Figure 3. Addition of H2A/H2B dimer to hexasomes produces canonical nucleosomes.

(A) Schematic representation of the 80-601-0 nucleosome and hexasome, highlighting the locations where H2B-S53C cross-links to DNA. Due to the absence of one H2A/H2B dimer, H2B cross-linking with hexasomes is limited to the TA-rich side of the Widom 601. (B) Histone mapping demonstrates that canonical nucleosomes can be generated by addition of H2A/H2B dimer to hexasomes. For reactions containing hexasomes plus H2A/H2B, the hexasomes (10 nM) were incubated for 2–3 min with H2A/H2B (20 nM) prior to labeling with APB. Nucleosome and hexasome alone were subjected to the same brief incubation. Following UV cross-linking and DNA extraction, the DNA was cleaved at the crosslinking site and the products separated on a denaturing gel alongside a sequencing ladder to determine the cross-linking position. Results are representative of three or more independent experiments. See also Figure 3—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.21356.006

Figure 3—figure supplement 1. Addition of H2A/H2B dimer to hexasomes produces canonical nucleosomes, regardless of flanking DNA location.

(A) Schematic representation of the 0-601-80 nucleosome and hexasome, highlighting the location where the hexasome lacks one H2A/H2B dimer. (B) Histone mapping experiments using 0-601-80 nucleosomes, hexasomes, and hexasomes plus excess H2A/H2B dimer. Reactions were carried out as described for Figure 3. Sequencing ladder markers (T, C, A, G) were run in the same gel as the samples. Representative of three or more experiments.

DOI: http://dx.doi.org/10.7554/eLife.21356.007