Ribonucleic acid and other polyanions facilitate chromatin assembly in vitro

Crude extracts of Drosophila embryos are a rich source of both DNA topoisomerase I and chromatin assembly activity [Nelson, T., Hsieh, T., & Brutlag, D.L. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5510-5514; Hseih, T., & Brutlag, D. L. (1980) Cell (Cambridge, Mass.) 21, 115-125]. Purified topoisomerase I from Drosophila embryos, however, is not sufficient for chromatin assembly. Rather, the ability of Drosophila embryo extracts to mediate chromatin assembly in vitro requires an anionic fraction which we demonstrate to be RNA. Exogenous natural and homopolymer RNAs, if of sufficient length, can also mediate chromatin assembly in vitro. The RNA acts stoichiometrically in assembly, being required in amounts at least equal in weight to the amount of histones present. Natural and homopolymer DNAs, whether single or double stranded, are inactive under the same conditions. The arginine-rich histones H3 and H4 or histone H4 alone is sufficient to produce nucleoprotein complexes with physiological numbers of supertwists in the DNA. Complexes containing these subsets of the core histones also resemble assembled complexes containing all four core histones with respect to some patterns of nuclease sensitivity, although complexes containing all four core histones more closely resemble native chromatin in nuclease digestions.