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. 2019 Aug 21;117(11):2217–2227. doi: 10.1016/j.bpj.2019.07.055

Figure 1.

Figure 1

Real-time assembly of single tetrasomes onto DNA with a 601 sequence. (A) Shown is the top view on ∼80 bp of DNA (dark gray) bound to a tetrameric protein core consisting of the histones H3 (orange) and H4 (cyan). This image was created by modifying the structural data of the Drosophila nucleosome from the Protein Data Bank (PDB) with the PDB identification code 2PYO (82) using the PyMOL Molecular Graphics System, Version 1.8 (Schrödinger, New York, NY). (B) Experimental assay based on FOMT is shown (49). A single DNA construct (black) containing a 601 sequence (red) at its center (DNAw/601) is attached to a coverslip (light blue) at one end and tethered to a superparamagnetic bead (light brown) at the other end. Above the flow cell, a cylindrically shaped permanent magnet (dark blue/red), with its axis precisely aligned with the DNA tether, exerts a constant force (F) on the bead while allowing its free rotation in the (x,y) plane (indicated by the black circular arrow). This enables the direct measurement of the DNA molecule’s length z and linking number Θ, which upon the assembly of a tetrasome (orange), are changed by Δz and ΔΘ (indicated by the red straight and circular arrows, respectively). Tetrasome assembly is induced by flushing in either (H3.1-H4)2 tetramers alone or preincubated histone/NAP1 complexes. Nonmagnetic beads attached to the flow cell surface serve as reference for drift correction. This figure is adapted from (37) (https://doi.org/10.1063/1.5009100), with the permission of AIP Publishing (Melville, NY). (C) Shown are partial time traces of the length z (in nm, top panel) and the linking number Θ (in turns, bottom panel) of a DNAw/601 molecule before and upon the assembly of a (H3.1-H4)2 tetrasome. The formation of a tetrasome simultaneously decreased both quantities in the form of a step identified using a step-finder algorithm (red lines) (Materials and Methods). About 60 s after assembly, free proteins were flushed out with measurement buffer (orange arrows) to prevent further histone binding. In this particular experiment, a tetrasome was assembled by flushing in NAP1/histone complexes (green arrows) in buffer A (Table 1). A corresponding time trace of a DNAw/601 molecule upon the assembly of a tetrasome from histone tetramers only is shown in Fig. S4. (D) Shown is a histogram of the changes in DNA length upon assembly and disassembly Δz(dis)ass (in nm, top panel) of single (H3.1-H4)2 tetrasomes (N = 27) and in DNA linking number upon assembly and disassembly ΔΘ(dis)ass (in turns, bottom panel) of single (H3.1-H4)2 tetrasomes (N = 27) in all buffers (Table 1), shown together with the mean spatial resolution corresponding to the average 1 SD (16 nm and 0.5 turns; green lines) from all experiments. Data beyond the resolution limits (hatched area) were excluded from the determination of the mean change Δz(dis)ass = 23 ± 5 nm (n = 25) and ΔΘ(dis)ass = 0.9 ± 0.2 turns (n = 26). To see this figure in color, go online.