Torsional stress generated during DNA replication and transcription has been suggested to facilitate nucleosome unwrapping and thereby the progression of polymerases. However, the propagation of twist in condensed chromatin remains yet unresolved. Here, we measure how force and torque impact chromatin fibers with a nucleosome repeat length of 167 and 197. We find that both types of fibers fold into a left-handed superhelix that can be stabilized by positive torsion. We observe that the structural changes induced by twist were reversible, indicating that chromatin has a large degree of elasticity. Our direct measurements of torque confirmed the hypothesis of chromatin fibers as a twist buffer. Using a statistical mechanics-based torsional spring model, we extracted values of the chromatin twist modulus and the linking number per stacked nucleosome that were in good agreement with values measured here experimentally. Overall, our findings indicate that the supercoiling generated by DNA-processing enzymes, predicted by the twin-supercoiled domain model, can be largely accommodated by the higher-order structure of chromatin.

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Eukaryotic genomes are hierarchically organized into protein-DNA assemblies for compaction into the nucleus. Nucleosomes, with the (H3-H4)2 tetrasome as a likely intermediate, are highly dynamic in nature by way of several different mechanisms. We have recently shown that tetrasomes spontaneously change the direction of their DNA wrapping between left- and right-handed conformations, which may prevent torque buildup in chromatin during active transcription or replication. DNA sequence has been shown to strongly affect nucleosome positioning throughout chromatin. It is not known, however, whether DNA sequence also impacts the dynamic properties of tetrasomes. To address this question, we examined tetrasomes assembled on a high-affinity DNA sequence using freely orbiting magnetic tweezers. In this context, we also studied the effects of mono- and divalent salts on the flipping dynamics. We found that neither DNA sequence nor altered buffer conditions affect overall tetrasome structure. In contrast, tetrasomes bound to high-affinity DNA sequences showed significantly altered flipping kinetics, predominantly via a reduction in the lifetime of the canonical state of left-handed wrapping. Increased mono- and divalent salt concentrations counteracted this behavior. Thus, our study indicates that high-affinity DNA sequences impact not only the positioning of the nucleosome but that they also endow the subnucleosomal tetrasome with enhanced conformational plasticity. This may provide a means to prevent histone loss upon exposure to torsional stress, thereby contributing to the integrity of chromatin at high-affinity sites.@en

The entire blueprint of all living things is encoded in their genomes, which consist of DNA strands. The genome of a complex organism like ourselves can be several meters long. One of the miracles of nature is that such DNA molecules can be stored in the micron-sized nucleus of eukaryotic cells. For this purpose, the relatively large genome of eukaryotes has to be tightly packed while still remaining accessible for vital cellular processes such as replication, transcription, and repair. This is achieved by the organization of the eukaryotic genome into a hierarchical nucleoprotein assembly termed chromatin. Its fundamental unit is the nucleosome, which comprises a short piece of DNA wrapped around a disk-shaped core of eight histone proteins in a left-handed superhelix. As such, nucleosomes constitute the first level of DNA compaction and are assigned a key role in the regulation of the genome to maintain the proper functioning and viability of eukaryotic cells. Hence, detailed knowledge of this fascinating complex is crucial for understanding fundamental processes of life. This thesis deals with investigations of the structure and dynamics of a nucleosomal substructure called tetrasome at the single-molecule level. @en

Nucleosomes consisting of a short piece of deoxyribonucleic acid (DNA) wrapped around an octamer of histone proteins form the fundamental unit of chromatin in eukaryotes. Their role in DNA compaction comes with regulatory functions that impact essential genomic processes such as replication, transcription, and repair. The assembly of nucleosomes obeys a precise pathway in which tetramers of histones H3 and H4 bind to the DNA first to form tetrasomes, and two dimers of histones H2A and H2B are subsequently incorporated to complete the complex. As viable intermediates, we previously showed that tetrasomes can spontaneously flip between a left-handed and right-handed conformation of DNA-wrapping. To pinpoint the underlying mechanism, here we investigated the role of the H3-H3 interface for tetramer flexibility in the flipping process at the single-molecule level. Using freely orbiting magnetic tweezers, we studied the assembly and structural dynamics of individual tetrasomes modified at the cysteines close to this interaction interface by iodoacetamide (IA) in real time. While such modification did not affect the structural properties of the tetrasomes, it caused a 3-fold change in their flipping kinetics. The results indicate that the IA-modification enhances the conformational plasticity of tetrasomes. Our findings suggest that subnucleosomal dynamics may be employed by chromatin as an intrinsic and adjustable mechanism to regulate DNA supercoiling.

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Eukaryotic DNA is tightly packed into a hierarchically ordered structure called chromatin in order to fit into the micron-scaled nucleus. The basic unit of chromatin is the nucleosome, which consists of a short piece of DNA wrapped around a core of eight histone proteins. In addition to their role in packaging DNA, nucleosomes impact the regulation of essential nuclear processes such as replication, transcription, and repair by controlling the accessibility of DNA. Thus, knowledge of this fundamental DNA–protein complex is crucial for understanding the mechanisms of gene control. While structural and biochemical studies over the past few decades have provided key insights into both the molecular composition and functional aspects of nucleosomes, these approaches necessarily average over large populations and times. In contrast, single-molecule methods are capable of revealing features of subpopulations and dynamic changes in the structure or function of biomolecules, rendering them a powerful complementary tool for probing mechanistic aspects of DNA–protein interactions. In this review, we highlight how these singlemolecule approaches have recently yielded new insights into nucleosomal and subnucleosomal structures and dynamics.@en