DNA Sequence Is a Major Determinant of Tetrasome Dynamics

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.Accepted Author ManuscriptBN/Nynke Dekker La

Modification of the histone tetramer at the H3-H3 interface impacts tetrasome conformations and dynamics

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.Accepted Author ManuscriptBN/Nynke Dekker La