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. This approach offers many advantages, such as the ability to combine histone variants and tail deletion mutants, and the opportunity to study the effect of individual histone tail modifications on nucleosome structure and function. We have previously described comprehensive protocols for the expression and purification of histones, for the refolding of the histone octamer, and for the reconstitution and purification of crystallization-grade mononucleosomes. The previously published version has now been amended, and steps that can be omitted or simplified if high degrees of purity and homogeneity are not an issue are indicated. The cloning strategies for the construction of plasmids containing multiple repeats of defined DNA sequences, and the subsequent large-scale isolation of definedsequence DNA for nucleosome reconstitution, are described in detail. We also describe 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 also described. A flow chart for all procedures involved in the preparation of ‘‘synthetic nucleosomes’’ is given in Fig. 1. Procedures described here are indicated in gray in Fig. 1.