Chromatin assembly in vitro with purified recombinant ACF and NAP-1.

Publisher Summary This chapter describes the methodology for the ATP-dependent assembly of periodic nucleosome arrays by using purified recombinant chromatin assembly factors. The assembly of DNA to chromatin is a critical step in the duplication and maintenance of the eukaryotic genome. Chromatin assembly is an ATP-dependent process and is mediated by the concerted action of core histone chaperones and ATP-dependent chromatin assembly machines. NAP-1, CAF-1, ASF-1, nucleoplasmin, and other histone chaperones play an essential role by escorting histones to the sites of nucleosome assembly. The reaction for chromatin assembly with plasmid DNA can be broken down in three steps: pre-binding of core histones with the chaperone, NAP-1; relaxation of the plasmid DNA (optional); and ATP-dependent nucleosome assembly. The assembled chromatin can be used directly in downstream assay reactions. Certain applications (for example, electron microscopy studies) may require further purification of the chromatin. The nucleosome arrays can be purified from chromatin assembly factors by sucrose gradient sedimentation or gel filtration. The assembly of chromatin with the linker histone H1 is discussed. H1 is efficiently incorporated in the nucleosome arrays, and these arrays possess structural and functional features of H1-containing native chromatin.

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