Author index

Binding of dexamethasone · receptors with isolated nuclei, DNA-cellulose and cellulose has been compared with respect to dependence on salt concentration and resistance to KCl extraction and DNAase I digestion. A solution of cytoplasmic dexamethasone-receptor complexes was prepared by the incubation of rat thymus cells with steroid at 3°C and breaking the cells by hypotonic lysis. Activation of the complexes was accomplished by warming the solution at 25°C for 15 min. Activation significantly increased the ability of dexamethasone · receptors to bind to nuclei and DNA-cellulose but not to cellulose. Dexamethasone-receptor complexes bound to nuclei at 3°C are completely resistant to extraction with 0.1 M KCl, 76% resistant to 0.2 M KCl and 20% resistant to 0.4 M KCl. Dexamethasone · receptors bound to DNA-cellulose are 45% resistant to extraction with 0.1 M and 0.2 M KCl and 29% resistant to 0.4 M KCl extraction. Cellulose-bound dexamethasone · receptors are not resistant to any of these extractions. DNAase I treatment releases 60% of the dexamethasone · receptors bound to DNA-cellulose but only 13% of those bound to nuclei, though at least 60% of the nuclear DNA is solubilized. The presence of 0.15 M KCl decreases binding of activated dexamethasone · receptors to nuclei by 73% but to DNA-cellulose by only 17%. Pretreatment of nuclei with 0.1–0.4 M KCl reduces their capacity to bind activated dexamethasone · receptors by 90% whereas similar treatment reduces the capacity of DNA-cellulose to bind dexamethasone · receptors by only 29%. Nuclei extracted with 0.1 M KCl appear to have a limited capacity to accept dexamethasone · receptors. These studies demonstrate that binding of dexamethasone · receptors to nuclei and DNA-cellulose differs by (a) the higher resistance of nuclear complexes to KCl and DNAase I treatment; (b) the much greater sensitivity of nuclei to KCl treatment.     

Effects of deoxyribonuclease I and micrococcal nuclease on the release of dexamethasone-receptor complex from nuclei of sensitive and insensitive hepatoma cell lines

(1) Fu5 cells were sensitive to the glucocorticoid inhibition of cell growth and the hormonal induction of tyrosine aminotransferase (but not fructose-1,6-bisphosphatase and glycogen synthase). AH-130 and AH-7974 cells were insensitive to both effects. (2) The release of [3H]dexamethasone radioactivity from the nuclei of Fu5 and AH-130 cells preincubated with [3H]dexamethasone increased as the KCl concentration increased from 0 to 0.4 M, with no significant difference between the two cell lines. (3) The radioactivity was more sensitively released in Fu5 nuclei than in AH-130 nuclei upon treatment with DNAase I. The release of radioactivity was always larger than the release of DNA in both cell nuclei. In contrast to DNAase I, micrococcal nuclease treatment did not show any difference between the two cell lines in the release of radioactivity from nuclei, always showing a release of radioactivity equal to that of DNA.     

Pancreatic DNAase cleavage sites in nuclei

The DNA of nuclei is cleaved by a variety of nucleases in such a way that the cuts on a given strand are always separated by an integral multiple of 10 nucleotides. However, the spacing between cutting sites on opposite strands is not known for any nuclease. In this paper, we describe the determination of the spacing, or stagger, between cuts on opposite strands produced by the action of pancreatic DNAase (DNAase I) on nuclei. When nuclei are digested with DNAase I and the resultant DNA is analyzed by gel electrophoresis without prior denaturation, a complex pattern of bands is observed. A method which gives better than 90% recovery of DNA from polyacrylamide gels was used to isolate the individual fractions corresponding to these bands. The structure of the fractions was then determined using single-strand-specific nuclease to digest single-stranded "tails" and using DNA polymerases to extend recessed 3'-OH termini of partially duplex regions. Our results show that each component consists of a double-stranded region terminating in single-stranded tails at both ends. Although both chains of every duplex are 10-n nucleotides long (n integer), the chains are never completely paired. The experiments with DNA polymerase show an abundance of structures in which the 3'-OH termini of these duplexes are recessed by 8 nucleotides, and by inference, there must be structures with 5'-P termini recessed by 2 or 12 nucleotides. Thus DNAase I acts on nuclei to produce DNA with staggered cuts on opposite strands, separated by (10-n + 8) and (10-n + 2) base pairs (with 5'-P and 3'-OH termini extending, respectively). Two classes of models of DNA folding in the nucleosome have been proposed by other investigators to account for the presence of DNAase I cleavage sites at 10-n intervals along each DNA chain. One class of models leads to the prediction that cuts should either be unstaggered or separated by 10 nucleotides, while the other class is consistent with staggers of 6 and 4 nucleotides. Neither prediction is verified by our data; however, all these models may be made consistent with the results by assuming that the enzyme's site of recognition on nucleosomal DNA is not the same as its site of cleavage.     

Non-histone proteins in fractionated chromatin before and after DNAse II treatment

Chromatin from spleen cells of normal, non-immunized mice and from mice 3 days after immunization with human immunoglobulin G was fractionated at increasing salt concentrations into three fractions: 0.35 M NaCl-soluble, 2 M NaCl-soluble and a residual fraction, dissociated in 2 M NaCl/5 M urea. The residual fraction of chromatin, homogeneous by ultracentrifugation and containing only 25% of the total chromatin DNA, was associated with proteins strongly labeled with [3H]tryptophan, [3H]methionine and [3H]leucine. This fraction was more sensitive to DNAase II treatment than was native, non-fractionated chromatin and it contained approx. 40% Mg2+-soluble DNA sequences. The template activity of the residual fraction was 6--7-times higher than that of non-fractionated chromatin. Fraction A, characteristic for non-immunized spleen cells, was present in three chromatin fractions and after DNAase II treatment it remained only in the residual fraction, which suggests that this fraction is associated with genes non-transcribed in non-immunized mice. Fractions I and B1 were found mainly in the residual fraction, and only in smaller amounts in the 0.35 M NaCl-soluble fraction. After DNAase II treatment, fractions I and B1 in chromatin from immunized mice disappeared, which suggests that these fractions may be associated with active transcribed sequences during the immune reaction.     

Effect of X-ray induced DNA damage on DNAase I hypersensitivity of SV40 chromatin: relation to elastic torsional strain in DNA.

The effect of X-irradiation on DNAase I hypersensitivity of SV40 minichromosomes within nuclei or free in solution was investigated. The susceptibility of the specific DNA sites in the control region of minichromosomes to DNAase I decreased in a dose dependent manner after irradiation of isolated nuclei. On the other hand, the irradiation of minichromosomes extracted from nuclei in 0.1 M NaCl-containing buffer almost did not affect the level of their hypersensitivity to DNAase I. This suggests that DNAase I hypersensitivity may be determined by two different mechanisms. One of them may be connected with elastic torsional strain within a fraction of minichromosomes and another seems to be determined by nucleosome free region. The first mechanism may be primarily responsible for the hypersensitivity of minichromosomes within nuclei. After irradiation of the intact cells, DNAase I hypersensitivity tested in nuclei substantially increased. This was connected with activation of endogeneous nucleases by X-irradiation which led to accumulation of single- and double-strand breaks superimposed to DNAase I induced breaks in the control region of SV40 DNA.     

Changes in chromatin structure at the replication fork. II The DNPs containing nascent DNA and a transient chromatin modification detected by DNAase I.

Discrete deoxyribonucleoproteins (DNPs) containing nascent and/or bulk DNA, were obtained by fractionating micrococcal nuclease digests of nuclei form 3H-thymidine pulse (15-20 sec) and 14C-thymidine long (16 h) labeled sea urchin embryos in polyacrylamide gels. One of these DNPs was shown to contain the micrococcal nuclease resistant 300 bp "large nascent DNA" described in Cell 14, 259-267, 1978. The bulk and nascent mononucleosome fractions provided evidence for the preferential digestion by micrococcal nuclease of nascent over bulk linker regions to yield mononucleosome cores with nascent DNA. DNAase I was used to probe whether any nascent DNA is in nucleosomes. Nascent as well as bulk single-stranded DNA fragments occurred in multiples of 10.4 bases with higher than random frequencies of certain fragment sizes (for instance 83 bases) as expected from a nucleosome structure. However, a striking background of nascent DNA between nascent DNA peaks was observed. This was eliminated by a pulse-chase treatment or by digestion of pulse-labeled nuclei with micrococcal nuclease together with DNAase I. One of several possible interpretations of these results suggests that a transient change in nucleosome structure may have created additional sites for the nicking of nascent DNA by DNAase I; the micrococcal nuclease sensitivity of the interpeak radioactivity suggest its origin from the linker region. Endogenous nuclease of sea urchin embryos cleaves chromatin DNA in a manner similar to that of DNAase I.     

Purification of 14C-labelled deoxyribonuclease II from HeLa S3 lysosomes and its use as a marker for the study of nuclear deoxyribonuclease II

Deoxyribonuclease II (DNAase II) in mammalian cells has generally been considered to be located in the lysosomes. Several recent studies have indicated that some DNAase II activity is present in purified nuclei; this, however, could have been due to some contamination of the nuclear fraction by lysosomes, or alternatively, it could have been caused by specific binding of lysosomal DNAase II to the nuclear fraction during isolation. Our previous studies have eliminated the possibility that lysosomal contamination was the cause of the presence of DNAase II in isolated nuclei. In this study I have purified (14)C-labelled lysosomal DNAase II and added it to cells during isolation of their nuclei. This study demonstrates that there is no specific binding of lysosomal DNAase II to the nuclear fraction and concludes that DNAase II activity observed in isolated nuclei represents an intrinsic activity that might be involved in nuclear DNA metabolism.     

DNAase I,DNAase II and staphylococcal nuclease cut at different,yet symmetrically located,sites in the nucleosome core

We have determined the relative location of pancreatic DNAase (DNAase I), spleen acid DNAase (DNAase II) and staphylococcal nuclease cleavage sites in the nucleosome core. Each of these three enzymes cleaves the DNA of chromatin at 10. n nucleotide intervals (n integer); this specificity presumably reflects the internal structure of the nucleosome. We have already reported that DNAase I cleaves nucleosomal DNA so that nearest adjacent cuts on opposite strands are staggered by 2 nucleotides, 3′ end extending (Sollner-Webb and Felsenfeld, 1977). Here we show that the nearest cuts made by DNAase II in nucleosomal DNA are staggered by 4 nucleotides, 3′ end extending, while cuts made by staphylococcal nuclease have a stagger of 2 nucleotides, 5′ end extending. The cutting sites of the three enzymes thus do not coincide. Each pair of staggered cuts, however, is symmetrically located about a common axis-that is, the “dyad axes” that bisect nearest pairs of cutting sites coincide for all three enzymes. This result is consistent with the presence of a true dyad axis in the nucleosome core.Our results support the conclusion that a structural feature of the nucleosome, having a 10 nucleotide periodicity, is the common recognition site for all three nucleases. The position of the cut is determined, however, by the individual characteristics of each enzyme. Sites potentially available to nuclease cleavage span a region of 4 nucleotides out of this 10 nucleotide repeat, and a large fraction of these sites are actually cut. Thus much of the nucleosomal DNA must in some sense be accessible to the environment.     

Distribution of tightly bound non-histone proteins in chromatin fractions obtained by DNAase II digestion

Digestion of pig liver chromatin with DNAse II afforded three different fractions which were characterized in terms of their DNA, RNA and tightly bound non-histone protein content, their DNA fragment size and their template activity. Two of these fractions are soluble after digestion with DNAase II and have been separated on the basis of their different solubility in MgCl2. A third fraction is not solubilized even after extensive digestion, although the size of its DNA is comparable to that of the enzyme solubilized fractions. The three fractions show qualitatively and quantitatively different distribution of tightly bound non-histone proteins, with specific protein components in each fraction; furthermore the non-solubilized fraction is greatly enriched in proteins tightly bound to DNA. From all the data obtained it can be suggested that the tightly bound proteins of the insoluble fraction may play, directly or indirectly, a role in maintaining an organized chromatin structure.     

Extraction of nuclear glucocorticoid-receptor complexes with pyridoxal phosphate

Rat thymic lymphocytes have saturable, specific receptors for glucocorticoids, which are localized predominantly in the nucleus following exposure of thymocytes to dexamethasone at 37°C. The present results demonstrate the dose-dependent extraction by pyridoxal phosphate of dexamethasone-receptor complexes from isolated thymocyte nuclei. On an equal molar basis, pyridoxal phosphate is considerably more effective than pyridoxal; pyridoxine, pyridoxamine phosphate and 5-deoxypyridoxal are ineffective. The release of the nuclear dexamethasone receptor complex is dependent on the integrity of the C4′ carboxaldehyde group of pyridoxal phosphate as evidenced by the inhibition of extraction of dexamethasone-receptor complexes by either hydroxylamine or semicarbazide. The dexamethasone which pyridoxal phosphate liberates from thymus nuclei is bound to a macromolecule which is of smaller size than unactivated cytoplasmic dexamethasone receptor.     

Interaction of dexamethasone-receptor complexes with rat liver nuclei and DNAs]

The role of DNAs in the nuclear binding of dexamethasone-receptor complexes (DRC) was studied. The cytosolic receptors from rat liver have a sedimentation coefficient of about 7S, the Stock's radius--of about 50 A and possess a high affinity to dexamethasone (Kas = 2,6 X 10(8) M-1). Their capacity is 3 X 10(-13) and 5.5--7.0 X 10(-12) mole of dexamethasone per mg cytosolic protein and mg DNA, respectively. DRC has the ability to bind to the nuclei of rat liver. DRC binding to nuclei is increased approximately 3-fold by temperature activation of cytosol. The nuclear acceptor sites are saturated at the level of 16.2 pmoles of bound DRC per mg nuclear DNA. Free DNA has the ability to compete with nuclei for binding with DRC. Temperature-activated DRC can bind both with homo- and heterologous DNAs. Secondary DRC-DNA complexes were isolated by means of gel filtration on Sepharose 4B. Thermal denaturation of DNA decreases its ability to bind DRC approximately 2-fold. DNAs of a similar nucleotide composition, i.e. DNA from rat liver (GC = 43 mole%) and DNA from Photobacterium belozerskii (GC = 44 mole%), have a close DRC-binding ability. At the same time, these DNAs bind about 1.5-fold less DRC, as compared to DNA from Pseudomonas aeruginosa (GC = 67 mole%) and about 1.5-fold more, than does DNA from T2 phage (GC = 35 mole%). Thus the positive correlation between the GC composition of DNA and its DRC-binding ability was established. Unique sequences (Cot greater than 600) bind several times less DRC than the reiterated sequences (also denaturated) (Cot = O--600) of rate liver DNA. Thus, DNA can be considered as a nuclear acceptor of DRC. It is assumed, that DRC is able to recognise in DNA certain short GC-rich sequences, distributed in the rate genome in a non-random fashion.     

ACTION OF MITOCHONDRIAL DNAASE I IN DESTROYING THE CAPACITY OF ISOLATED CELL NUCLEI TO FORM GELS

1. DNA prepared from non-gelable rat liver nuclei isolated in the presence of disrupted mitochondria at pH 6.0, has been compared with DNA obtained from gelable nuclei isolated at pH 4.0. The DNA of the non-gelable nuclei is partially depolymerized relative to the DNA of the gelable nuclei. 2. It has been found that sufficiently small quantities of crystallized DNAase I can cleave a very large part of the DNA of gelable nuclei isolated at pH 4 from the residual protein of these nuclei without causing extensive depolymerization of the DNA. At the same time the gelable nuclei are rendered non-gelable. 3. Partially purified DNAase II can also render gelable nuclei isolated at pH 4 non-gelable, and in so doing presumably also cleaves the DNA from the residual protein of the nuclei. 4. Mitochondrial DNAase I appears to be the enzyme responsible to a large extent for the cleavage of DNA from the residual protein of gelable rat liver cell nuclei with concomitant destruction of the gel-forming capability of these nuclei, when the nuclei are subjected to the action of disrupted mitochondria at pH 6.0 during the isolation procedure. 5. Mitochondrial DNAase II does not appear to exert appreciable action on nuclei during the course of isolation of the nuclei at pH 6.0 in the presence of disrupted mitochondria. 6. It is probable that DNAase I is not the sole enzyme responsible for destroying the gelability of nuclei isolated at pH 6.0 in the presence of disrupted mitochondria. Protease may be involved. 7. Sodium dodecyl sulfate at pH 6.0–6.3 cleaves the DNA of isolated gelable nuclei from the residual protein of these nuclei over a period of 2 to 3 hours. At pH 7.0–7.5, however, there is negligible cleavage over a period of 96 hours. 8. If non-gelable nuclei are isolated at pH 6.0 in the presence of disrupted mitochondria, DNA subsequently can be removed from them by the use of detergent at pH values ranging from 6.0–7.5 without the necessity of incubation in the detergent solution, since the DNA had already been detached from the residual protein by the action of the mitochondrial enzyme system during isolation of the nuclei.     

Evidence for the existence of a stable association between nascent DNA and the nuclear membrane of HeLa cells.

Nascent DNA-nuclear membrane complexes isolated from HeLa cells and solubilized in a sodium dodecyl sulfate-urea solution were examined by gel electrophoresis, column chromatography, isopycnic centrifugation, and by extraction with chloroform/methanol. Radioactivity attributable to [3H]DNA co-migrated with three protein peaks during electrophoresis. This radioactivity was eliminated by prior treatment with DNAase. In addition, all of the radioactivity attributable to nascent DNA eluted with a specific protein on Sepharose 4B columns. This DNA - protein complex banded at a density of 1.58 gm/cm3 in sucrose-CsCl gradients. Treatment with DNAase, phospholipase A and C, and dilute alkali disrupted the complex. Moreover, 93% of the radioactivity attributable to protein and 70% of that attributable to DNA could be extracted from the complex with a chloroform/methanol solution. The results suggest that nascent DNA may be in a stable association with a proteolipid moiety of the nuclear membrane.     

Studies on the binding of d-α-tocopherol to rat liver nuclei

The present study describes the nature and characteristics of the intranuclear binding sites of [³H]d-α-tocopherol in rat liver. When radioactively labeled d-α-tocopherol was intravenously administered to rats, approximately 55% of the nuclear radioactivity was associated with an intranuclear nucleoprotein complex. This complex, which was extractable by high concentrations of NaCl, was characterized by equilibrium density ultracentrifugation on a 30 to 60% linear sucrose gradient. About 50% of the high-salt-extracted radioactivity was coprecipitable with macromolecules by 10% ice-cold trichloroacetic acid (TCA). This TCA-precipitable radioactivity was completely ethanol soluble. Alkaline conditions favored the solubilization of the vitamin-receptor complex. Among various enzymes tested, only Pronase and trypsin were capable of dissociating the vitamin-receptor complex. Both ionic (sodium dodecyl sulfate) and nonionic (Triton X-100) detergents solubilized α-tocopherol from the nuclei and concomitantly released some of the associated macromolecules. In addition, treatment of nuclei with low concentrations of Triton X-100 showed that about 30% of the nuclear bound α-tocopherol is associated with inner core sites in the nucleoprotein complex with very high affinity for the vitamin. Dissociation of the nucleoprotein complex (chromatin) by high-salt solubilization and subsequent partial reassociation of the components by salting out procedures revealed the high affinity association of α-tocopherol with the reconstituted DNA-protein complex. Subfractionation of this complex further revealed that α-tocopherol is predominantly associated with the fraction containing phenol-soluble nonhistone proteins having a high affinity for DNA. In vitro binding studies also showed that there are specific saturable binding sites for d-α-tocopherol in rat liver nuclei.     

Analysis of cellular nucleoproteins by the nucleoprotein-celite chromatography method. III. Two types of DNA-nuclear matrix interaction

There are two types of DNA-nuclear matrix interactions in animal cells as revealed by the release of DNA from isolated nuclei by three successive gradients: NaCl, LiCl-urea and temperature. Nuclei were treated with dissociating agents while being adsorbed on the Celite columns. "Weak" DNA-matrix interactions which dissociate in 1.5 M LiCl-3 M urea at 2 degrees appear to be sensitive to ethidium bromide and resistant to exogeneous nucleases (DNAase I, DNAase II and micrococcal nuclease), to DNA-damaging agents, including alkylators and gamma-irradiation, and also to psoralen-induced cross-links. "Strong" DNA-matrix interactions proved to be very different. They dissociate in 4 M LiCl-8 M urea at approximately 90 degrees, are very sensitive to DNAase I and other nucleases, slightly sensitive to chemicals and irradiation at doses stimulating single-stranded DNA breaks, but resistant to ethidium bromide. DNA strand separation seems to be necessary prerequisite for DNA release from its "strong" complex with nuclear matrix. A model for the topological link between DNA and the nuclear matrix involved in DNA replication complex is discussed.     

Binding of glucocorticoid-receptor complexes with the acceptor zones of nuclei and effect of glucocorticoids on RNA synthesis in hormone-sensitive and hormone-resistant cell populations

The binding of free radioactive glucocorticoid and the glucocorticoid-receptor complex to rat liver nuclei was studied in vitro. The binding is non-saturated and independent of preliminary injection of the "cold" hormone. In the course of DNA hydrolysis the amount of the radioactive hormone bound to the chromatin moiety in vivo remains practically unchanged relatively to the initial radioactivity of the protein. The liberation of the nuclei into a cell-free medium and the effect of DNAase I on the nuclei are associated with the redistribution of the hormone-receptor complex in the chromatin molecule and with the appearance of new, previously masked acceptor zones of the hormone binding. During the first 1-2 hours following the hormone injection the endogenous RNA-synthesizing activity of the nuclei is decreased. The increase of RNA synthesis in liver nuclei occurs not earlier than 3 hours after the injection. In Zajdela hepatoma nuclei the repression of RNA synthesis persists as long as 3 hours after the injection of dexamethasone. When RNA synthesis is determined in the nuclei in the presence of exogenous RNA-polymerase of E. coli in vitro, the increase in nuclear RNA synthesis can be observed beginning with the 30th min after the hormone injection. It is assumed that this effect is due to conformational changes in the chromatin structure, which are concomitant with the initial steps of association of the hormone-receptor complex.     

Localization of oxidized nocotinamide--adenine dinucleotide glycohydrolase in the mouse liver nuclear envelope.

NAD+ glycohydrolase activity located in the nuclear envelope was maximally solubilized by treatment with 0.1--0.2% Triton X-100. The residual activity largely represents the chromatin-associated NAD+ glycohydrolase. Under these conditions the phospholipids were extensively solubilized (over 90%) while leaving the nuclei physically stable, although the nuclear membranes were removed, as shown by electron microscopy. After Triton X-100 treatment, deoxyribonuclease I did not significantly affect the residual NAD+ glycohydrolase activity, although the DNA was completely broken down. This enzyme activity can be released from the nuclear pellet by incubation with phospholipase C. For comparative studies, the glucose 6-phosphatase activity, known to be present in the nuclear envelope, was investigated. Treatment with 0.01% Triton X-100 released 10--20% of the phospholipids, but without solubilizing either glucose 6-phosphatase or NAD+ glycohydrolase. Higher Triton X-100 concentrations (0.1--1.0%) inhibited glucose 6-phosphatase, but not NAD+ glycohydrolase activity. NAD+ glycohydrolase is apparently present in a latent form in the nuclear envelope. Glucose 6-phosphatase, However, shows no such latency.     

Aggregation of fragmented chromatin associated with the synthesis of products of its treatment with nuclease

Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low salt buffer. With an increase of nuclear chromatin degradation with DNAse I or micrococcal nuclease, solubilization of deoxyribonucleoprotein (DNP) by a low salt buffer increases, reaching a maximum upon hydrolysis with 2-4% nuclear DNA and then decreases appreciably after extensive treatment with nucleases. Soluble fragmented chromatin aggregates in the course of treatment with DNAase. I. Addition to gel chromatin preparations of exogenous products of nuclease treatment of isolated nuclei leads to its aggregation. Pretreatment of nuclear chromatin with RNAase prevents solubilization of DNP by low ionic strength solutions. Some experimental data obtained with the use of severe nuclease treatment are discussed; for a correct interpretation of these data the aggregation of fragmented chromatin by products of its nuclease degradation should be taken into consideration.