ASSEMBLY OF NUCLEOSOMAL DNA IN A CELL-FREE-EXTRACT FROM WILD-TYPE ANDTOP1(-) STRAINS OF USTILAGO-MAYDIS

An in vitro nucleosome assembly system has been established from cell- free extracts of the fungus Ustilago maydis. The extract catalyzed DNA supercoiling in the absence of exogenously added co-factors such as A TP and MgCl2 and was inhibited by moderate concentrations (200 mM) of KCl or NaCl. DNA supercoiling occurs via the formation of nucleosomes. Similar extracts, displaying the same activity, were prepared from Sa ccharomyces cerevisiae and Candida albicans, suggesting that the extra ct preparation protocol may be useful for many lower eukaryotic system s. An extract prepared from a strain of U. maydis lacking topoisomeras e I failed to catalyze nucleosome assembly, clearly implicating this e nzyme in this process. Addition of purified topoisomerase I, and, to a lesser extent, topoisomerase II, to the top1(-) extract regenerated t he supercoiling activity. Our results provide a method for preparing a ssembly extracts from organisms, that are particularly amenable to gen etic manipulation.