Structural studies of the nucleosome, assembled with different histones, DNA fragments and HMGN proteins, and derivatized with ruthenium anticancer agents

14 Introduction . 16 1.1. Chromatin Architecture . 17 1.1.1. Nucleosome Core Particle . 17 1.1.2. Centromeric Nucleosome Core Particle . 22 1.1.3. Telomeres. 24 1.1.4. Higher Order Chromatin . 25 1.2. Chromatin Regulation . 26 1.2.1. Nucleosome Organization and Dynamics . 26 1.2.2. Histone Variants . 28 1.2.3. Histone Post-translational Modification . 30 1.2.4. Nucleosome Remodelling Complex . 32 1.3. High Mobility Group Protein Family . 32 1.3.1. High Mobility Group A Protein . 33 1.3.2. High Mobility Group B Protein . 33 1.3.3. High Mobility Group N Protein . 34 1.4. Chromatin as anticancer drug target. 37 1.5. Scope of this thesis . 38 Materials and Methods . 40 2.1. Palindromic DNA Production . 40 4 2.1.1. DNA constructs and Plasmids . 41 2.1.2. Cell transformation . 41 2.1.3. Plasmid extraction . 42 2.1.4. EcoRV Digestion and Purification of DNA Insert. 43 2.1.5. Large Scale Dephosphorylation . 43 2.1. 6. HinfI/EcoRI Digestion and Purification of Half-palindromic Repeats by FPLC . 44 2.1. 7. Ligation and Purification of palindromic DNA . 44 2.2. Histone Octamer Preparation . 45 2.2.1. Cell transformation . 45 2.2.2. Histone protein Overexpression . 45 2.2.3. Inclusion Body Preparation . 46 2.2.4. FPLC Purification of Histones . 46 2.2.5. Histone Octamer Refolding . 47 2.2.6. FPLC Purification of Histone Octamer. 47 2.3. Nucleosome Core Particle Reconstitution . 47 2.3.1. Off-centered NCP Reconstitution . 47 2.3.2. Centered NCP Reconstitution . 48 2.3.3. NCP Purification . 48 2.4. HMGN Production . 48 2.5. Crystal Preparation . 49 2.6. Crystal Processing . 52 2.6.1. Crystallographic Data Collection . 52 2.6. 2. Crystallographic Data Analysis . 52 2.7. Electrophoretic Mobility Shift Assay (EMSA) . 52 2.8. Western Blot . 53 2.9. Crosslinking . 53 2.10. Fluorescence Assay . 53 5 Nucleosome Core Particle Production and Crystallization . 55 3.1. Palindromic DNA Production . 55 3.2. Histone Octamer Production . 56 3.3. Nucleosome Core Particle Reconstitution . 59 3.4. Nucleosome Core Particle Crystallization . 62 Nucleosome Core Particle Structure . 63 4.1. Overall NCP Structure . 63 4.1.1. Xenopus laevis NCP145 and Human NCP145 structure . 68 4.1.2. Human NCP146b structure . 74 Ruthenium Agent Interaction with Nucleosome Core Particle . 77 5.1. Result . 77 HMGN Interaction with Nucleosome Core Particle . 87 6.1. Result . 87 6.1.1. HMGN triggers variant-specific conformational changes in nucleosome structure without apparent loss of histone proteins . 87 6.1.2. Higher stoichiometry of HMGN is observed on S2 species . 89 6.1.3. Binding assessment of HMGN NBD truncation series to Nucleosome Core Particle . 91 6.1.4. Identification of the NBD’s histone binding partner . 94 6.1.5. Crystallization of HMGN-NCP Complex . 98 6.1. 6. Crystallization of an HMGN NBD-NCP Complex . 100 6.1. 7. Searching for NCP’s divalent ion-free crystallization buffers . 104 Discussion . 105 7.1. Human and Xenopus laevis Nucleosomes adopt nearly identical structures . 105 7.2. Steric hindrace governs RAPTA compound-NCP interactions. 106 7.3. Arene ring and PTA ligand confer site selectivity for RAPTA binding . 107 7.4. RAPTA compound association to Xenopus laevis NCP and human NCP . 111 7.5. RAPTA binding sites overlap with a nuclear protein binding site on the NCP surface 113 6 7.6. HMGN binding to Nucleosome . 114 7.7. NBD Binding Characterization . 116 7.8. Crystallization Strategy . 119 Appendix . 124 References . 128