Figure 5.

Nucleosome positioning. The positioning of the nucleosome objects on the DNA fragment is dominated by the Widom-601 positioning sequence. (a) The theoretical length of the DNA arms protruding from the octamer, consisting of a (H3–H4)₂ tetramer (turquoise) and two H2A-H2B dimers (red and yellow) when positioned on the 147 bp long Widom- 601 sequence. The α-side corresponds for 464 bp nucleosomes to the longer and for 599 bp nucleosomes to the shorter DNA arm. This illustration displays the approximate length ratios between nucleosome core and protruding DNA arms. As nucleosome cores appear on the AFM images roughly twice the theoretical size (Table 2), ratios between the DNA arms were corrected (see Methods AFM data evaluation). (b) Cumulative frequency plot of the sa-ratio_c of all nucleosome samples reconstituted on 464 bp and 599 bp DNA. The cumulative frequency values of sa-ratio_c, were normalized and binned (bin wide 0.01). (c) shows the median values with 95% confidence interval (CI) of the sa- ratio_c for the samples in (b). The theoretical values for positioning on the Widom-601 sequences are indicated by black arrows. For (b and c) only objects with a sa-ratio_c of at least 0.3 were considered: n_wt/464 = 443, n_{wt +H1/464} = 443, n_mut/464 = 439, n_{mut +H1/464} = 439, n_wt/599 = 174, n_{wt +H1/599} = 185, n_mut/599 = 183, n_{mut +H1/599} = 162. The shift between wt and mut for both 464 bp, and 599 bp DNA fragments indicate an asymmetric unwrapping of the α-side for the mutants.