(A) Coomassie-stained SDS gel of protein complexes used for these studies. (B) At a 1:10 INO80:nucleosome ratio, the sliding of 0N100 nucleosomes by hINO80 is highly processive, with appearance of centred nucleosomes very early in the time course and long before most of the nucleosomes have been shifted at all from the ends. This suggests a small proportion of the enzyme is in a highly active state. The data in Figure 3 show that the proportion of this increases as the ratio to nucleosomes increases that we interpret to be two hINO80 complexes binding to each nucleosome. At early time points at low ratios of hINO80:nucleosomes it is evident that discrete bands appear at intermediate positions between the end- and centrally-positioned nucleosomes. Using different length overhangs, we observed one intermediate band for each additional 20 bp of overhang, corresponding to a shift towards the centre of 10 bp each time. Hence, the bands are spaced approximately 10 bp apart. At first, we thought this might represent a step size for the protein as suggested by studies on other remodellers, but we observe a similar banding pattern if we heat a sample of our nucleosomes in the absence of enzyme revealing that this is an intrinsic property of the nucleosomes themselves rather than of the enzyme catalysed reaction (see right end panel). We prepare end-positioned nucleosomes by reconstitution on the strongly positioning Widom 601 sequence (Lowary and Widom, 1998). The most plausible explanation for the ‘intermediate’ bands is, therefore, that phasing of the DNA sequence within the Widom 601 sequence favours positions every 10 bp (i.e. one turn of the duplex) to bring regions into phase that favour being placed on the inside or outside of the nucleosome wrap, a property of DNA sequences that has been recognised for over three decades (Drew and Travers, 1985). The intermediate bands we observe could therefore correspond to either a ‘step-size’ or simply pausing of nucleosomes at preferred sites during translocation by multiple smaller steps between these sites. Since nucleosomes wrap just under 150 bp of duplex around the core, and INO80 slides mono-nucleosomes to the centre of DNA fragments (Udugama et al., 2011), it would require an overhang of around 300 bp to completely shift an end-positioned nucleosome away from the influence of the Widom 601 sequence. (C) hINO80 can centre nucleosomes with short overhangs. Nucleosomes with 20, 40, 60, 80 and 100 bp overhangs all shift towards the centre.