Chromatin assembly in yeast cell-free extracts.

A simple method for preparing chromatin assembly extracts has not been available for budding yeast. Here I describe such a method in detail. The assembly extract, a crude 100,000g supernatant, is prepared from cells disrupted in a manual or motorized grinder while they are frozen. The core histones and all soluble protein factors required for chromatin assembly under physiological conditions are present in the extract. Assembly is sensitive to mutation of lysine residues in the amino-terminal tail of histone H4 whose acetylation is associated with nucleosome deposition in vivo. The reaction is ATP dependent, and assembly-driven DNA supercoiling occurs with the same efficiency as in extracts from mammalian somatic cells. This simple system offers a unique opportunity to analyze chromatin metabolism by a combined biochemical and genetic approach that is not feasible for any other model organism.