Effect of nickel(II) on DNA-protein binding,thymidine incorporation,and sedimentation pattern of chromatin fractions from intact mammalian cells

Nuclear uptake and chromatin binding of nickel(II) was investigated in Chinese hamster ovary (CHO) cells. The cytoplasmic:nuclear ratio of nickel immediately following treatment was 5:1, but by 24 and 48 hours this ratio decreased to 4:l and 2:1, respectively, indicating that nickel is retained longer in the nucleus than cytoplasmic nickel. Chromatin was fractionated by sonication and centrifugation into fast-sedimenting, magnesium-insoluble, or magnesiumsoluble components. The magnesium-insoluble portion bound more nickel ions and retained the metal longer than either the magnesium-soluble or the fastsedimenting fractions. Treatment of cells with nickel chloride (NiCl₂) decreased the amount of DNA in the magnesium-insoluble fraction but increased the amount of DNA in the fast- sedimenting chromatin fraction. The magnesium-insoluble fraction isolated from nickel-treated cells contained approximately ten times more [35-S]-methionine–labeled protein per milligram DNA compared with untreated cells. The magnesium-soluble and the fast-sedimenting fractions isolated from the nickel-treated cells did not exhibit a similar increase in [35-S]-methionine–labeled protein per milligram of DNA. Nickel treatment suppressed [14-C]-thymidine incorporation into total DNA by 30% compared with untreated cells. However, the magnesium-insoluble chromatin fraction from nickel-treated cells had a tenfold to 20-fold increase in thymidine incorporation, while the other chromatin fractions did not exhibit an increase in thymidine incorporation. These findings indicate that nickel induced widespread alterations in chromatin conformation and preferentially interacted with an Mg-insoluble component of chromatin.     

DNA-protein cross-links induced by nickel compounds in intact cultured mammalian cells

The carcinogenic activity of crystalline NiS has been attributed to phagocytosis and intracellular dissolution of the particles to yield Ni2+ which is thought to enter the nucleus and damage DNA. In this study the extent and type of DNA damage in Chinese hamster ovary CHO cells treated with various nickel compounds was assessed by alkaline elution. Both insoluble (crystalline NiS) and soluble (NiCl2) nickel compounds induced single strand breaks and DNA protein cross-links. The single strand breaks were repaired relatively quickly but the DNA-protein cross-links were present and still accumulating 24 h after exposure to nickel. Single strand breakage occurred at both non-cytotoxic and cytotoxic concentrations of nickel, however, DNA-protein cross-linking was absent when cells were exposed to toxic nickel levels. The concentration of nickel that induced DNA-protein cross-linking correlated with those metal concentrations that reversibly inhibited cellular replication.     

Steroid-induced nuclear binding of glucocorticoid receptors in intact hepatoma cells

Some of the early steps of steroid hormone action have been studied in cultured hepatoma cells, in which glucocorticoids induce tyrosine aminotransferase. The hypothesis that inducer steroids promote the binding of specific cytoplasmic receptors to the cell nucleus has been examined in intact cells.Binding of steroids such as dexamethasone and cortisol results in a loss of most of the receptor sites from the cytoplasm. This coincides with the binding of an equivalent number of steroid molecules in the nucleus. Both processes occur concomitantly, even when their kinetics are altered by reducing the temperature. When the inducer is removed from the culture, steroid dissociates from the nucleus while the level of cytoplasmic receptor returns to normal, even if protein or RNA synthesis is inhibited. These results suggest that nuclear binding of glucocorticoids is due to the association with the nucleus of the cytoplasmic receptor-steroid complex itself and make it unlikely that the receptor acts as a mere carrier for the intracellular transfer of the steroid.Steroids that differ in their effects on tyrosine aminotransferase induction were also studied. In contrast to those bound with inducer steroids, receptors complexed with the anti-inducer progesterone did not leave the cytosol. Further, a suboptimal inducer (deoxycorticosterone) produced an intermediate level of depletion. Thus, the biological effect of different classes of steroids can be related to their capacity to promote nuclear binding of the receptor. These data support a model proposed earlier, according to which the receptor is an allosteric regulatory protein directly involved in the hormone action, under the control of specific steroid ligands. They further suggest that the conformational state influenced by the inducer is such that a nuclear binding site on the receptor is exposed.Evidence is also presented that a distinct reaction takes place between the binding of the steroid to the receptor and the association of the complex with the nucleus. At 0 °C, this change is rate-limiting. It could correspond to the “activation” of receptor-steroid complexes known to be required for binding of the complexes by isolated nuclei, and thus represent an additional step in hormone action.     

Effects of inhibition of protein synthesis on DNA replication in cultured mammalian cells

We have investigated the effects of inhibiting protein synthesis on the overall rate of DNA synthesis and on the rate of replication fork movement in mammalian cells. In order to test the validity of using [³H]thymidine incorporation as a measure of the overall rate of DNA synthesis during inhibition of protein synthesis, we have directly measured the size and specific radioactivity of the cells' [³H]dTTP pool. In three different mammalian cell lines (mouse L, Chinese hamster ovary, and HeLa) nearly complete inhibition of protein synthesis has little effect on pool size (±26%) and even less effect on its specific radioactivity (±11%). Thus [³H]thymidine incorporation can be used to measure accurately changes in rate of DNA synthesis resulting from inhibition of protein synthesis.Using the assay of [³H]thymidine incorporation to measure rate of DNA synthesis, and the assay of [¹⁴C]leucine or [¹⁴C]valine incorporation to measure rate of protein synthesis, we have found that eight different methods of inhibiting protein synthesis (cycloheximide, puromycin, emetine, pactamycin, 2,4-dinitrophenol, the amino acid analogs canavanine and 5-methyl tryptophan, and a temperature-sensitive leucyl-transfer tRNA synthetase) all cause reduction in rate of DNA synthesis in mouse L, Chinese hamster ovary, or HeLa cells within two hours to a fairly constant plateau level which is approximately the same as the inhibited rate of protein synthesis.We have used DNA fiber autoradiography to measure accurately the rate of replication fork movement. The rate of movement is reduced at every replication fork within 15 minutes after inhibiting protein synthesis. For the first 30 to 60 minutes after inhibiting protein synthesis, the decline in rate of fork movement (measured by fiber autoradiography) satisfactorily accounts for the decline in rate of DNA synthesis (measured by [³H]thymidine incorporation). At longer times after inhibiting protein synthesis, inhibition of fork movement rate does not entirely account for inhibition of overall DNA synthesis. Indirect measurements by us and direct measurements suggest that the additional inhibition is the result of decline in the frequency of initiation of new replicons.     

A Leuconostoc lactis protein with homology to ribosomal protein S1 shares common epitopes and common DNA binding properties with a mammalian DNA binding nuclear factor

A mouse testis cDNA expression library (Clontech) was screened with a synthetic oligonucleotide ligand containing CT-rich motifs derived from the rat skeletal muscle actin gene promoter. These motifs bind nuclear proteins, and seem to be involved in the regulation of the gene. Analysis of isolated clones, which expressed proteins that specifically bind the oligonucleotide, indicated that they were derived from a single gene. This gene was identified as a contaminant of bacterial origin (Leuconostoc lactis). The cloned gene from L. lactis encodes a protein with significant homology to bacterial ribosomal protein S1, which we designated LrpS1-L. Band shift analysis and competition experiments indicated that both the bacterial protein and a mouse nuclear protein specifically bind to the same CT-rich motif of the skeletal muscle actin promoter. Furthermore, antibodies against the recombinant bacterial protein interfered with the formation of complex between the CT-rich element and the mouse nuclear protein. These results indicate that the bacterial LrpS1-L protein and the mammalian protein bind the same CT-rich motif and share common antigenic epitopes.     

The binding mode of a mammalian (boar) protamine to DNA

The binding modes of mammalian and fish protamines to DNA were studied by reconstitution experiments from dansylated protamines and DNA, using fluorescence spectroscopy, thermal denaturation and sedimentation. Both boar and fish protamines showed strong positive cooperativity in binding to DNA. Binding parameters of the protamines were determined in 0.1 M NaCl, 50 mM Tricine-HCl, pH 7.4, at 37 degrees C: in the boar protamine, the cooperative binding constant (Kc) = 3.4 X 10(6) M-1 and the cooperative factor (q) = 667, in the fish protamine, Kc = 1.8 X 10(7) M-1 and q = 304. The boar protamines bound to DNA with two functional domains, but the fish protamines bound directly to DNA as a single linear molecule.     

In situ localization of mitochondrial DNA replication in intact mammalian cells

Nearly all of the known activities required for mitochondrial DNA (mtDNA) replication and expression are nuclear-encoded gene products, necessitating communication between these two physically distinct intracellular compartments. A significant amount of both general and specific biochemical information about mtDNA replication in mammalian cells has been known for almost two decades. Early studies achieved selective incorporation of the thymidine analog 5-Bromo-2-deoxy-Uridine (BrdU) into mtDNA of thymidine kinase-deficient (TK[-]) cells. We have revisited this approach from a cellular perspective to determine whether there exist spatiotemporal constraints on mtDNA replication. Laser-scanning confocal microscopy was used to selectively detect mtDNA synthesis in situ in cultured mammalian cells using an immunocytochemical double-labeling approach to visualize the incorporation of BrdU into mtDNA of dye-labeled mitochondria. In situ detection of BrdU-incorporated mtDNA was feasible after a minimum of 1- 2 h treatment with BrdU, consistent with previous biochemical studies that determined the time required for completion of a round of mtDNA replication. Interestingly, the pattern of BrdU incorporation into the mtDNA of cultured mammalian cells consistently radiated outward from a perinuclear position, suggesting that mtDNA replication first occurs in the vicinity of nuclear-provided materials. Newly replicated mtDNA then appears to rapidly distribute throughout the dynamic cellular mitochondrial network.     

Influence of copper(II) and magnesium(II) ions on the ciprofloxacin binding to DNA

The influence of magnesium(II) and copper(II) ions on the binding of ciprofloxacin to double stranded calf thymus DNA was studied by fluorescence emission spectroscopy, ultraviolet- and circular dichroism (CD) spectroscopy. The interaction of ciprofloxacin and copper(II) ions was followed by strong fluorescence quenching which was almost unaffected by the presence of DNA. On the other hand, only a slight decrease in fluorescence emission intensity, which was enhanced in the presence of DNA, was observed for ciprofloxacin interaction with magnesium(II) ions. Furthermore, magnesium(II) ions increase the thermal stability of the DNA, while, in the presence of ciprofloxacin, the degree of stabilisation is smaller. In contrast, copper(II) ions destabilise double helical DNA to heat, while ciprofloxacin slightly affects only the second transition of the biphasic melting curve of calf thymus DNA. Magnesium(II) ions at 25 degrees C induce conformational transitions of DNA at concentrations of 1.5 mM and 2.5 M, as monitored by CD. On the other hand copper(II) ions induce only one conformational transition, at a concentration of 12.7 microM. At higher concentrations of copper(II) ions (c>700 microM) DNA starts to precipitate. Significant changes in the CD spectra of DNA were observed after addition of ciprofloxacin to a solution containing DNA and copper(II) ions, but not to DNA and magnesium(II) ions. Based on our spectroscopic results, we propose that copper(II) ions are not directly involved into ciprofloxacin binding to DNA via phosphate groups as it has been suggested for magnesium(II) ions.     

Photosensitivity of DNA replication and respiration to haematoporphyrin derivative (photofrin II) in mammalian CV-1 cells

DNA synthesis, as well as respiration, has been studied in CV-1 cells incubated with 5 or 25 micrograms/cm3 haematoporphyrin derivative Photofrin II (PF II) for 1, 24 or 48 h and then irradiated with various doses of UVA light (365 nm). The impairments of DNA synthesis increased with the duration of incubation with the porphyrin, its concentration and the dose of irradiation. The cellular consumption of oxygen is also inhibited by the treatment, but less severely. In the case of the higher PF II concentration (25 micrograms/cm3), the impairment of DNA synthesis after illumination seems to be mainly due to 3HTdR transport inhibition. This effect can be related to plasma membrane damage as shown by lactate dehydrogenase leakage. At 5 micrograms/cm3 PF II, DNA synthesis inhibition is observed even after short exposure to PF II and light without 3HTdR transport impairment. In that case, DNA and/or mitochondrial photodamage may explain the inhibition.     

Study of propidium iodide binding to DNA in intact cells by flow cytometry

We studied thein situ binding of propidium iodide to DNA in fixed human lymphocytes, using flow cytometry. Experimental data of fluorescence emission vs dye concentration and vs cell concentration were obtained. Data were interpreted by means of two different mathematical models specific for the staining reaction, and the binding parameters were obtained by “best-fitting” of the data. A model based on two classes of binding sites with different affinity constants gave the most satisfactory fitting. The accessibility of thein situ chromatin turned out to be reduced with respect to the nonin situ accessibility for ethidium bromide as reported in the literature. The present study shows the usefulness of the flow-cytometric technique for probing DNA structure in intact cells.     

Radiation-induced binding of DNA from irradiated mammalian cells to hydroxyapatite columns

In experiments designed to measure radiation-induced DNA damage using the DNA unwinding-hydroxyapatite chromatography technique, we observed that under some experimental conditions a significant proportion of the test DNA became tightly bound to the hydroxyapatite (HA) and could not be released even with a high concentration of phosphate buffer. Approximately 5-10% of DNA from unirradiated cells binds to the HA. With increasing radiation doses in air, the fraction of bound DNA increases, reaching about 30% at about 35 Gy. The binding exhibits many of the characteristics of a radiation-induced cell lesion: the proportion of DNA retained by the HA is less when cells are irradiated under hypoxic conditions or in the presence of the thiol radioprotector dithiothreitol; and the binding decreases when an incubation period is allowed between irradiation and harvest of the cells for assay. Studies to determine the nature of the lesion responsible for the binding demonstrated that lesion production requires a component found in cells since no binding was observed with irradiated isolated DNA or nuclear matrix; the binding is not a result of the production of DNA-protein crosslinks; and the bound DNA is single-stranded, based on its sensitivity to nuclease S1. Because of the dose dependence of the binding of DNA to HA, the slopes of the dose-response curves for DNA damage determined with this assay depend on the method used to calculate the fraction of double-stranded DNA. Our demonstration that the bound DNA is single-stranded guides the choice of the method for data analysis.     

Multi-site binding of human nuclear protein to the Alu-family repeated DNA

Nuclear protein which selectively binds to the Alu-family DNA repeat (AFR, Blur8) is partially purified from human HeLa cells using a gel retention assay. At low protein concentrations only a single complex of the protein with AFR is formed (CII). Increasing protein concentrations lead to the gradual disappearance of CII, being replaced by complexes with higher (CI) and lower (CIII, CIV) electrophoretic mobilities. Differential binding of AFR restriction subfragments indicates that multiple protein-binding sites are present within AFR. We discuss two models explaining the anomalous electrophoretic mobility of CII by DNA bending or looping upon cooperative multi-site binding of the protein to AFR.     

Nickel(II) binding to Cap43 protein fragments

Cap43 protein has been tested for metal binding domains. The protein, specifically induced by nickel compounds in cultured human cells, had a new mono-histidinic motif consisting of 10 amino acids repeated three times in the C-terminus. The 20-Ac-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(356)-Thr-Ser-Glu-Gly(360) - peptide 1) and the 30-Ac-TRSRSHTSEG-TRSRSHTSEG-TRSRSHTSEG (Thr(341)-Arg-Ser-Arg-Ser-His(346)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(356)-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-Ser-His(366)-Thr-Ser-Glu-Gly(370) - peptide 2) amino acids sequence has been analyzed as a site for Ni(II) binding. A combined pH-metric and spectroscopic (UV-visible, CD, NMR) studies of Ni(II) binding to both fragments were performed. The 20-amino acid peptide can bind one and two metal ions while the 30-amino acid fragment one, two and three metal ions. At physiological pH, depending on the metal to ligand molar ratio, peptide 1 forms the Ni(2)L species while peptide 2 the NiL, Ni(2)L and Ni(3)L complexes where each metal ion is coordinated to the imidazole nitrogen atom of the histidine residue of the 10-amino acid fragment. Octahedral complexes at pH 8-9 and planar 4N complexes with (N(Im), 3N(-)) bonding mode at pH above 9, are formed. This work supports the existence of an interesting binding site at the COOH-terminal domain of the Cap43 protein.     

Synthesis, characterization, crystal structures and DNA binding studies of nickel(II) hydrazone complexes

A series of octahedral and square-planar Ni(II) complexes have been synthesized from two different types of hydrazone ligands. The isolated complexes have been characterized by means of analytical and spectroscopic techniques. The structures of two of the complexes have been determined by single crystal X-ray diffraction study. The binding modes of the complexes with DNA and their ability to bind DNA have been investigated by UV-Vis absorption titration, ethidium bromide fluorescence displacement experiments, and viscometry measurements and cyclic voltammetry studies. The experimental results show that the mode of binding of the complexes to DNA is combination of different mode of interaction.     

Effects of polarity on dioxygen binding to cobalt(II) porphyrins

Some picket fence porphyrinatocobalt(II) complexes which contain a replaced polar or non-polar group in their fences were newly prepared and characterized, and their oxygen affinities were measured spectrophotometrically. The O₂ affinities of the complexes containing a replaced polar group other than amido linkages are appreciably reduced as compared with those of the complexes with a similar non-polar group, regardless of the charge sign of the polarity in the cavity. On the other hand, solvent polarity affects the O₂ affinity of the complex with a polar group which is accessible to the coordinated dioxygen molecule, while solvation effects of the corresponding complex without such a group are little. On the basis of these results the relationships between O₂ affinity and pocket polarity or solvent polarity are discussed.