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In living cells, DNA is wrapped around proteins called histones in the form of chromatin fibers which limit its accessibility to proteins and protein complexes involved in DNA transcription, replication, recombination and repair. These processes occur throughout the life of a cell, and therefore chromatin structure must change to allow the genetic information of the DNA to be processed. The classical biochemical and biophysical methods used in chromatin research are population-averaged methods, which assess properties of the whole population of macromolecules. They are neither capable of detecting possible heterogeneities among individual molecules nor of observing transitional structural changes in real-time. On the other hand, recently developed single-molecule methods allow observation of individual molecules in real-time, thus providing molecular parameters important for understanding structural dynamics. Single molecule techniques can be sorted into several groups: (i) imaging methods (AFM), (ii) fluorescence methods, used to study structural changes, either spontaneous or occurring during biochemical processes like enzymatic action, and (iii) methods that allow application and measurements of force. The latter probe the mechanical response of biomacromolecules to applied stretching force or torque. Applying single-molecule techniques to the study of chromatin is especially advantageous in view of the complexity of its structure and the enormous heterogeneity in terms of post-synthetic modifications. Nucleosome assembly and transcription are related because they address the broader issue of how cellular machineries deal with the organization of DNA into chromatin structure. The answer to these questions will lead to a better understanding of whether the enzymatic machineries, by themselves being molecular motors, can deal with chromatin structure, or whether they need the help of external factors to do so. Specifically, we aim at understanding the behaviour of the chromatin fiber upon external application of tension and/or torsion to mimic similar conditions created by physiological processes in vivo . My work is aimed at studying the dynamics of chromatin fibers and transcription through nucleosomes with the use of home-built magnetic tweezers (MT). In this instrumental set-up, a single DNA molecule is attached at one of its termini to the surface of the observation chamber and at the other terminus to a magnetic bead. Manipulation of the magnetic bead with the help of external magnets allows the introduction of positive or negative supercoiling in the DNA molecule, as well as stretching it with a defined force. This set-up was used to approach the following issues: (1) Nucleosome assembly in real-time on topologically-constrained DNA molecules using Magnetic Tweezers Assembly was achieved using chicken erythrocyte core histones and histone chaperone protein Nap1 under constant low force. We observed partial assembly when the DNA was topologically-constrained and complete assembly on unconstrained (nicked) DNA tethers. To verify that the lack of full nucleosome assembly on topologically-constrained tethers was due to compensatory accumulation of positive supercoiling in the rest of the template, we performed experiments in which we mechanically relieved the positive supercoiling by rotating the external magnetic field at certain time points of the assembly process. Such rotation did lead to complete saturation of the template with nucleosomes. (2) Effect of histone H2A.Z on transcription depending on the DNA sequence Recent observations have shown that some histone variants that are deposited in nonreplicating chromatin are found in genes that are actively transcribed. Although the phenomenology of the deposition process is more or less understood, the structural consequences of the presence of these variants are unclear. My work addresses the issue of whether the 'active' variants H3.3 and H2A.Z directly affect the ability of reconstituted nucleosomes to be transcribed. We used nucleosomal particles reconstituted with human recombinant core histones and naturally occurring nucleosome positioning sequences. T7 RNA polymerase was used as a model enzyme to transcribe reconstituted nucleosomes containing either canonical human recombinant histones, or two histone variants, H2A.Z or H3.3, whose presence has been associated with active transcription. H2A.Z-containing nucleosomes were refractive to transcription, with the actual level of transcription determined by the sequence of the underlying DNA template. These results underscore the interplay between the presence of H2A.Z and the DNA sequence in determining transcription through nucleosomes. (3) Fate of nucleosome during transcription elongation using magnetic tweezers We used an array of nucleosomes reconstituted on 18 tandem repeats of nucleosomal positioning DNA containing 208 bp. For transcription through nucleosomes we used a nucleosomal array construct and T7 RNA polymerase molecules freely moving along the DNA tether. Bulk transcription experiments were carried out to confirm that transcription occurred under our experimental conditions. In the MT experiments, transcription on the freely moving polymerase construct was achieved using transcription buffer, T7 RNA polymerase, RNase A and all four NTPs. We observed a net extension in the DNA length during transcription due to nucleosome disassembly. When analyzing the step size of both upward steps and downward steps, a dominant peak at ~50 nm was observed which may be due to the release of entire octamer.
