University of Dundee DOCTOR OF PHILOSOPHY Characterisation of the (H3-H4)2-tetramer and its interaction with histone chaperones Bowman, Andrew Award date: 2010 Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 28. 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Characterisation of the (H3-H4)2-tetramer and its interaction with histone chaperones Andrew James Bowman A thesis submitted for the degree of Doctor of Philosophy University of Dundee 10th September 2010 2 Table of Contents List of figures and tables .................................................................................................. 5 Abbreviations .................................................................................................................... 7 Abstract.............................................................................................................................. 9 1. Introduction............................................................................................................... 11 1.1 The nucleosome core particle, higher order chromatin structure and the dynamic nature of nucleosomes.................................................................................... 11 1.1.1 Structure of the NCP................................................................................. 11 1.1.2 Higher order chromatin structure.............................................................. 15 1.1.3 Non canonical nucleosomes and the dynamic nature of the NCP ............ 17 1.1.4 Nucleosome positioning and associated free energies of histone-DNA interactions................................................................................................................ 19 1.1.5 Summary................................................................................................... 22 1.2 Pathways for altering chromatin structure and composition............................. 23 1.2.1 Post translation modification of histones.................................................. 23 1.2.2 ATP-dependent remodelling of chromatin ............................................... 27 1.2.3 Histone variants ........................................................................................ 29 1.3 Histone chaperones in the regulation of chromatin .......................................... 31 1.3.1 The structure of histone chaperones and their interaction with core histones ……………………………………………………………………………31 1.3.2 Histone chaperones in chromatin replication............................................ 37 1.3.3 The role of histone chaperones in transcription........................................ 39 1.3.4 The role of histones in DNA damage and repair ...................................... 42 1.4 Aims and objectives.......................................................................................... 42 2. Materials and Methods.............................................................................................. 44 2.1 Protein expression and purification .................................................................. 44 2.1.1 Expression of recombinant histones ......................................................... 44 2.1.2 Expression and purification of deuterated histones .................................. 44 2.1.3 Refolding of histone octamer and tetramer............................................... 45 2.1.4 Expression and purification of recombinant histone-chaperones ............. 45 2.1.5 Sequential affinity purification for single labelling a dimer..................... 47 2.2 Spin labelling of proteins.................................................................................. 47 2.2.1 Labelling of histones................................................................................. 47 2.2.2 Labelling of Asf1 ...................................................................................... 48 2.3 Preparation of proteins for EPR........................................................................ 48 2.3.1 Preparation of histone octamer and tetramer ............................................ 48 2.3.2 Preparation of labelled H3 in complex with Asf1 K41R1........................ 48 3 2.3.3 Preparation of labelled histones in complex with unlabelled chaperones 49 2.4 Analytical gel filtration..................................................................................... 49 1. 2.4.1 Histones in complex with chaperones.............................................. 49 2.4.2 Histone tetramer........................................................................................ 50 2.5 Protein crosslinking .......................................................................................... 50 2.5.1 Site directed sulfydryl reactive crosslinking............................................. 50 2.5.2 Amine reactive crosslinking ..................................................................... 51 2.6 In vitro tetrasome reconstitution and template preparation .............................. 52 2.6.1 Amplification of tetrasomal templates by PCR ........................................ 52 2.6.2 Cloning of a 601 tetrasomal array construct............................................. 52 2.6.3 Reconstitution by salt dialysis .................................................................. 53 2.6.4 Reconstitution by Nap1............................................................................. 53 2.7 Acetyl transferase assay.................................................................................... 54 2.8 Circular dichroism ............................................................................................ 54 2.9 Electron paramagnetic resonance & molecular dynamics simulations............. 54 2.10 Buffers and reagents ......................................................................................... 55 3. Characterising the interaction between the core histones and their chaperones Nap1, Vps75 and Asf1................................................................................................................. 57 3.1 Introduction............................................................................................................. 57 3.2 Results............................................................................................................... 58 3.2.1 Gel filtration analysis of Nap1 with core histones.................................... 58 3.2.2 Gel filtration analysis of Vps75 and Asf1 with H3 and H4...................... 61 3.2.3 A directed crosslinking reporter assay for probing tetramerisation of H3- H4 ……………………………………………………………………………64 3.2.4 Probing the conformation of H3-H4 when in complex with Nap1, Vps75 and Asf1 ……………………………………………………………………………67 3.2.5 Long range distance extraction from labelled histones in complex with Nap1, Vps75 and Asf1.............................................................................................. 68 3.2.6 Nap1 and Vps75 can utilise H3 and H4 trapped in their tetrameric conformation............................................................................................................. 70 3.2.7 Strategies for singly labelling a dimer ...................................................... 75 3.3 Discussion......................................................................................................... 77 3.3.1 Summary................................................................................................... 77 3.3.2 Implications in replication dependent chromatin reconfiguration............ 78 3.3.3 Implications in replication independent chromatin reconfiguration......... 80 3.3.4 Symmetry as a requirement for (H3-H4)2-tetramer interaction................ 83 4. Probing the structure of the (H3-H4)2-tetramer using SDSL and pulsed EPR......... 85 4.1 Introduction....................................................................................................... 85 4.2 Results..............................................................................................................