Assembly of nucleosomal DNA in a cell-free extract from wild-type and top1- 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 ATP 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 Saccharomyces cerevisiae and Candida albicans, suggesting that the extract preparation protocol may be useful for many lower eukaryotic systems. An extract prepared from a strain of U. maydis lacking topoisomerase I failed to catalyze nucleosome assembly, clearly implicating this enzyme in this process. Addition of purified topoisomerase I, and, to a lesser extent, topoisomerase II, to the top1- extract regenerated the supercoiling activity. Our results provide a method for preparing assembly extracts from organisms, that are particularly amenable to genetic manipulation.
D002176:Candida albicans / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004271:DNA, Fungal / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D008836:Micrococcal Nuclease / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009707:Nucleosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D014588:Ustilago / Q000378:metabolism*
D004270:DNA, Circular; D004271:DNA, Fungal; D004278:DNA, Superhelical; D009707:Nucleosomes; D008836:Micrococcal Nuclease; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D004264:DNA Topoisomerases, Type I