Nuclear architecture, intranuclear DNA distribution, and nuclease digestion

G0, G1, and mammalian cells and nuclei were shortly digested with either micrococcal nuclease or DNAse I, both before and after mild fixation, either before (G0) or after (G1) partial hepatectomy. Cells were Feulgen stained and examined by high resolution light microscopy. In metabolically active G1 nuclei, intranuclear DNA appears organized at least in two distinct domains, whereby the highly dispersed one is large enough to be detected at the resolution of the light microscope and appears preferentially attacked by limited DNAse I digestion. The action of the enzyme is readily apparent only in the nuclei that are first digested and then fixed. Spectroscopic characterization of the same nuclei reveals that the fixation causes a sizeable removal of proteins, mostly in the soluble chromatin subfraction. Results are discussed in terms of two control levels for gene expression and for higher order DNA structure.     

Non-random binding of a chemical carcinogen to the DNA in chromatin

The distribution of a liver carcinogen (N-hydroxy-2-aminofluorene) along the DNA of chromatin has been studied using two nucleases as probes for the structure of chromatin. Rats were injected with the carcinogen and killed at various times after the injection. The nuclei of the liver were prepared and digested with Staphylococcal nuclease or pancreatic nuclease DNAse I. We show that the carcinogen is non randomly distributed along the DNA of chromatin since it binds preferentially to the regions of chromatin digested by the Staphylococcal nuclease whereas it is preferentially bound to the DNAse I resistant fraction. Our results also indicate that the two nucleases do not recognize exactly the same region of chromatin.     

Core nucleosomes by digestion of reconstructed histone-DNA complexes.

Reconstructed complexes of the inner histones (H2A, H2B, H3, H4) and a variety of DNAs were digested with micrococcal nuclease to yield very homogeneous populations of core nucleosomes (nu 1). Nucleosomes containing Micrococcus luteus DNA (72% G+C); chicken DNA (43% G+C), Clostridium perfringens DNA (29% G+C); or poly(A-dT.poly(dA-dT) have been examined by circular dichroism, thermaldetenaturation, electron microscopy, and DNAse I digestion. Circular dichroism spectra of all particles show a typically suppressed ellipticity at 260--280 nm and a prominent alpha-helix signal at 222 nm. All particles show biphasic melting except nu 1 (dA-dT), which show three prominent melting transitions at ionic strength less than or equal to 1 mM. DNAse I digestion of nu 1 (dA-dT) produces a ladder of DNA fragments fiffering in lengthy by one base residue. nu 1 (dA-dT) contain 146 base pairs of DNA and exhibit an average DNA helix pitch of 10.4-10.5 bases per turn. There appear to be two regions of different DNA pitch wihtin nu 1 (dA-dT). It is suggested that the two regions of DNA pitch might correspond to the two regions of the melting profiles.     

Effect of antibody excess on the size, stoichiometry, and DNAse resistance of DNA anti-DNA immune complexes

The binding of antibodies to DNA was examined under conditions of increasing antibody excess. DNA anti-DNA immune complexes (IC) formed at increasing antibody to DNA ratios were digested with excess DNAse I, and the DNAse-resistant (protected) IC were analyzed. With increasing antibody excess, the size of the IC that were resistant to DNAse digestion increased, and the size of the protected DNA within the IC also increased. This suggested that IgG molecules could bind in close proximity along the DNA molecule, preventing access of DNAse to the DNA between adjacent IgG. To further define the binding of adjacent IgG, DNAse digested IC containing one or two IgG were isolated, and the DNA contained within these IC was analyzed on DNA sequencing gels. Binding of a single IgG to DNA resulted in the protection of a DNA fragment 35 to 45 base pairs (bp) long, corresponding to the distance between binding sites of a single IgG molecule. Binding of two IgG to DNA protected a DNA fragment 50 to 60 bp long, 1 1/2 times the size of the fragment protected by one IgG. These data suggest that in conditions of Ab excess, IgG molecules can interdigitate along the DNA molecule, resulting in small, stable, DNAse-resistant IC of high antibody density.     

Nuclease-induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide

A flow cytometric analysis of DNA structural changes induced by cleavage with nucleases was performed on isolated HeLa nuclei by assessing changes in stainability with the DNA-specific fluorochrome propidium iodide (PI). After mild digestion with DNAse I, micrococcal nuclease, or with the single-strand-specific S1 and Neurospora crassa nucleases, fluorescence intensity of nuclei stained with PI increased by about 15-30% above the value of undigested control samples. No significant modifications were observed with the restriction enzymes Eco RI, Alu I, and Not I. The DNAse I-induced increase in fluorescence intensity was also observed with the non-intercalating dye Hoechst 33258, but not with mithramycin. Nuclease-induced fluorescence intensity changes as determined with PI were found to be dependent on the dye concentration. A constant increase (about 20%) was measured at dye/DNA-P ratios greater than 0.11. Below this value (2 micrograms/ml PI), the fluorescence intensity of digested samples was 15-30% lower than that of undigested controls. This behaviour towards intercalating dyes is similar to that of the relaxed (nicked) vs. the supercoiled (intact) form of circular DNA. These results suggest that conformation- but not sequence-specific nucleases induce a relaxation of DNA supercoils.     

Alterations of neuronal nuclear matrix and chromatin structure after irradiation under aerobic and anoxic conditions

This study was undertaken to determine if structural alterations of the bulk chromatin and the amount of protein associated with the nuclear matrix in cerebellar neurons depend on radiation dose and a cell's state of oxygenation. After irradiation with 2.5 to 25.0 Gy under both aerobic and anoxic conditions, the sensitivity of the neuronal chromatin to m. nuclease digestion increase linearly with dose up to about 5 Gy, beyond which there was no further increase. The same increase in accessibility of chromatin to micrococcal nuclease digestion was observed when neuronal nuclei were irradiated at 4 degrees C. Neuronal nuclei were stained with propidium iodide (PI) for DNA and with fluorescein isothiocyanate (FITC) for protein, both before and after complete digestion with DNase I, and analyzed by flow cytometry. There was no change in either the PI (P greater than 0.4) or the FITC (P greater than 0.9) fluorescence of undigested nuclei after irradiation. For the DNase I digested nuclei, the PI fluorescence was unchanged after irradiation (P greater than 0.4), but the FITC fluorescence increased significantly (P less than 0.02). This increase in the FITC fluorescence was linear with dose up to about 5 Gy, beyond which there was no further increase. The flow cytometry results from DNase I digested nuclei were identical for neurons irradiated under aerobic or anoxic conditions, indicating that this phenomenon is oxygen independent. This increase in FITC fluorescence after irradiation was inhibited at ice-cold temperatures and probably reflects an increase in protein content at the nuclear matrix that requires metabolism. This may explain our previously observed resistance of nuclear matrix-associated DNA to digestion by DNase I. This protein increase at the nuclear matrix appears to follow "saturation" kinetics identical to that previously reported for repair of DNA strand breaks in cerebellar neurons. However, the exact molecular nature of this process and its role in DNA repair or cell survival remains to be determined.     

The sperm nuclear matrix is required for paternal DNA replication

The mammalian sperm nucleus provides an excellent model for studying the relationship between the formation of nuclear structure and the initiation of DNA replication. We previously demonstrated that mammalian sperm nuclei contain a nuclear matrix that organizes the DNA into loop domains in a manner similar to that of somatic cells. In this study, we tested the minimal components of the sperm nucleus that are necessary for the formation of the male pronucleus and for the initiation of DNA synthesis. We extracted mouse sperm nuclei with high salt and dithiothreitol to remove the protamines in order to form nuclear halos. These were then treated with either restriction endonucleases to release the DNA not directly associated with the nuclear matrix or with DNAse I to digest all the DNA. The treated sperm nuclei were injected into oocytes, and the paternal pronuclear formation and DNA synthesis was monitored. We found that restriction digested sperm nuclear halos were capable of forming paternal pronuclei and initiating DNA synthesis. However, when isolated mouse sperm DNA or sperm DNA reconstituted with the nuclear matrices were injected into oocytes, no paternal pronuclear formation or DNA synthesis was observed. These data suggest that the in situ nuclear matrix attachment organization of sperm DNA is required for mouse paternal pronuclear DNA synthesis.     

Higher order chromatin structure determines double-nucleosome periodicity of DNA fragmentation

Double-nucleosome periodicity of DNA fragmentation with DNAse I in the nuclei of cells differing in size of the linker DNA length and lysine-rich histone composition was analyzed by means of nondenaturing agarose gel electrophoresis. DNAse I revealed this type of periodicity in rat thymus and CHO cell nuclei as well as in erythrocyte nuclei. It has been deduced that the so-called nucleodisome structure is also typical of cells possessing a usual DNA repeat length (200 bp or less) and lysine-rich histone H1. Two probably related events are important for establishing a clear double-nucleosome periodicity of DNA fragmentation: the replacement of H1 histone by a specific arginine-rich histone fraction (H5 histone in the case of erythrocyte) and the increase of the linker DNA length. The results are interpreted in terms of supranucleosomal organization of chromatin which may determine the dinucleosome periodicity of DNA fragmentation due to a specific packing of nucleosomes.     

Developmental changes in DNAse I digestibility and RNA template activity of neuronal nuclei relative to the postnatal appearance of a short DNA repeat length

Neurons of the rat cerebral hemispheres are known to undergo a postnatal shift to a short DNA repeat length. In the present study we report that rat neuronal nuclei are more sensitive to digestion with DNAse I when isolated at a developmental stage after the shift in neuronal DNA repeat length compared to nuclei isolated before the shift. This observation may suggest that a decondensation of neuronal chromatin accompanies the postnatal shift in DNA repeat length. We have also found that neuronal nuclei isolated after the shift to a short DNA repeat length demonstrate an increased ability to synthesize RNA in vitro.Abbreviations used op base pairs - PCA perchloric acid - RSB reticulocyte standard buffer - TCA trichloroacetic acid - PMSF phenylmethylsulfonylfuoride     

DNase I sensitive site in the core region of the human beta-globin origin of replication

HeLa cells were synchronized at late G1, early S, and late S phase of the cell cycle by nocodazole treatment. The cells were permeabilized with Triton X-100, digested with DNAse I, and extracted with 0.2 M ammonium sulfate to remove the digested chromatin. DNA was isolated from the residual chromatin attached to the nuclear matrix, digested with Hind III, and subjected to hybridization with [(32)P] labeled probe located upstream of the core region of the human beta-globin replication origin. The hybridization pattern revealed the existence of a DNase I sensitive site in the core region of the beta-globin replicator. The results suggest that association with the nuclear matrix induce alteration in the chromatin structure of the origin of replication that represents a more open chromatin configuration.     

Comparative heterokaryon study of cellular senescence and the serum-deprived state

Autoradiographic patterns of DNA replication in serum-deprived human diploid fibroblast-like cells (HDFC) and “senescent” HDFC have been compared in two types of heterokaryons. Each was fused to low passage, proliferating HDFC and, in separate experiments, to HeLa cells. Sequential 1 h pulses with [³H]thymidine were initiated at short intervals following fusion. In all hybridizations serum-deprived and senescent cells behaved identically. Upon fusion to HeLa cells, DNA synthesis in the quiescent nuclei occurred in a wave between 3 and 30 h after fusion. When either serum-deprived or senescent HDFC were fused to young proliferating HDFC, the nuclei of the latter were blocked from entering the S phase if fusion occurred at least 3 h before the G/S boundary. These findings are consistent with the interpretation that one or more crucial steps in G0 occurs 3 h before the G1/S interface. That young serum-deprived (G0) HDFC behave identically to senescent cells in these hybridization studies suggests that the mechanism of arrest in each state might share a final common pathway, and a model based on these observations is proposed.     

Association of DNA with the nuclear lamina in Ehrlich ascites tumor cells

We have studied in vitro binding of DNA to nuclear lamina structures isolated from Ehrlich ascites tumor cells. At low ionic strength in the presence of Mg++, they bind considerable amounts of mouse and bacterial DNA, forming complexes stable in 2 M NaCl. Single-stranded DNA and pulse-labeled DNA show higher binding efficiencies than native uniformly labeled DNA. When mixing occurs in 2 M NaCl, complex formation is inhibited. When nuclei are digested with DNAse I under conditions that favor chromatin condensation, DNA associated with matrices subsequently prepared from such nuclei is markedly enriched in satellite DNA. If digestion is carried out with DNAse II while nuclei are decondensed in EDTA, no enrichment in satellite DNA is observed. Preparations of purified, high-molecular weight, double-stranded DNA contain variable amounts of fast-sedimenting aggregates, which are insoluble in 2 M NaCl but are dispersed by DNA fragmentation or denaturation. These results point at some artifacts inherent in studies of DNA bound to residual nuclear structures in vivo and suggest conditions expected to avoid these artifacts. Further, using controlled digestion with DNAse II, we have studied the in vivo association of DNA with nuclear lamina isolated from Ehrlich ascites tumor cells. In the course of DNA fragmentation from above 50 kbp to about 20 kbp average size, the following events were observed. The DNA of high molecular weight (much longer than 50 kbp) behaved as if tightly bound to the nuclear lamina, as judged by sedimentation in sucrose and metrizamide density gradients, electron microscopy, and retention on glass fiber filters. As the size of DNA decreased, it was progressively detached from the nuclear lamina, and at about 20 kbp average length practically all DNA was released. The last 1-4% of DNA, although cosedimenting with the nuclear lamina in sucrose gradients, behaved as free DNA, banding at 1.14 g/cm3 in metrizamide density gradients and showing less than 4% retention on filters. At no stage of digestion did the DNA cosedimenting with nuclear lamina show changes in satellite DNA content relative to that of total DNA or enrichment in newly replicated DNA. It was shown, however, that digestion of nuclear lamina-DNA complex with EcoRI or Hae III led to the formation of DNA-protein aggregates, which banded at 1.35 g/cm3 in high salt containing metrizamide density gradients and which were strongly enriched in satellite DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA-tropic inhibitors of nuclease action on DNA in liver nuclei

Distamycin is a potent, wide-spectrum inhibitor of the breaking of both free and intranuclear DNA with DNAse I and with own nuclear nucleases. It compares very favourably with actinomycin D, proflavin and ethidium bromide, especially in the inhibition of DNAse I action in the nuclei. This seems likely to be due to partial overlapping of the binding sites of the nuclei with chromatin proteins in contrast to distamycin that interacts with a minor furrow of DNA being blocked to a less extent by proteins. The DNA-tropic agents under test exert no qualitative effect on the kinetics of intranuclear DNA splitting by DNAse I. Carminomycin and bleomycin are the least effective inhibitors in all the systems depicted.     

Effect of chromatin decondensation on the intranuclear matrix

We have studied the effect of chromatin condensation on the morphology of the residual structures isolated from rat liver nuclei. DNAse I digestion followed by high salt extraction of nuclei in the presence of Mg++ yields residual structures consisting of a dense peripheral layer surrounding an internal network, similar to those described by Berezney and Coffey [6]. These structures are stable at low ionic strength in the presence of EDTA. When nuclei swollen in EDTA are digested with DNAse II in the presence of EDTA, structures devoid of internal network are obtained even without subsequent treatment with high salt. When swollen nuclei are exposed to Mg++ a specific recondensation of chromatin takes place. The residual structures from recondensed nuclei are similar to those isolated from control nuclei in the presence of Mg++. The results suggest that the integrity and stability of the intranuclear matrix are acquired in the course of the isolation procedure and this is favoured by chromatin condensation.     

Comparative analysis of proteins selectively released from cell nuclei of different eukaryotic species under conditions of mild hydrolysis by nucleases

Nuclei of cultured Chinese hamster fibroblasts, dechoryonized eggs ofDrosophila melanogaster, calf thymocytes and mouse spleen were digested with micrococcal nuclease or DNAse I under conditions when about 15–20% of nuclear DNA is hydrolyzed. Proteins released from the nuclei with this DNA were analyzed by electrophoresis in polyacrylamide gels in the presence of SDS and the most prominent components were purified in each case. It is shown that these components derived from different species have different electrophoretic mobilities but are very similar in the amino acid composition characterized by high glycine content and a low lysine/arginine ratio. Molecular weights of two of those components, from mouse spleen and thymocytes, were estimated and are shown to be 25,500 and 23,000, respectively. Notwithstanding their different molecular weights they have the same N-terminal amino acid-arginine.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Distinctive features of dinoflagellate chromatin. Absence of nucleosomes in a primitive species Prorocentrum micans E

The absence of nucleosome-like structures from purified nuclei of the primitive dinoflagellate Prorocentrum micans was demonstrated by three means. i) Electron microscopy revealed mostly thin, smooth 6-nm nucleofilaments in chromatin incubated at various ionic strengths and either fixed in 0.1% glutaraldehyde or unfixed. No "beads-on-a-string" structure was found. ii) Analysis of nuclear proteins showed that low amounts of basic proteins were present (basis proteins: DNA less than 0.1), the two major one with molecular weights 12 000 and 13 000 and that histones characteristic of eucaryotes were absent. iii) Digestion of the nuclei with micrococcal endonuclease of DNase I did not result in partially digested DNA fragment repeats. Only about 10% of the bulk of the nuclear DNA was digested by micrococcal endonuclease. The high molecular weight of the remainder suggests particular protection against this type of nuclease. In the light of these distinctive nuclear features, we discuss the evolutionary position of the dinoflagellate protists with respect to the procaryotes and eucaryotes.     

Multiple structural features are responsible for the nuclease sensitivity of the active ovalbumin gene

The ovalbumin gene in chick oviduct nuclei or nucleosomes is digested preferentially by either DNase I or staphylococcal nuclease. Staphylococcal nuclease preferentially cuts between and within core particles of the oviduct ovalbumin gene; thus, the ovalbumin gene is more quickly degraded to mononucleosomes and the DNA within these monomers is digested to a nonhybridizable size significantly faster than the chicken globin gene. Mono- and oligonucleosomes generated by partial staphylococcal nuclease digestion at 0 degrees C, but not at 37 degrees C, retain equal sensitivity to DNase I. Most of this sensitivity persists when histone H1 and most of the non-histone chromosomal proteins are removed with 0.6 M NaCl. On the basis of these observations, we propose that nuclease sensitivity of the oviduct ovalbumin gene is due to covalent modifications of the core histones and that this sensitivity is amplified by interaction of other chromosomal proteins with these modified histones.