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Evidence of 2 levels of dna folding in histone depleted hela interphase nuclei

, : Evidence of 2 levels of dna folding in histone depleted hela interphase nuclei. Journal of Molecular Biology 156(2): 309-324

The long-range order of the DNA in interphase nuclei was investigated by sedimentation studies. Two discreet sedimentation forms were characterized for histone-depleted HeLa nuclei. Type I structures, obvtained by extraction of isolated nuclei with a buffer containing 2 M-NaCl, have an s value of 18,000S. Histone-depletion with the polyanions dextran sulfate/heparin produces slightly slower type I structures of 15,000 S. When the thiols, .beta.-mercaptoethanol or dithiothreitol, or the metal chelators, 1,10-phenanthroline or neocuproine, are included during histone extraction, the interphase DNA is further unfolded, generating type II structures of 8500 S. Both forms are destroyed by proteolytic agents, but are insensitive to RNAase A. Fluorescence studies of type I and type II structures show that both retain the spherical shape of nuclei, with type II structures having a more expanded DNA halo than that of type I. Metalloprotein interactions apparently are important in 1 level of the DNA folding of type I structures, since the chelators 1,10-phenanthroline and neocuproine bring about formation of type II structures. Metal-depleted nuclei, prepared in buffers containing .beta.-mercaptoethanol, provide a means for identifying the metal involved. Such nuclei generate type II structures upon histone-depletion. Addition of as little as 1 .times. 10-6 M-CuSO4 or 1 .times. 10-6 M-CaCl2 to metal-depleted nuclei restores the capacity to generate type I structures. The restoration of the type I complex is reversible following exposure of nuclei to CuSO4, but is irreversible after CaCl2 treatment. One level of DNA compaction in histone-depleted nuclei is probably stabilized by either Cu or Ca.

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