Protocol

Reconstitution of Nucleosomal Arrays Using Recombinant Drosophila ACF and NAP1

Adapted from Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, 2nd edition, by Michael F. Carey, Craig L. Peterson, and Stephen T. Smale. CSHL Press, Cold Spring Harbor, NY, USA, 2009.

INTRODUCTION

The goal of chromatin assembly procedures is to prepare extended nucleosomal arrays from cloned DNA templates and purified core and linker histones. The assembled chromatin should be highly defined in its protein content and resemble bulk chromatin isolated from living cell nuclei in terms of periodicity and nucleosome positioning. This protocol describes how to assemble minichromosome templates in an ATP-dependent fashion from circular plasmid DNA and purified core histones. This system can also be used to assemble minichromosomes from linear DNA (plasmid and ) and can also incorporate proteins other than core histones (linker histone H1, HMG17, and DNA-binding transcription factors). The products of the chromatin assembly reaction can be used directly (or after purification) in assays to study transcription, DNA replication, recombination, and repair. The system uses purified recombinant Drosophila chromatin assembly factors ACF (ATP-utilizing chromatin assembly and remodeling factor) and NAP1 (nucleosome assembly protein 1).