Nucleosome periodicity in HeLa cell chromatin as probed by micrococcal nuclease.

Abstract

When HeLa cell nuclei were treated with micrococcal nuclease (nucleate 3-oligonucleotidohydrolase, EC 3.1.4.7), lysed, and centrifuged, the supernatant from early digests contained two predominant classes of polynucleosomes of repeat size 8N and 16N. With increasing digestion time, the 16N polynucleosome appeared to be cleaved to the 8N species and finally to the basic subunit of chromatin. The size of the polynucleosomes has been determined by DNA analysis and on polyacrylamide electrophoretic gels of native chromatin particles. Thel6Npolynucleosome appears to be a unique higher ordered structural component of HeLa cell chromatin. Our recent report, showing that the nuclear protein-modifying enzyme poly(ADP-ribose) polymerase increases in specific activity progressively with increasing nucleosome repeat size up to 8-1ON, has been extended in the present study. Activity was also elevated in the polynucleosomes of the 16N structure preferentially cleaved by micrococcal nuclease, although specific activity of the enzyme was highest in octanucleosomes. Acceptors for poly(ADP-ribose) have also been determined in these particles.Despite recent advancement in our understanding of the structure of the nucleosome core particle, similar information concerning the higher order structure of chromatin remains largely undetermined (1)(2)(3). At low ionic strength the nucleofilament with diameter of 100 A is a single chain of nucleosomes (4-7). In the presence of monovalent and divalent cations, the diameter of the nucleofilament is increased to 250400 A (4, 5, 7). Based on electron micrographic data, Finch and Klug (4) postulated a solenoid model of chromatin.Kinetic studies performed on the action of micrococcal nuclease (nucleate 3'-oligonucleotidohydrolase, EC 3.1.4.7) on interphase chromatin and metaphase chromosomes (8-10) have supported the theory of random cleavage by the enzyme at uniformly susceptible sites on linker regions of polynucleosomes. However, it has also been shown that newly replicated chromatin is relatively more susceptible to the action of micrococcal nuclease (11).Using electron microscopic studies and sedimentation analysis on the chromatin produced after the digestion of lymphocyte nuclei by micrococcal nuclease, Hozier et al. (7) have postulated that chromatin is organized into superbeads containing six to ten nucleosomes. However, ambiguity about these structures still remains since Hozier et al. (7) were unable to isolate and resolve them on sucrose gradients. In kinetic studies performed in the work below, it has clearly been shown that micrococcal nuclease cleaves HeLa chromatin at a periodicity of eight nucleosomes. In addition to sedimentation analysis, we have been able to characterize these structures by (i) native chromatin gels, (ai) determination of the size of the DNA, and (Mi) the presence of a full complement of nuclear histones.These observations on nucleosome periodicity stemmed directly from our previous observation that the specific activity of a chromatin-bound enzyme, ...

Differences of supranucleosomal organization in different kinds of chromatin: cell type-specific globular subunits containing different numbers of nucleosomes.

“…(b) From electron microscopic observations of spread chromatin preparations and from studies of the products of limited digestion with micrococcal nuclease (16,20,24) other authors have proposed that the nucleosomal chain forms a discontinuous periodic structure appearing as a higher order chain of tightly adjoining globular aggregates ("superbeads," 42; "nucleomers," 25). This also results in the appearance ofa higher order fibril, which displays an overall contour diameter of -30 nm in many cell types (3,8,19,21,22,25,37,39,44,52,54,66,67,68). This mode of supranucleosomal organization into higher order granules is similar to the organization that has been described for circular forms ofchromatin, such as the SV40-"minichromosomes" (12,23,35,59) and nontranscribed amplified rDNA chromatin of certain oocytes (46).…”

“…Each type of chromatin shows a distribution with a significant maximum specific for this type of chromatin . The mean values for nucleosomal contents have been determined to be 48 for sea urchin sperm (A, n = 150), 20 for chicken erythrocyte (8, n = 450), and 8 for chicken (C, n = 150) and rat (D, n = 150) liver . Agarose gel electrophoresis of higher order chromatin particles and size determinations of DNA contained in supranucleosomal chromatin particles from chicken erythrocytes and sea urchin sperm .…”

Upon the observation of superbeads in chromatin

“…Another higher order structure of chromatin which on the basis of electronmicroscope data emphasizes the existence of discrete globular particles (=superbeads) of about 250 0 A, has been presented (8), (9), (10). Further biochemical evidence in favour of the latter model came from the observation that nucleases were able to recognize and preferentially digest these irregularities (or superbeads) of 0 the 250 A fiber (11), (12). This was demonstrated by sucrose gradient centrifugation of chromatin extracts obtained after a brief nuclease digestion.…”

DNase I sensitivity of ribosomal genes in isolated nucleosome core particles

“…Staphylococcal nuclease digests bulk chromatin in five stages (5). The first stage is cleavage between groups of about eight nucleosomes (6)(7)(8). The second stage involves preferential cleavage between nucleosomes to produce an oligomeric series of particles from which an oligomeric series of DNA molecules can be extracted.…”