting ................................................................................ 113 2.3.6 Dot Blot procedure ............................................................................ 114 2.3.7 Chromatin immunoprecipitation ....................................................... 114 2.3.7.1 Chromatin preparation ............................................................... 114 2.3.7.2 Immunoprecipitation .................................................................. 115 2.3.8 Immunoprecipitation of BrdU labelled, purified DNA ..................... 115 2.3.9 Immunoprecipitation of BrdU labelled chromatin ............................ 116 2.3.10 Immunoprecipitation of EdU labelled, naked DNA using 5’-BMA azide ................................................................................................... 116 2.3.11 Immunoprecipitation of EdU labelled chromatin using 5’-BMA azide ........................................................................................................... 117 5 Table of contents 2.3.12 DNA mediated chromatin pull-down of EdU labelled chromatin using biotin-TEG azide ............................................................................... 118 2.4 Mass spectrometry methods ........................................................... 118 2.4.1 Filter aided sample preparation (FASP) method ............................... 118 2.4.2 Peptides purification using ZipTip .................................................... 119 2.4.3 Mass spectrometry sample preparation and protein identification after FASP method ..................................................................................... 119 2.5 Cell biology methods ....................................................................... 120 2.5.1 Tissue culture techniques................................................................... 120 2.5.1.1 Cryopreservation ........................................................................ 120 2.5.1.2 Resuscitation............................................................................... 121 2.5.1.3 Cell cycle synchronisation using double thymidine block .......... 121 2.5.1.4 Stable isotope labelling with amino acids in cell culture (SILAC) method ........................................................................................ 121 2.5.2 Detection of DNA synthesis by fluorescence microscopy ................ 122 2.5.2.1 BrdU labelling ............................................................................ 122 2.5.2.2 EdU labelling.............................................................................. 122 2.5.3 Flow cytometry analysis .................................................................... 123 2.5.3.1 Analysis of DNA content and DNA synthesis using propidium iodide and BrdU ......................................................................... 123 2.5.3.2 Analysis of DNA content and DNA synthesis using 7-AAD and 5’- BMA ............................................................................................ 124 2.5.3.3 Analysis of DNA content and DNA synthesis using 7-AAD and 6- carboxyfluorescein-TEG azide ................................................... 124 CHAPTER 3 DEVELOPMENT OF DNA MEDIATED CHROMATIN PULL-DOWN TECHNIQUE ......................................................................... 125 3.1 Introduction ..................................................................................... 125 3.2 Preliminary steps for development of Dm-ChP method .............. 125 6 Table of contents 3.2.1 Optimisation of the cross-link step .................................................... 125 3.2.2 Preparation of chromatin enriched fraction ....................................... 126 3.2.3 Optimisation of sonication step ......................................................... 128 3.2.4 Protein detection after DNA-protein cross-link reversal ................... 129 3.3 Chromatin pull-down of replication proteins ............................... 130 3.3.1 Immunoprecipitation of Mcm2 .......................................................... 130 3.4 Immunoprecipitation of BrdU labelled DNA ................................ 131 3.4.1 Detection of BrdU incorporation in HeLa cells................................. 131 3.4.2 Immunoprecipitation of BrdU labelled, naked DNA ........................ 133 3.4.3 Immunoprecipitation of BrdU labelled DNA from chromatin fraction ........................................................................................................... 136 3.5 DNA mediated chromatin pull-down methodology ...................... 138 3.5.1 Detection of EdU incorporation in HeLa cells .................................. 138 3.6 Capturing of EdU labelled DNA from chromatin enriched fraction ........................................................................................................... 140 3.6.1 Immunoprecipitation of EdU labelled, naked DNA using 5’-BMA azide ................................................................................................... 141 3.6.2 Immunoprecipitation of EdU labelled DNA from chromatin fraction using 5’-BMA azide .......................................................................... 143 3.6.3 Capture of EdU labelled, naked DNA using biotin-TEG azide ........ 144 3.6.4 Capture of EdU labelled DNA from chromatin fraction ................... 147 CHAPTER 4 CHARACTERISATION OF DNA MEDIATED CHROMATIN PULL-DOWN TECHNOLOGY .......................................... 149 4.1 Introduction ..................................................................................... 149 4.2 Requirements for Dm-ChP ............................................................. 149 4.3 Specificity of Dm-ChP ..................................................................... 150 4.4 Sensitivity of Dm-ChP ..................................................................... 152 4.5 Linearity and resolution of Dm-ChP ............................................. 155 7 Table of contents 4.6 Saturation of streptavidin-coated resin during Dm-ChP ............ 156 4.7 Conclusions ...................................................................................... 159 4.8 Proteomic analysis of labelled chromatin recovered after Dm-ChP ........................................................................................................... 160 4.8.1 Introduction ....................................................................................... 160 4.8.2 Detection of chromatin associated proteins after Dm-ChP ............... 161 4.8.3 Preparation of protein complexes for mass spectrometry analysis using FASP method ..................................................................................... 162 4.8.4 Functional classification of Dm-ChP proteome ................................ 163 4.8.5 Identification of non-histone proteins associated with EdU labelled chromatin by western blotting ........................................................... 164 4.8.6 Conclusions ....................................................................................... 165 CHAPTER 5 ASSESSMENT OF PROTEIN DYNAMICS DURING DNA SYNTHESIS AND CHROMATIN MATURATION .................................... 167 5.1 Introduction ..................................................................................... 167 5.2 Dm-ChP studies of DNA replicating at different times during S phase .............................................................................................. 167 5.2.1 Introduction and SILAC methodology .............................................. 167 5.2.2 Proteome analysis of early and late replicating chromatin using SILAC approach............................................................................................. 168 5.2.3 Characterisation of proteins associated with DNA synthesised at different time during S phase ............................................................ 174 5.2.4 Conclusions ....................................................................................... 178 5.3 Dm-ChP studies of protein dynamics during chromatin maturation ........................................................................................ 179 5.3.1 Introduction ....................................................................................... 179 5.3.2 Proteins associated with active replication forks ............................... 180 5.3.3 Conclusions ....................................................................................... 183 8 Table of contents 5.4 Dm-ChP studies of protein dynamics during chromatin maturation after DNA damage ....................................................... 184 5.4.1 Introduction ....................................................................................... 184 5.4.2 Proteins associated with stalled replication forks .............................. 184 5.4.3 Conclusions ....................................................................................... 187 5.5 Preliminary attempt to capture the replisome .............................. 188 CHAPTER 6 DISCUSSION ............................................................................ 192 6.1 Development of DNA mediated chromatin pull-down technique .......................................................................................................... .192 6.2 Validation of Dm-ChP technique ................................................... 194 6.3 Dm-ChP studies of DNA replicating at different times during S phase .............................................................................................. 197 6.4 Dm-ChP studies of protein dynamics during chromatin maturation ........................................................................................ 198 6.5 Dm-ChP studies of protein dynamics during chromatin maturation upon replication stress ................................................ 201 6.6 Overall conclusions and future perspectives ................................. 202 REFERENCES ................................................................................................. 204 APPENDIX 1 .................................................................................................... 234 APPENDIX 2 .................................................................................................... 234 APPENDIX 3 .................................................................................................... 242 APPENDIX 4 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