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

Figure 3.

Figure 3

Kinetics and energetics of single tetrasomes on DNA with a 601 sequence. (A) Shown are histograms of the dwell times of a single NAP1-loaded (H3.1-H4)2 tetrasome on a DNAw/601 molecule in the left- (left panel) and right-handed conformation (right panel) in buffer A (Tables 1 and 2). Exponential fits (red lines) yielded a mean dwell time of τD,left = 37 ± 2 s (N = 371) and τD,right = 28 + 2/−1 s (N = 373), respectively (68% confidence level for estimated values). (B) is the same as in (A) but now in buffer B (Tables 1 and 2). Exponential fits (red lines) yielded a mean dwell time of τD,left = 62 ± 3 s (N = 559) and τD,right = 33 ± 1 s (N = 564), respectively (68% confidence level for estimated values). (C) Shown are histograms of the dwell times of a single NAP1-loaded (H3.1-H4)2 tetrasome on a DNAw/601 molecule in the left-handed (top panel) and right-handed conformation (bottom panel) in buffer C (Tables 1 and 2). Exponential fits (red lines) yielded a mean dwell time of τD,left = 141 + 11/−10 s (N = 187) and τD,right = 30 ± 2 s (N = 188), respectively (68% confidence level for estimated values). All data in (A)–(C) were obtained by dwell-time analysis of the DNA linking number time traces filtered by averaging over 3.3 s (N = 330) (Materials and Methods). (D) Schematic energy diagrams of single NAP1-loaded tetrasomes on DNAw/601 in all buffer conditions are based on the dwell-time values and the probabilities obtained from the linking number distributions (Tables 2, S11, and S13–S15). The solid black lines illustrate the energy levels in buffer A (Table 1) for a NAP1-loaded (H3.1-H4)2 tetrasome on DNArandom, whereas the solid blue lines depict the energetics for a NAP1-loaded (H3.1-H4)2 tetrasome on DNAw/601. The green and orange dashed lines show the energy levels for a NAP1-loaded (H3.1-H4)2 tetrasome on DNAw/601 in buffers B and C, respectively. The free energy differences (ΔE) between the left- and right-handed conformations of tetrasomes on DNAw/601, with the respective energies Eleft and Eright, are considerably decreased compared to tetrasomes loaded onto DNArandom (Table 2). The heights of the energy barriers ΔG∗left and ΔG∗right are estimated from the rates kl->r and kr->l, respectively. For tetrasomes on DNAw/601 relative to DNArandom in buffer A, ΔG∗left decreases strongly, whereas ΔG∗right is largely unchanged. In the presence of mono- and divalent salts in buffers B and C, tetrasomes on DNAw/601 become considerably longer lived in the left-handed conformation (i.e., ΔG∗left is higher) compared with tetrasomes on DNAw/601 in buffer A, whereas the right-handed state (hence ΔG∗right) remains essentially unaltered. To see this figure in color, go online.