Recent insights from single-molecule studies into nucleosome structure and dynamics

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.BN/Nynke Dekker La

Comparing the Assembly and Handedness Dynamics of (H3.3-H4)2 Tetrasomes to Canonical Tetrasomes

Eukaryotic nucleosomes consists of an (H3-H4)2 tetramer and two H2A-H2B dimers, around which 147 bp of DNA are wrapped in 1.7 left-handed helical turns. During chromatin assembly, the (H3-H4)2 tetramer binds first, forming a tetrasome that likely constitutes an important intermediate during ongoing transcription. We recently showed that (H3-H4)2 tetrasomes spontaneously switch between a left- and right-handed wrapped state of the DNA, a phenomenon that may serve to buffer changes in DNA torque induced by RNA polymerase in transcription. Within nucleosomes of actively transcribed genes, however, canonical H3 is progressively replaced by its variant H3.3. Consequently, one may ask if and how the DNA chirality dynamics of tetrasomes is altered by H3.3. Recent findings that H3.3-containing nucleosomes result in less stable and less condensed chromatin further underline the need to study the microscopic underpinnings of H3.3-containing tetrasomes and nucleosomes. Here we report real-time single-molecule studies of (H3.3-H4)2 tetrasome dynamics using Freely Orbiting Magnetic Tweezers and Electromagnetic Torque Tweezers. We find that the assembly of H3.3-containing tetrasomes and nucleosomes by the histone chaperone Nucleosome Assembly Protein 1 (NAP1) occurs in an identical manner to that of H3-containing tetrasomes and nucleosomes. Likewise, the flipping behavior of DNA handedness in tetrasomes is not impacted by the presence of H3.3. We also examine the effect of free NAP1, H3.3, and H4 in solution on flipping behavior and conclude that the probability for a tetrasome to occupy the left-handed state is only slightly enhanced by the presence of free protein. These data demonstrate that the incorporation of H3.3 does not alter the structural dynamics of tetrasomes, and hence that the preferred incorporation of this histone variant in transcriptionally active regions does not result from its enhanced ability to accommodate torsional stress, but rather may be linked to specific chaperone or remodeler requirements or communication with the nuclear environment.BN/BionanoscienceApplied Science

Nucleosome Assembly Dynamics Involve Spontaneous Fluctuations in the Handedness of Tetrasomes

DNA wrapping around histone octamers generates nucleosomes, the basic compaction unit of eukaryotic chromatin. Nucleosome stability is carefully tuned to maintain DNA accessibility in transcription, replication, and repair. Using freely orbiting magnetic tweezers, which measure the twist and length of single DNA molecules, we monitor the real-time loading of tetramers or complete histone octamers onto DNA by Nucleosome Assembly Protein-1 (NAP1). Remarkably, we find that tetrasomes exhibit spontaneous flipping between a preferentially occupied left-handed state (?Lk = ?0.73) and a right-handed state (?Lk = +1.0), separated by a free energy difference of 2.3 kBT (1.5 kcal/mol). This flipping occurs without concomitant changes in DNA end-to-end length. The application of weak positive torque converts left-handed tetrasomes into right-handed tetrasomes, whereas nucleosomes display more gradual conformational changes. Our findings reveal unexpected dynamical rearrangements of the nucleosomal structure, suggesting that chromatin can serve as a “twist reservoir,” offering a mechanistic explanation for the regulation of DNA supercoiling in chromatin.BN/BionanoscienceApplied Science