Reconstitution of Nucleosome Core Particles from Recombinant Histones and DNA

The ability to prepare nucleosome core particles (NCPs), or nucleosomal arrays, from recombinant histone proteins and defined-sequence DNA has become a requirement in many projects that address the role of histone modifications, histone variants, or histone mutations in nucleosome and chromatin structure. The cloning strategies for the construction of plasmids containing multiple repeats of defined DNA sequences, and the subsequent large-scale isolation of defined sequence DNA for nucleosome reconstitution are described. This chapter also describes adapted procedures to prepare nucleosomes with histones from other species, and for the refolding and reconstitution of (H2A– H2B) dimers and (H3–H4)2 tetramers. Methods to reconstitute nucleosomes from different histone subcomplexes are described. A flow chart for all procedures involved in the preparation of synthetic nucleosomes is also presented.