Extended Data Fig. 5: GAF-DBD preferentially re-visits the same cognate site on individual hsp70 nucleosomes.

a, Representative single-molecule trajectory for Site C binding. b, Representative single-molecule trajectories showing repetitive visits to the same binding site on a single nucleosome. Upper trace shows repetitive visits to Site A; middle trace, Site B; lower trace, Site C. The bottom trace shows repeated binding events with zero Cy3-Cy5 FRET and low (0.24) Cy3-Cy7 FRET, consistent with binding to a distal part of the Cy7-labeled motif similar to Site B. The difference in Cy3-Cy5 FRET could be due to heterogeneous phasing of the hsp70 nucleosome as suggested by the Cy5-Cy7 FRET histogram in c. Black asterisks mark transient Cy3-only periods, potentially caused by ultra-short-range 1D diffusion on the nucleosome, resulting in a temporary loss of FRET. Green asterisks indicate binding events to non-cognate sites on the nucleosome. c, Pie charts showing, for all binding events at site A (left pie chart, N = 65), B (middle, N = 45) or C (right, N = 25), the fraction of events that were followed by a second binding to site A, B or C. For example, if a nucleosome is visited by GAF-DBD three times – first at Site A, second also at Site A, and third at Site B – then an A-A rebinding event and an A-B rebinding event have occurred on this nucleosome. d, Cy5-Cy7 FRET histogram for the hsp70 nucleosome (N = 377).