Abstract
Nucleosomes formed by DNA wrapping around histone octamers are the basic compaction unit of eukaryotic chromatin. Besides packaging DNA, they also play an essential role in genome regulation by controlling DNA accessibility. Their assembly follows a precise pathway in which first tetramers of H3 and H4 histones bind to DNA, forming tetrasomes, and then two dimers of H2A and H2B histones complete the formation of full nucleosomes. Here, we present our single-molecule studies on the assembly of tetrasomes by directly measuring the length, twist, and torque of single DNA molecules using Freely Orbiting Magnetic Tweezers (FOMT) and electro-Magnetic Torque Tweezers (eMTT). By this means, we have investigated both the replication-coupled histone H3.1 and its replication-independent variant histone H3.3. Our results indicate that tetrasomes exhibit a spontaneous flipping between a preferentially occupied left-handed state (ΔLk = −0.73) and a less populated right-handed state (ΔLk = +1.0), while weak torques convert them from one state into the other. In addition, the stepwise assembly of full nucleosomes suggests that tetrasomes undergoing changes in handedness remain viable intermediates. We further show how studies performed on tetrasomes that are artificially modified at their H3-H3 interface provide more insight into their handedness dynamics. Our findings reveal dynamical rearrangements of the nucleosomal structure that illustrate how chromatin could regulate DNA-supercoiling.
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