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Abstract

We have studied the structure of tandemly repetitive α-satellite chromatin (α-chromatin) in African green monkey cells (CV-1 line), using restriction endonucleases and staphylococcal nuclease as probes.While more than 80% of the 172-base-pair (bp) α-DNA repeats have a HindIII site, less than 15% of the α-DNA repeats have an EcoRI site, and most of the latter α-repeats are highly clustered within the CV-1 genome. EcoRI and HindIII solubilize approximately 8% and 2% of the α-chromatin, respectively, under the conditions used. EcoRI is thus approximately 30 times more effective than HindIII in solubilizing α-chromatin, with relation to the respective cutting frequencies of HindIII and EcoRI on α-DNA.EcoRI and HindIII solubilize largely non-overlapping subsets of α-chromatin. The DNA size distributions of both EcoRI- and HindIII-solubilized α-chromatin particles peak at α-monomers. These DNA size distributions are established early in digestion and remain strikingly constant throughout the digestion with either EcoRI or HindIII. Approximately one in every four of both EcoRI- and HindIII-solubilized α-chromatin particles is an α-monomer.Two-dimensional (deoxyribonucleoprotein → DNA) electrophoretic analysis of the EcoRI-solubilized, sucrose gradient-fractionated α-oligonucleosomes shows that they do not contain “hidden” EcoRI cuts. Moreover, although the EcoRI-solubilized α-oligonucleosomes contain one EcoRI site in every 172-bp α-DNA repeat, they are completely resistant to redigestion with EcoRI. This striking difference between the EcoRI-accessible EcoRI sites flanking an EcoRI-solubilized α-oligonucleosome and completely EcoRI-resistant internal EcoRI sites in the same α-oligonucleosome indicates either that the flanking EcoRI sites occur within a modified chromatin structure or that an altered nucleosome arrangement in the vicinity of a flanking EcoRI site is responsible for its location in the nuclease-sensitive internucleosomal (linker) region.Analogous redigestions of the EcoRI-solubilized α-oligonucleosomes with either HindIII, MboII or HaeIII (both before and after selective removal of histone H1 by an exchange onto tRNA) produce a self-consistent pattern of restriction site accessibilities. Taken together, these data strongly suggest a preferred nucleosome arrangement within the EcoRI-solubilized subset of α-oligonucleosomes, with the centers of most of the nucleosomal cores being ∼20 bp and ∼50 bp away from the nearest EcoRI and HindIII sites, respectively, within the 172-bp α-DNA repeat. However, as noted above, the clearly preferred pattern of nucleosome arrangement within the EcoRI-solubilized α-oligonucleosomes is invariably violated at the ends of every such α-oligonucleosomal particle, suggesting at least a partially statistical origin of this apparently non-random nucleosome arrangement. We discuss the relative contributions of deterministic and statistical factors to the observed patterns of nucleosome arrangement on the α-DNA and also possible influence of specific non-histone components, such as a hypothetical DNA-binding protein that may recognize and bind the 172-bp α-DNA repeat.