A model for particulate structure in chromatin

Digestion of chromatin with DNase (nucleate 3'-oligonucleotidohydrolase, EC 3.1.4.7) releases 11-12S nucleoprotein particles. After extensive nuclease digestion, the DNA in these particles consists of a collection of eight discrete DNA fragments. When these nuclease-particles are treated with trypsin (EC 3.4.21.4), only 20 to 30 amino-acid residues are cleaved from histone Nterminals, the histone C-terminal segments being resistant. The resulting 5S nucleoprotein particles have now been shown on acrylamide gels to consist of a series of eight discrete DNA-containing bands. Four of these bands contain C-terminal cleavage fragments from four histones (III, IV, Ib2, and Ilbi) tightly bound to them; a fifth contains fragments from only histones III and IV. The remaining three bands contain only DNA. Since these protein-free DNA bands were resistant to nuclease prior to trypsin treatment, they were presumably associated with histone N-terminal segments in the native structure.Trypsin, therefore, appears to split nuclease-particlps, releasing two subfractions of DNA-one associated with protein, the other not. The data is compatible with a model in which the majority of DNA in the eukaryotic nucleus is folded into hairpin loops of double-stranded helix, each created by the concerted cross-linking action of 6 to 10 histones which interact to form a trypsin-resistant complex composed, for the most part, of all four major histones. These loops may further fold upon themselves to form the "nu" bodies that have been visualized by electron microscopy.The DNA in chromatin is digested by staphylococcal nuclease (nucleate 3'-oligonucleotidohydrolase, EC 3.1.4.7) into eight discrete, limit-digest DNA fragments (1, 2), which range in size between 45 and 145 base pairs. Yet, partial staphylococcal nuclease digestion results in the appearance of transient DNA fragments of 200 base pairs or integer multiples of that size (3,4). A reasonable interpretation of these findings is that these larger transient segments are generated by a regular arrangement of very accessible, DNase-sensitive sites, while the eight bands of the limit-digest are generated by the further digestion of less accessible but discrete sites within each 200-base-pair repeat. Recent electron microscope data has shown that "bead-like" structures occur at regular intervals of about 200 base pairs along the extended chromosome fiber (5). It is thus tempting to consider that the initial nuclease cut is between these so-called "nu" bodies (5), and that further digestion introduces a limited number of specific cuts within each "nu" body.Exhaustive treatment of chick erythrocyte chromatin with trypsin (EC 3.4.21.4) results in the complete digestion of histones I and V and the cleavage of only 20 to 30 amino-acid residues from the positively charged N-terminal parts of histones III, IV, IIb2, and (possibly) IHbl (2). In this comAbbreviations: ST, produced by treatment with staphylococcal nuclease followed by trypsin; TS, trypsin followed by nuclease. munication I...

Section: Nh2supporting

confidence: 90%

Release of discrete subunits after nuclease and trypsin digestion of chromatin.

Weintraub¹

1975

“…However, a simple supercoil loop of about 680 A in length (200 base pairs) of DNA duplex would by far exceed the size of a nucleosome. Our results are, therefore, better accounted for by a model of nucleosome structure in which the DNA would be folded (kinked, 30), rather than by models in which the DNA would be regularly wound in a superhelical form (29,31).…”

Section: Experiments and Resultsmentioning

confidence: 89%

“…Alternatively, a combination of intranucleosomal unwinding and supercoiling could cause the torsional alteration. Current models of nucleosome structure involve wrapping of the DNA around a protein core (28)(29)(30)(31). However, a simple supercoil loop of about 680 A in length (200 base pairs) of DNA duplex would by far exceed the size of a nucleosome.…”

Section: Experiments and Resultsmentioning

confidence: 99%

Folding of the DNA double helix in chromatin-like structures from simian virus 40.

Germond¹,

Hirt²,

Oudet³

et al. 1975

578

341

Relaxed circular, covalently closed simian virus 40 DNA molecules were associated with the four histones that are present in virions. In electron micrographs the resulting complexes appear twisted, with globular structures (nucleosomes) along the DNA. Incubation with an untwisting extract converts the twisted complexes to relaxed structures. Extraction of the DNA from the relaxed complexes yields supercoiled molecules. The number of superhelical turns in these molecules corresponds to the number of nucleosomes per DNA molecule in the complexes.In eukaryotic nuclei, the fundamental structure of chromatin fibers appears to be a flexible chain composed of globular particles connected by DNA filaments (1, 2). In these particles, termed nucleosomes (2), about 200 base pairs of DNA are associated with the four histonies F2a,, F2a2, F2b, and F3 (2-7). Such a repeating unit structure can be formed in vitro by association of the four histonies and linear l)NAs (2).In the nucleosomes the DNA is under constraint, since it is compacted about 5-fold compared to its length in the extended double helical form (2). The nature of this constraint can be studied by the association of histones to covalently closed circular DNA molecules, since supercoiling or unwinding of the DNA within the nucleosome (luring its formation would alter the supercoiling of the rest of the molecule (8). In addition, from the known thermodynamic prolerties of superhelical DNAs (8-10), the influence of the degree of superhelicity on the formation of nucleosome structures can provide information on whether the formation of a nucleosome is equivalent to an unwinding or winding of the double helix. Simian virus 40 (SV40) D)NA is particularly attractive for such a study for two reasons. First, two circular covalently closed allomorphic forms of this DNA are available, the superhelical DNA I and the relaxed circular DNA Ir which results from the incubation of DNA I with an untwisting extract (FE) (l1). This extract is thought to introduce a single-strand nick into superhelical DNA and to reseal the nick after the torsional tension in the double helix has been relieved. Second, SV40 DNA and the four histones are associated in vivo anld the complexes can be isolated from virions (12, 13) and from infected cells. In the latter case, the complex appears as a compactecl structure with about 20 nueleosomes (14).We

“…Subunits from higher eukaryotes contain two copies each of histones H2A, H2B, H3, and H4 associated with about 140 base pairs of DNA (12,14). Recent neutron diffraction studies (15) have confirmed earlier proposals (10,11) that these histones form a "core" about which the unit length of DNA is wrapped. Lysine-rich histones HI (and H5 in erythrocytes) appear to be associated primarily with the "spacer" DNA, 30-60 base pairs long, between globular subunits (14,16,17).…”

mentioning

confidence: 84%

“…Interphase chromatin fibrils of higher eukaryotes have a "beaded string" structure consisting of repeated arrays of globular particles (about 100 A diameter) connected by thin nucleoprotein filaments (9)(10)(11)(12). Nucleases cleave these interconnecting regions, producing individual "subunits" and larger oligomeric nucleoprotein units that can be readily isolated (12,13).…”

mentioning

confidence: 99%

Chromatin subunits from baker's yeast: isolation and partial characterization.

Nelson¹,

Beltz²,

Rill³

1977

The organization of proteins along DNA in chromatin of Saccharomyces cerevisiae (baker's yeast) was examined by analyzing the DNA and nucleoprotein products obtained after digestion of yeast nuclei with staphylococcal nuclease. Yeast DNA is digested in situ at regularly spaced cleavage sites about 160 Before the transcriptional apparatus of yeast can be related with certainty to that of higher cells, similarities in genome organization must be established. Interphase chromatin fibrils of higher eukaryotes have a "beaded string" structure consisting of repeated arrays of globular particles (about 100 A diameter) connected by thin nucleoprotein filaments (9-12). Nucleases cleave these interconnecting regions, producing individual "subunits" and larger oligomeric nucleoprotein units that can be readily isolated (12, 13). Subunits from higher eukaryotes contain two copies each of histones H2A, H2B, H3, and H4 associated with about 140 base pairs of DNA (12,14). Recent neutron diffraction studies (15) have confirmed earlier proposals (10, 11) that these histones form a "core" about which the unit length of DNA is wrapped. Lysine-rich histones HI (and H5 in erythrocytes) appear to be associated primarily with the "spacer" DNA, 30-60 base pairs long, between globular subunits (14,16,17).Yeast chromatin potentially may have a subunit structure since yeast nuclei contain several major basic proteins that electrophorese similarly to histones (18,19). In fact, staphylococcal nuclease digests yeast chromatin at discrete intervals, indicating that proteins are arranged along the DNA in-a periodic manner (18,20,21). However, some caution is warranted in interpreting these results because of reports that yeast lacks histones Hi and H3 (22) and that yeast ribosomal proteins may be coisolated with or otherwise mistaken for histones (23).As described below, we have confirmed the periodic structure of yeast chromatin and have isolated and partially characterized monomeric subunits and oligomer units from yeast that appear to be nearly identical to subunits from higher eukaryotes.MATERIALS AND METHODS Isolation and Digestion of Yeast Nuclei. Saccharomyces cerevisiae strain A8209B was obtained from Dr. Gerald Fink (Cornell University). Yeast cells and protoplasts were prepared as described by deKloet et al. (24). Prior to isolation of nuclei, protoplasts (OD6Wj nm = 1-1.5) were incubated for 45 min at 300 in 20 mM KH2PO4 (pH 6.2)/2 mM MgCl2/2% glucose/ 0.3% casamino acids/20% sorbitol to permit return to a metabolically active state (24). The protoplasts were then rapidly cooled to 40 and washed with cold 20% sorbitol. Nuclei were isolated (at 0-40) by a method modified slightly from that of Blobel and Potter (25). Protoplasts were washed once in TKMC buffer (50 mM Tris-HCI, pH 7.5/25 mM KCI/5 mM MgCI2/1 mM CaCI2) containing 0.25 M sucrose, then twice in the same solution containing 0.5% Triton X-100. The crude nuclear pellet was purified by centrifugation through dense sucrose in TKMC buffer plus 0.1 mM phenylmethylsulfo...