The homing endonuclease I-CreI uses three metals, one of which is shared between the two active sites

Homing endonucleases, like restriction enzymes, cleave double-stranded DNA at specific target sites. The cleavage mechanism(s) utilized by LAGLIDADG endonucleases have been difficult to elucidate; their active sites are divergent, and only one low resolution cocrystal structure has been determined. Here we report two high resolution structures of the dimeric I-CreI homing endonuclease bound to DNA: a substrate complex with calcium and a product complex with magnesium. The bound metals in both complexes are verified by manganese anomalous difference maps. The active sites are positioned close together to facilitate cleavage across the DNA minor groove; each contains one metal ion bound between a conserved aspartate (Asp 20) and a single scissile phosphate. A third metal ion bridges the two active sites. This divalent cation is bound between aspartate residues from the active site of each subunit and is in simultaneous contact with the scissile phosphates of both DNA strands. A metal-bound water molecule acts as the nucleophile and is part of an extensive network of ordered water molecules that are positioned by enzyme side chains. These structures illustrate a unique variant of a two-metal endonuclease mechanism is employed by the highly divergent LAGLIDADG enzyme family.     

Calcification in insects: The dwelling-tube and midgut of machaerotid larvae (Homoptera)

The dwelling-tubes of machaerotid larvae consist of a mineralized organic scaffolding of mucofibrils. The mineral component accounts for 85 per cent of the dry weight and is composed of calcium, ferrous iron, manganese, magnesium, potassium, sodium, phosphate, carbonate, and chloride and of these the major ions are calcium and carbonate. Ferric iron in the form of ferritin is probably also present.Calcium, manganese, magnesium, and phosphate are derived from spherites secreted by a specialized region of the midgut. Calcium and phosphate are present in the spherites, probably as amorphous tricalcium phosphate. Subsequent to secretion the spherites are slowly dissolved and the calcium is incorporated into the dwelling-tube as calcium carbonate. Thus it appears that within the dwelling-tube calcium phosphate is converted to calcium carbonate.Ferritin and ferrous iron are secreted by another specialized region of the midgut and are also incorporated into the dwelling-tube.     

Phospholipids in plant and animal chromatin

Isolated hepatic nuclei and hepatic chromatin have been analysed for their DNA, RNA, protein and phospholipid content. The protein/DNA ratio is 3 for nuclei and 1.95 for chromatin extracted from Triton X-100 treated nuclei. The phospholipids, (2.36 +/- 0.91 (S.D.) per cent of the total nuclear material), are lost during the chromatin preparation mainly during the Triton X-100 washings of the nuclei. Nevertheless, 10 per cent of the total nuclear phospholipids remain bound to the chromatin. The comparative analysis of both nuclei and chromatin shows a difference in phospholipids and fatty acid composition. Thus, the chromatin-associated phospholipid cannot be attributed simply to contaminating nuclear membrane. This is supported by the autoradiographic study of semi-thin sections of interphase nuclei from root apices of Vicia faba in which [3H] ethanolamine is clearly localized in the chromatin and nucleolar regions of the nuclei.     

The Movement of Calcium in Germinating Pea Seeds

Pea seeds contain less calcium than phosphorus, potassium ormagnesium; more than half of this calcium is located in thetesta. Peas at either end of a pod have more calcium than thosein the middle. When pea seeds are allowed to germinate in water,less than 30 per cent of the cotyledonary calcium moved to thegrowing axis during the first 15 days of germination, whereas70–90 per cent of magnesium, potassium and phosphate wasexported. Various attempts to increase the amount of calciumexported were not successful. When radioactive calcium was appliedto the cotyledons, essentially no movement to the axis was observedunder conditions where extensive movement of radioactive phosphateoccurred.     

In vitro demonstration of putative nuclear 3,5,3'-triiodothyronine receptors in isolated liver nuclei of Singi fish, Heteropneustes fossilis (Bloch)

Putative thyroid hormone (TH) receptors have been demonstrated in the isolated liver nuclei of Singi fish, Heteropneustes fossilis (Bloch), and their binding characteristics have been examined. Nuclear T3 saturation analyses were carried out in vitro at 27 degrees C in a sucrose-Tris-HCl buffer (pH 7.5) containing calcium (2 mM), magnesium (3 mM) and 2-mercaptoethanol (5 mM). After incubation the bound and free hormones were separated by centrifugation and the nuclei were treated with Triton X-100 (final concentration 0.25%) to reduce the non-specific binding. The binding was saturable and reached equilibrium by 20 minutes of incubation and was also stable for 2 hours. The binding was reversible and the rate of dissociation was more or less equal to the rate of association. The binding was linearly increased with the increased concentrations of the DNA (nuclei). Scatchard analyses of the equilibrium binding data revealed that only one class of binding sites for T3 did exist in the hepatic nuclei of Singi fish. The affinity of these sites or the mean dissociation constant (Kd = 0.20 +/- 0.07 x 10(-10) M) and the mean maximum binding capacity (MBC = 0.17 +/- 0.04 pmol/mg DNA) were in reasonable agreement with the values reported for other teleost fishes.     

The binding of calcium to human fibronectin

The binding of calcium to human plasma fibronectin has been measured by equilibrium dialysis at 25° in 0.1 M NaCl 50mM Tris HCL, pH 7.4. Curve fitting of the binding data indicates that fibronectin has two strong calcium binding sites per chain (Mr 220,000), KD = 1.3 mM and approximately 12 weak sites, KD = 2.3 mM. No significant displacement of bound calcium by magnesium was observed at magnesium concentrations up to 1 mM. Calcium binding to a pair of tryptic fragments of fibronectin (Mr ? 160,000 and 180,000) that bind to gelatin has also been investigated. These fragments have a single class of calcium binding sites, with 2.2 sites per chain, KD = 1.1 mM. Negligible calcium binding to tryptic fragments derived from other regions of the fibronectin molecule was observed.     

Relation of the biological activity of double helical interferon inducers and their penetration into the cell and suppression of protein synthesis

The quantities of 125I-ds-inductors of interferon penetrating into the cells of transplantable cultures such as M19 (human fibroblast cells) and L-929 (mouse line) were not significant i.e. 10.5-4 per cent of the drug added. Under conditions of transfection with calcium phosphate and in complex with DEAE dextran the quantities of the inductors adhering to the cells and their contents in the cytoplasmic and nuclear fractions markedly increased. During the transfection with calcium phosphate up to 50 per cent of the applied inductor bound to the cells and its content in the cytoplasm and nuclei reached at least 10 per cent. After penetration into the cells poly I.poly C probably maintained its native structure and appeared to be firmly bound to the nuclear material. Preliminarily hydrolyzed inductors showed no such penetrating capacity. Contrary to the human fibroblast cells, in the mouse cells L-929 treated with the ds-inductors there was observed inhibition of the total protein synthesis which was probably due to activation of enzymes such as 2-5A-synthetase and proteinkinase. Increased penetration of the ds-inductors into the cells was accompanied by a marked (from 10- to 1000-fold) rise in their antiviral activity and a 2-4-fold rise in their interferon-inducing activity. It was concluded that there was immediate dependence of ds-inductor biological activity manifestation on the level of the inductor penetration into the cells.     

DNA protection by aminothiols: study of the cysteamine - DNA interaction by vibrational spectroscopy

Infrared linear dichroism measurements and Roman scattering spectra show that the cysteamine molecule binds strongly to the DNA stabilizing the double helix in a B geometry conformation. The B→A conformational transition is not observed for a cysteamine/DNA ratio of one cysteamine molecule per two phosphate sites. No evidence of interaction has been found between the radioprotector and the DNA bases. A model is proposed in which the cysteamine molecule is bound by its two ends through electrostatic interaction to two consecutive phosphate groups along the same DNA strand.     

Circular dichroism (CD) studies on yeast enolase: activation by divalent cations

The effect of divalent cations on the near ultraviolet circular dichroism (CD) spectrum of yeast enolase showed that calcium, magnesium, and nickel ions produced identical changes. This was interpreted as indicating that the cations bound to the same sites on the enzyme and produced identical changes in tertiary structure. There was no effect of magnesium ion on the far ultraviolet spectrum. Evidently magnesium ion has no effect on the secondary structure. Substrate bound to the enzyme when the above cations were present although calcium permits no enzymatic activity. The CD spectral difference produced by the substrate was nearly the reverse of that produced by the metal ions. Glycolic acid phosphate, a competitive inhibitor lacking carbon-3, produced no effect, indicating carbon-3 was necessary for the CD spectral changes. The CD and visible absorption spectra of nickel and cobalt bound to various sites on the enzyme showed that the binding sites were octahedral or distorted octahedral in coordination and that the ligands appeared to be oxyligands: water molecules, hydroxyl or carboxyl groups. Examination of the effects of substrate and two compounds thought to be "transition state analogues" showed that these perturbed the "conformational" sites of the enzyme. The "catalytic" and "inhibitory" sites did not appear to be very CD active.     

Self-association of calcium and magnesium complexes of dentin phosphophoryn

Self-association of rat dentin phosphophoryn in the presence of calcium and magnesium ions was examined by chemical cross-linking and electron microscopy. Highly phosphorylated phosphophoryn (HP) binds a maximum of 1.33 calcium ions or 1.07 magnesium ions per organic phosphate residue at pH 7.4-8.0. The Ca-HP complexes are predominantly linear when the calcium content of the complex is less than about 65% of the saturation level. At higher calcium levels, the protein has a folded conformation, and transient protein-protein interactions occur. The equilibrium mixture of monomers and oligomers is predominantly monomeric unless the protein is saturated with calcium. The saturated Ca-HP complex forms discrete high molecular weight particles about 25 nm in diameter. The particles are electrically neutral and generally occur in clusters. Mg-HP complexes appear predominantly linear by electron microscopy at all concentrations of bound magnesium up to about 99% of the saturation level; however, protein-protein interaction is measurable when the magnesium content is as little as 65% of the saturation level. At saturation, Mg-HP complexes form high molecular weight particles which are negatively charged. Because of the negative charge, these particles form a stable colloidal suspension and have a rather stellate configuration.     

Functional groups at the catalytic site of F1 adenosine triphosphatase

The protection of F1 ATPase by inorganic phosphate, ADP, ATP, and magnesium ion against inactivation by 1-fluoro-2,4-dinitrobenzene, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, respectively, has been investigated. Dissociation equilibrium constants and rate constants for the labeling reactions have been deduced from a quantitative treatment of the kinetic data. Comparison of these dissociation constants with each other and with the corresponding literature values indicates that the essential Tyr, Arg, Lys, and Glu or Asp residues are indeed located at the catalytic site of the enzyme. Examination of the rate constants for the labeling reactions in the presence of excess inorganic phosphate, ADP, ATP, or magnesium ion, respectively, suggests that the essential phenol and amino groups are located nearer to the bound inorganic phosphate or the gamma-phosphate group than to the alpha- or beta-phosphate group of the bound ATP, that the essential guanidinium group is located nearer to the alpha- or beta-phosphate group than to the gamma-phosphate group of the bound ATP or the bound inorganic phosphate, and that the essential carboxylate group is located slightly farther away but complexed with magnesium ion which it shares with the bound inorganic phosphate. A mechanism consistent with these topographical relationships is proposed for the catalytic hydrolysis and synthesis of ATP.     

Phosphodiesterase Found in Young Wheat Roots

Phosphodiesterase has been found in the particulate and soluble fractions from young wheat roots. The intracellular distribution of this enzyme was studied by using RNA, oligo DNA and DNPP as the substrates. When oligo DNA was used, 50 to 60 per cent of PPDase activity was found in the soluble fraction and 30 to 40 per cent in the microsomal fraction. Besides magnesium ion, calcium, cobalt, manganese and nickel ions were effective for its activity. The pH optimum of the enzyme was found at 6.0. This PPDase produced 5′-nucleotides from RNA at pH 6.9 on addition of magnesium chloride.     

The similarity of DNA sequences remaining bound to scaffold upon nuclease treatment of interphase nuclei and metaphase chromosomes.

The fragments of DNA attached to protein skeleton of interphase nuclei or metaphase chromosomes were obtained. Both the method involving restriction endonuclease treatment/1,2/and a novel procedure based on mild staphylococcal nuclease digestion were used. In the latter case, DNA fragments remaining bound to nuclei or chromosomes are not enriched in satellite but only in abundant middle repetitive DNA. The shorter the fragments of attached DNA, the higher the content of middle repetitive DNA in the fraction. It has a slightly higher density in a CsCl gradient comparing to the main DNA. The yield of attached DNA, its distribution in a CsCl density gradient, and its renaturation properties are essentially the same for interphase and metaphase chromosomes. The average size of DNA loops was found to be equal to approximately 60 kb for both metaphase chromosomes and interphase nuclei. The conclusion has been drawn that the bulk of attachment sites of DNP fibrils to axial chromosomal structures remains unchanged during the cell cycle.     

The absence of histone in the bacterium Escherichia coli. I. Preparation and analysis of nucleoprotein extract

The deoxyribonucleic acid (DNA) from Escherichia coli has been isolated as an extract containing about 50 per cent by weight protein. The protein component differs both in composition and chemical behaviour from histone which occurs in combination with the DNA in most cells of higher organisms. Although this result suggests the absence of histone-like protein, it is not clear whether the bacterial protein found is naturally bound to the bacterial DNA in the cell or becomes attached to the DNA during the course of isolation.     

On the association of DNA primase activity with the nuclear matrix in HeLa S3 cells

We have reinvestigated the association of DNA primase activity with the nuclear matrix prepared from exponentially growing HeLa S3 cells. We have found that 25–30 per cent of the nuclear primase activity resists extraction with 2 M NaCl and digestion with Dnase I. Unlike previous investigations, done with the same cell line, the results showed that nuclear matrix-bound DNA primase activity represented less than 10 per cent of the total cell activity. Association of high levels of primase activity with the nuclear matrix was strictly dependent on a 37°C incubation of isolated nuclei prior to subfractionation. Evidence was obtained that the method used for preparing nuclei can have a dramatic effect on the amount of primase activity which is recovered both in the postnuclear supernatant and in isolated nuclei, thus seriously affecting the interpretation of the results about the quantity of DNA primase activity bound to the nuclear matrix.     

Interpretation of 25Mg spin relaxation in Mg-DNA solutions: temperature variation and chemical exchange effects.

The temperature dependencies of line shapes and spin-lattice relaxation times T1 have been measured for 25Mg in dilute solutions of Na-DNA/NaCl containing varying amounts of added magnesium(II) ions. The 25Mg spectrum is clearly non-Lorentzian, due to the presence of motions modulating the quadrupolar interaction that are slow compared to the inverse of the Larmor frequency. The weakly temperature-dependent line shapes and relaxation rates appear to be influenced by the relatively slow exchange of the Mg2+ ions between the DNA surface and the aqueous bulk phase. The observed temperature dependencies depend on the ratio of total magnesium to DNA phosphate, Mg/P. The line shape as well as the temperature dependence of the line width at half height can be qualitatively reproduced with a two-site discrete exchange model for the quadrupolar relaxation of a spin 5/2 nucleus in isotropic solution. The calculations give a value of the lifetime for magnesium bound to DNA of 4 ms at room temperature. Previously reported temperature-dependent 43Ca relaxation measurements in DNA solution can be reproduced under the assumption of a mean lifetime of bound calcium that is not larger than 2 ms but not smaller than 50 microseconds at room temperature. The temperature variation of T1 for 25Mg has been calculated, giving some qualitative agreement with the data. The correlation time for bound 25Mg has been found to be about 40 ns at room temperature.     

Incorporation and replication of foreign metaphase chromosomes in cultured mammalian cells

It is shown that the dyes used to produce banding patterns on chromosomes, quinacrine and Giemsa, are bound to DNA, and not to non-histone protein, the other chromosomal component remaining after acetic acid fixation. Studies on fixed nuclei and on extracted DNA in gelatine films show that the amount of dye bound is not affected by whether the DNA is native or denatured, and is not directly related to the amount of DNA present. Quinacrine is bound to the DNA ionically. With Giemsa, a new magenta compound is formed in situ, consisting of two molecules of methylene blue and one of eosin; this compound is attached to the chromosome by hydrogen bonds. Both quinacrine and the magenta compound formed from Giemsa appear to be attached to DNA molecules at two separate points, and the available evidence suggests that the amount of dye bound is related to the concentration of the DNA. It is suggested that the dye molecules bridge longitudinally separated sites brought into close proximity by folding of the DNA, and that the spatial arrangement of sites in the chromosome is influenced by non-histone proteins. It is concluded that chromosome banding is thus a consequence of the reduction of dye binding in those regions where the DNA chains become sufficiently dispersed to prevent bridging by the dye molecules. Possible indirect effects of base composition and repetition on dye binding at certain chromosomal sites are discussed.     

Some chemical properties of isolated pea nucleoli

Isolated nuclei and nucleoli of ungerminated pea embryos have been analyzed chemically for their content of DNA, RNA, zinc, iron, phosphorus, and protein sulfhydryl groups. The values obtained cannot be considered to represent the whole of the living nucleolar body as an undetermined amount of material is extracted from nucleoli in the course of their isolation. Only negligible amounts of DNA have been found in the isolated nucleoli; most of the DNA released on disruption of nuclei appears in a fraction showing very few structures under the light microscope. RNA is more concentrated in the nucleolus than in the nucleus or cytoplasm, but since nucleolar protein is 6 per cent of nuclear and less than 1 per cent of cytoplasmic protein, the total amount of nucleolar RNA is comparatively small. None of the other components listed occurs in high concentration in either nucleus or nucleolus.