Volume 109, Issue 8, Pages 1676-1685 (October 2015) Nucleosome Core Particle Disassembly and Assembly Kinetics Studied Using Single- Molecule Fluorescence  Noa Plavner Hazan, Toma E. Tomov, Roman Tsukanov, Miran Liber, Yaron Berger, Rula Masoud, Katalin Toth, Joerg Langowski, Eyal Nir  Biophysical Journal  Volume 109, Issue 8, Pages 1676-1685 (October 2015) DOI: 10.1016/j.bpj.2015.07.004 Copyright © 2015 Biophysical Society Terms and Conditions

Figure 1 smFRET/ALEX analyses of NCPs at three different NCP and NaCl concentrations, including a schematic of the NCP and an E/S histogram measured in each condition. (A) Measurement at 3 pM labeled NCP and 5 mM NaCl, with no unlabeled NCP present in the solution. Only an HF population, corresponding to intact NCP, was observed, and the NCP complex remained intact for the 2 h duration of the experiment. (B) Measurement at 3 pM labeled NCP at 80 mM NaCl with no unlabeled NCP present in the solution. The HF population decreased with time after the introduction of the NaCl, and an LF population, corresponding to free DNA, increased. (C) Measurement at 3 pM labeled NCP, 1 nM unlabeled NCP, and 750 mM NaCl. The HF population decreased with time and a low LF population increased. An MF population increased and then decreased with time. The MF population may correspond to NCP with one DNA arm unwrapped (3). To see this figure in color, go online. Biophysical Journal 2015 109, 1676-1685DOI: (10.1016/j.bpj.2015.07.004) Copyright © 2015 Biophysical Society Terms and Conditions

Figure 2 The fraction of HF population measured 900–1200 s after introduction of the indicated concentration of NaCl and at 1 nM unlabeled NCP concentration. The increase in NCP stability from 150 to 650 mM NaCl is a result of salt-induced stabilization of the dimers and possibly tetramers (see main text). Biophysical Journal 2015 109, 1676-1685DOI: (10.1016/j.bpj.2015.07.004) Copyright © 2015 Biophysical Society Terms and Conditions

Figure 3 Salt-induced NCP dissociation at 3 pM NCP concentration. (A) NCP dissociation kinetic profiles measured at different NaCl concentrations. At time zero, 3 pM NCP was introduced into buffered solution containing different NaCl concentrations. The HF population fractions, calculated every 300 s from smFRET/ALEX data, are plotted as a function of time. The solid lines are fits of the data using a first-order dissociation model (Eq. S4 in the Supporting Material). (B) Plot of the dissociation rate constants (kHF → LF) as a function of NaCl concentration. To see this figure in color, go online. Biophysical Journal 2015 109, 1676-1685DOI: (10.1016/j.bpj.2015.07.004) Copyright © 2015 Biophysical Society Terms and Conditions

Figure 4 Nucleosome dissociation and dependence of NCP stability on NCP concentration. (A) HF fraction as a function of time after addition to 80 mM NaCl at different NCP concentrations. (B) HF fraction as a function of time after addition to 300 mM NaCl in different NCP concentrations. The solid lines are fits to a two-state model. (C) Association rates calculated from the two-state fits. (D) Fraction of HF in equilibrium at different NCP concentrations. To see this figure in color, go online. Biophysical Journal 2015 109, 1676-1685DOI: (10.1016/j.bpj.2015.07.004) Copyright © 2015 Biophysical Society Terms and Conditions

Figure 5 Salt-induced NCP dissociation at 1 nM NCP concentration. (Left) HF fraction as a function of time after addition to the indicated NaCl concentration. (Right) Fraction of HF in equilibrium as a function of NaCl concentration. To see this figure in color, go online. Biophysical Journal 2015 109, 1676-1685DOI: (10.1016/j.bpj.2015.07.004) Copyright © 2015 Biophysical Society Terms and Conditions

Figure 6 Nucleosome dynamics in high salt concentration. (A) Fractions of HF, MF, and LF as a function of time after addition to the indicated NaCl concentration at 1 nM NCP. (B) Fractions of HF, MF, and LF as a function of time after addition to 900 mM NaCl at the indicated NCP concentrations. The lines are to guide the eye, and representative fitting of the kinetic curves is shown in Figs. 7 A and S8. (C) The fractions of HF, MF, and LF at time 5000 s taken from the graphs in (A) and (B). Examples of fitting of the kinetic curves are shown in Figs. 7 and S8). To see this figure in color, go online. Biophysical Journal 2015 109, 1676-1685DOI: (10.1016/j.bpj.2015.07.004) Copyright © 2015 Biophysical Society Terms and Conditions

Figure 7 Nucleosome dynamics at high salt concentration. (A) Typical reaction profiles demonstrating the decrease of the HF population, the increase followed by a decrease of the MF intermediate population, and the increase of the LF population. More examples are given in Fig. S8. (B and C) Summaries of the calculated rate constants measured at 1 nM NCP (B) and at 900 mM NaCl (C). To see this figure in color, go online. Biophysical Journal 2015 109, 1676-1685DOI: (10.1016/j.bpj.2015.07.004) Copyright © 2015 Biophysical Society Terms and Conditions