Figure 2.

Destabilization of nucleosomes by purified recombinant dNDF and human NDF (hNDF). (A) Schematic diagram of hNDF and dNDF. The drawing is roughly to scale. Specific features are described in the text. (B) Purification of bacterially synthesized N-terminally His6-tagged dNDF and hNDF. (C) Recombinant dNDF and hNDF facilitate the acetylation of nucleosomal H3K56 by p300. This assay was performed as in Figure 1A. (D) Loss of DNA supercoiling in chromatin reveals destabilization of nucleosomes by hNDF. Chromatin was reconstituted with plasmid DNA and purified core histones by the salt dialysis method and then incubated (at 0.3 µM nucleosome concentration) with purified recombinant hNDF and topoisomerase I (Topo I) as indicated. Samples were deproteinized and subjected to 0.8% agarose gel electrophoresis in the absence or presence of 10 ng/mL chloroquine. DNA was visualized by staining with ethidium bromide. As a control, reactions were carried out in parallel with plasmid DNA.