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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

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