Reconstitution of chromatin: assembly of the nucleosome

The order of reassociation of the four histones H2a, H2b, H3 and H4 to the DNA during the reconstitution of chromatin was determined. At each step of the reconstitution the DNA and associated histones were separated from the free histones by centrifugation in a glycerol gradient. The unbound and reassociated histones were analysed by gel electrophoresis and the histone-DNA complexes characterized by circular dichroism and electron microscopy. We show that H3 and H4 bind first to the DNA between 1.2 M NaCl and 0.85 M NaCl and impose a nucleosome like structure; in a second step histones H2a and H2b are placed around this kernel to complete the nucleosome.