a, Histogram of FRET levels for the initial position and pause positions of [WT H4, 40 bp] nucleosomes upon remodelling by ACF. The histogram was fitted by multiple Gaussian peaks (black line) and the peak values were used to compute the average translocation distances between pauses. The translocation distances could be quantified using a calibration curve of FRET efficiency versus exit-side linker DNA length24,26, yielding a 7.0 bp step size between the initial position and the first pause and a 3.4 bp step size between the first pause and the second pause. b, Step sizes for various mononucleosomes and dinucleosomes. The dinucleosome constructs, [WT H4, 40 bp, WT H4] and [WT H4, 78 bp, WT H4], are each composed of one FRET-labelled nucleosome and one unlabelled nucleosome, spaced by 40 bp and 78 bp of internucleosomal linker DNA, respectively. The flanking linker DNA is 3 bp on the side of the FRET-labelled nucleosome and 40 bp on the side of the unlabelled nucleosome. The unlabelled nucleosome contains 2-nt ssDNA gaps at the SHL ± two sites to prevent translocation. Data are mean ± s.e.m. derived from at least 100 remodelling traces from three independent experiments. c, Dwell-time distributions for the first translocation phase, tT1, and the first pause phase, tP1, for nucleosomes with different lengths of linker DNA. [ACF] = 10 nM and [ATP] = 20 μM.