Eukaryotic genomes are densely compacted into chromatin, so that they can be contained in the nucleus. Despite the tight packaging genes need to be accessible for normal metabolic activities to occur, such as transcription, repair and replication. These processes are regulated by a vast number of proteins but also by the level of compaction of chromatin. The translocation of motor proteins along DNA produces torsional stress which in turn alters chromatin compaction both upstream and downstream. Few single-molecule studies have investigated the behaviour of nucleosomes when subjected to torsion. The inability to measure the applied torque though represented a major limitation to those reports. The implementation of the rotor bead assay, which allows to directly measure the torque applied in magnetic tweezers experiments, has been hindered by a difficult sample preparation procedure. In order to overcome this limitation an efficient protocol for the insertion of chemical or structural modifications in long DNA substrates was developed. This was then further expanded to allow the introduction of labels in multiple loci and/or both strands and has been used successfully in a number of studies. Furthermore this is the first report of tensile experiments performed on nucleosomes with a histone variant. H2AvD nucleosomes were studied due to the interest in the biological role of H2A.Z-family proteins. Interestingly, the variant nucleosomes appear to bind less DNA and to be evicted from the DNA at lower forces than those observed for canonical nucleosomes. These findings show an important role for the H2A-H2B dimers in the mechanical stability of nucleosomes. Furthermore these results are in agreement with recently proposed models of a dynamic nucleosome, in contrast to the long-standing view of nucleosomes as static structures.

Eukaryotic genomes are densely compacted into chromatin, so that they can be contained in the nucleus. Despite the tight packaging genes need to be accessible for normal metabolic activities to occur, such as transcription, repair and replication. These processes are regulated by a vast number of proteins but also by the level of compaction of chromatin. The translocation of motor proteins along DNA produces torsional stress which in turn alters chromatin compaction both upstream and downstream. Few single-molecule studies have investigated the behaviour of nucleosomes when subjected to torsion. The inability to measure the applied torque though represented a major limitation to those reports. The implementation of the rotor bead assay, which allows to directly measure the torque applied in magnetic tweezers experiments, has been hindered by a difficult sample preparation procedure. In order to overcome this limitation an efficient protocol for the insertion of chemical or structural modifications in long DNA substrates was developed. This was then further expanded to allow the introduction of labels in multiple loci and/or both strands and has been used successfully in a number of studies. Furthermore this is the first report of tensile experiments performed on nucleosomes with a histone variant. H2AvD nucleosomes were studied due to the interest in the biological role of H2A.Z-family proteins. Interestingly, the variant nucleosomes appear to bind less DNA and to be evicted from the DNA at lower forces than those observed for canonical nucleosomes. These findings show an important role for the H2A-H2B dimers in the mechanical stability of nucleosomes. Furthermore these results are in agreement with recently proposed models of a dynamic nucleosome, in contrast to the long-standing view of nucleosomes as static structures.:Abstract Table of contents 1 Introduction 1.1 The transforming principle 1.2 Chromatin 1.2.1 Nucleosomes 1.2.2 The 30 nm fibre: a mirage? 1.2.3 Histone code 1.3 Histone variant H2A.Z 1.3.1 H2A.Z and transcription 1.4 Single molecule studies of chromatin 1.4.1 Chromatin under tension 1.4.2 Open nucleosome 1.4.3 Twisted chromatin 1.5 Single molecule techniques 1.5.1 Atomic force microscopy 1.5.2 Foerster resonance energy transfer 1.5.3 Magnetic tweezers 1.5.4 Worm-like chain model 2 Aims of the project 3 Cut and paste method for internal DNA labelling 3.1 Introduction 3.2 Experimental design 3.3 Results 3.3.1 Sequence design and cloning 3.3.2 Labelling and religation efficiency 3.3.3 Structural modifications 3.3.4 Labelling of multiple loci 3.3.5 Opposite-strand labelling 3.4 Discussion 4 Reconstituting chromatin 4.1 Long array of NPSs 4.1.1 Polymer physics applied to molecular cloning 4.1.2 Preventing homologous recombination 4.2 Expression and purification of histone proteins 4.2.1 Protein expression 4.2.2 Inclusions bodies 4.2.3 Histone purification 4.2.4 Octamer reconstitution and isolation 4.2.5 H2AvD 4.3 Reconstitution of nucleosomal arrays and biochemical analysis 4.3.1 Reconstitution procedure 4.3.2 Biochemical analysis 4.4 Tweezers construct with nucleosomes 5 Eviction of nucleosomes 5.1 Nucleosome eviction 5.1.1 A two-stage process 5.1.2 Chromatin fibres 5.1.3 Reassembly of nucleosomes 5.1.4 Distinct populations within nucleosome eviction events 5.1.5 Nicked and supercoilable nucleosomal arrays 5.2 Eviction of H2AvD-nucleosomes 5.2.1 H2AvD-nucleosomes bind less inner turn DNA 5.2.2 H2AvD-nucleosomes evict at lower forces 5.2.3 Likelihood of nucleosome reassembly 5.2.4 Gradual weakening of nucleosomes 5.2.5 Analysis software NucleoStep 5.3 Towards a rotor-bead assay on chromatin 5.4 Discussion 5.4.1 Nucleosome eviction in two stages 5.4.2 The fate of dimers in single molecule experiments 5.4.3 Structural origin and biological relevance of the mechanical properties of H2AvD-nucleosomal core particles 5.4.4 Monolithic or dynamic nucleosomes 6 Conclusions Bibliography Appendix 6.1 Internal labelling Procedure 6.1.1 Cloning 6.1.2 Nicking & cutting 6.1.3 The replace reaction 6.1.4 Purification 6.1.5 Ligation (optional) 6.1.6 Opposite strand labelling 6.1.7 Assessing the results of the labelling reaction 6.2 Chromatin reconstitution 6.2.1 Long array of NPSs 6.2.2 Expression and purification of histone proteins 6.2.3 Reconstitution of nucleosomal arrays and biochemical characterization 6.2.4 Simple Phenol:chloroform isolation of DNA 6.3 Magnetic tweezers experiments 6.3.1 Flow cell assembly 6.3.2 Functionalization of flow cells 6.3.3 Magnetic tweezers and rotor bead measurements 6.3.4 Force calibration List of Figures List of Tables List of publications Acknowledgements Declaration of originality