Tungsten particle-induced nicking of supercoiled plasmid DNA.

Small particles of metallic tungsten, known also as tungsten microprojectiles, are routinely used for biotechnological purposes. In such applications, tungsten was observed to affect the integrity of plasmid DNA. Here we present evidence that interaction between tungsten particles and intact circular plasmids pU19, pUC119, and ColE1 may result in generation of a limited number of single-strand DNA breaks. As a consequence, supercoiled DNA is converted into its open circular form and no fragmentation products can be detected. The rate of the tungsten-mediated reaction depends on pH but is not influenced by ascorbate, Tris, or EDTA. No DNA nicking can be observed when the tungsten particles are replaced by substances that can be leached out from these particles with water or incubation buffers. Likewise, commercial sodium tungstate, tungsten (VI) oxide, and tungsten (VI) chloride and products of its decomposition remain DNA undamaged. Native plasmid DNA molecules, upon adsorption on the surface of tungsten microparticles, may undergo some nicking without a need for participation of external catalysts.

Relaxation, linearization and fragmentation of supercoiled circular DNA by tungsten microprojectiles.

The aim of the study was to characterize DNA lesions caused by microprojectile bombardment and by the post-bombardment presence of tungsten particles in transformed cells. For the sake of simplicity, plasmid DNA was used as a target for bombardment with naked tungsten particles. Unexpectedly extensive DNA degradation was observed under standard bombardment conditions. However, no further DNA fragmentation occurred under post-bombardment conditions, simulated by incubation of plasmid DNA with a suspension of tungsten particles. Instead, relaxation and linearization of supercoiled circular plasmids (pAHC25 and others) took place. It is concluded that the observed linearization (a single site double-strand break in DNA circle) results from the ability of tungsten to catalyse the hydrolysis of phosphodiester bonds in torsionally strained sites of native DNA selectively.

The activity of calmodulin is altered by phosphorylation: modulation of calmodulin function by the site of phosphate incorporation.

Calmodulin transduces Ca2+ signals by binding to and activating essential regulatory enzymes. The large number of intracellular targets for calmodulin raises the possibility that mechanisms in addition to Ca2+ may modulate calmodulin activity. Phosphocalmodulin is found in cells and tissues, and calmodulin phosphorylation is enhanced by several mitogens. Phosphorylation of calmodulin on serine/threonine residues by casein kinase II decreased its ability to activate Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II). The major effect was a 2.5-fold increase in the concentration at which half-maximal velocity (K0.5) was attained, with no apparent alteration in the Vmax, or the K0.5 for Ca2+. In contrast, calmodulin phosphorylated on tyrosine residues by the insulin receptor kinase produced an increase in the Vmax, with no alteration in the affinity for CaM-kinase II or the K0.5 for Ca2+. Direct determination by surface plasmon resonance of the dissociation constants with a synthetic peptide corresponding to the calmodulin-binding domain of CaM-kinase II revealed that phosphorylation on serine/threonine residues of calmodulin significantly decreased its affinity for the peptide, while tyrosine phosphorylation had no effect on binding. In contrast to CaM-kinase II, neither serine/threonine nor tyrosine phosphorylation of calmodulin altered its ability to activate calcineurin. These data indicate that phosphorylation of calmodulin differentially modifies its interaction with individual target enzymes. Moreover, the amino acid residues phosphorylated provide an additional level of control. These results demonstrate that phosphorylation is an in vitro regulatory mechanism in the targeting of calmodulin responses and, coupled with the stoichiometric phosphorylation of calmodulin in rat hepatocytes, suggest that it may be relevant in intact cells.

Assessment of the interaction between calmodulin and casein kinase II.

Calmodulin is an in vitro substrate for casein kinase II and is also reported to inhibit casein kinase II activity in rat liver nuclear extracts. Here we demonstrate that in the presence or absence of Ca2+, calmodulin did not significantly alter either in vitro casein kinase II activity or autophosphorylation of its beta-subunit. In contrast, Ca2+ inhibited in a dose-dependent manner both casein kinase II activity and casein kinase II-catalysed calmodulin phosphorylation. These data indicate that calmodulin does not directly inhibit casein kinase II activity, but casein kinase II is sensitive to physiologic Ca2+ concentrations.

Inhibition of Euglena gracilis and wheat germ zinc RNA polymerases II by 1,10-phenanthroline acting as a chelating agent.

Copper complexes of 1,10-phenanthroline (OP-Cu) hydrolyze DNA [D'Aurora, V., Stern, A. M., & Sigman, D. S. (1978) Biochem. Biophys. Res. Commun. 80, 1025-1032; Marshall Pope, L., Reich, K. A., Graham, D. R., & Sigman, D. S. (1982) J. Biol. Chem. 257, 12121-12128]. This reaction has been studied to determine whether the 1,10-phenanthroline (OP) inhibition of the activity of RNA and DNA polymerases is the result of template hydrolysis or the chelation of a metal associated with and essential to the function of these enzymes. Addition of 4',6-diamino-2-phenylindole dihydrochloride (DAPI) to DNA generates a fluorescence signal with a linear increase of the intensity over a broad range of DNA concentrations from 0 to 100 micrograms/mL. The progress of hydrolysis of DNA by DNase I or OP (2 mM) is monitored by the time-dependent decrease in DAPI-induced fluorescence. In the presence of OP, the rate of hydrolysis increases as the Cu2+ concentration in the reaction mixture rises from 10(-8) to 10(-5) M. The rate differs for each nucleic acid template used; hydrolysis of poly(dA-dT) greater than denatured DNA greater than double-stranded DNA. However, millimolar amounts of OP do not hydrolyze the template even in the presence of Cu2+ (10(-6) M) when DNA is complexed with either Escherichia coli DNA polymerase I or Euglena gracilis or wheat germ RNA polymerase II. Under the same conditions, OP inhibits the activity of both varieties of RNA polymerase II with pKi's of 3.4 and 3.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Zinc deficiency and the Euglena gracilis chromatin: formation of an alpha-amanitin-resistant RNA polymerase II.

Both the single DNA-dependent RNA polymerase found in zinc-deficient (-Zn) Euglena gracilis and the RNA polymerase III from zinc-sufficient (+Zn) cells have been isolated by methods previously used to purify polymerases I and II [Falchuk, K. H., Mazus, B., Ulpino, L., & Vallee, B. L. (1976) Biochemistry 15, 4468; Falchuk, K. H., Mazus, B., Ulpino, L., & Vallee, B. L. (1977) Biochem. Biophys. Res. Commun. 74, 1206]. Like class II polymerases, the enzyme from -Zn organisms elutes from DNA-cellulose and phosphocellulose with 0.6 M NaCl and 0.35 M NH4Cl, respectively. It is inhibited by 8-hydroxyquinoline, 8-hydroxyquinoline-5-sulfonic acid, alpha,alpha'-bipyridyl, dipicolinic acid, and 1,10-phenanthroline (OP); 4,7-phenanthroline, the nonchelating analogue, does not inhibit. The pKI(OP) of this enzyme is identical with that of polymerase II but distinct from those of polymerases I and III. Elemental analysis confirms that zinc is the functional metal while copper, manganese, iron, and magnesium are absent. However, the -Zn enzyme is at least 4 orders of magnitude more resistant to alpha-amanitin (alpha-A) than the class II polymerase. Further, its response to alpha-A is unlike that of either polymerase I or polymerase III. Thus, -Zn cells contain a single, alpha-amanitin-resistant (alpha-Ar) RNA polymerase, whose behavior otherwise resembles that of the alpha-amanitin-sensitive polymerase II.

Histone formation, gene expression, and zinc deficiency in Euglena gracilis.

Histones, and other basic proteins, have been isolated from zinc-sufficient (+Zn) Euglena gracilis by standard chromatographic methods. These cells contain 2.46 micrograms of histones and 1.96 micrograms of DNA per 10(6) organisms. Each of the histones, H1, H3, H2A, H2B, and H4, is present in both log- and stationary-phase +Zn cells and has been characterized according to its electrophoretic mobility and molecular weight. H1 has been further identified on the basis of its amino acid composition and its cross-reactivity with calf thymus histone H1 antibodies. Similarly, H3 has been recognized as well by its specific reaction with an H3 antibody. In contrast, log-phase zinc-deficient (-Zn) cells contain H1 and H3 while H2A, H2B, and H4 are absent. All of the histones vanish in stationary-phase-Zn organisms. The DNA content increases as the -Zn cells progress from log to stationary phase, reaching a value of 4.40 micrograms/10(6) cells, double that of comparable stationary-phase +Zn organisms. A 2000-3000-dalton polypeptide whose electrophoretic behavior differs from that of the known histones constitutes over 90% of the total basic proteins of -Zn cells. On addition of zinc to stationary -Zn cells, cell division resumes, and all the histones and other basic proteins reappear. Together with previous results, the data demonstrate that zinc significantly affects the metabolism of all major chromatin components, i.e., the RNA polymerases, DNA, and histones of E. gracilis [Vallee, B.L., & Falchuk, K.H. (1981) Philos. Trans. R. Soc. London, Ser. B 294, 185-197]. The implications of these effects of zinc on chromatin structure and function are discussed.

Phosphorylation in vitro and in vivo of the wheat embryo RNA polymerase II.

One of the large subunits (220 000 daltons) of the wheat embryo RNA polymerase II was demonstrated to undergo phosphorylation with [gamma-32P]ATP in a reaction catalysed by a homologous protein kinase preparation. The same subunit was also observed to be phosphorylated in vivo, at the onset of germination. The phosphorylation resulted in a moderate increase of the RNA polymerase activity.

Euglena gracilis DNA dependent RNA polymerase II: a zinc metalloenzyme.

Zinc is essential for cellular proliferation. Zinc deficiency of Euglena gracilis results in arrest of cell division and deranges nucleic acid and protein metabolism pointing to a decisive role of zinc in transcription and translation. We have, therefore, investigated the role of zinc in the function of the DNA-dependent RNA polymerases of this organism. Two RNA polymerases from zinc sufficient organisms were purified first by affinity chromatography on a DNA cellulose column and subsequently separated on diethylaminoethyl (DEAE)-Sephadex A-25. The two fractions were characterized as polymerase I and II by their elution pattern from DEAE-Sephadex and sensitivity to alpha-amanitin. RNA polymerase II has a provisional molecular weight of 700 000 and contains an average of 2.2 g=atoms of zinc per mol of enzyme, but not Mn, Cu, or Fe, as measured by microwave emission spectroscopy. Chelating agents, such as 1,10-phenanthroline, 8-hydroxyquinoline, 8-hydroxyquinoline-5-sulfonic acid, and lomofungin, inhibit activity. In contrast, the nonchelating analogues, 1,7-and 4,7-phenanthroline, do not affect activity. Inhibition by 1,10-phenanthroline is instantaneous and fully reversible by dilution. 1,10-Phenanthroline also inhibits RNA polymerase I, suggesting a role of zinc in its function. The demonstration that RNA polymerase II is a zinc enzyme indicates the involvement of zinc in eukaryotic RNA synthesis and serves as a further basis for the definition of the role of this element in eukaryotic cell growth, division, and differentiation.

RNA polymerases I and II in germinating wheat embryo.

1. The RNA polymerase I was practically absent in the resting embryos and appeared several hours after the beginning of imbibition, whereas the level of polymerase II was high in the resting embryos and did not increase significantly during the imbibition phase. 2. Incorporation in vivo of [14C]valine into polymerase I and II indicated that the synthesis of RNA polymerase I is initiated in germinating embryos much earlier than that of RNA polymerase II. 3. It is suggested that RNA polymerase II is stored in resting wheat embryos to support mRNA synthesis at the onset of germination, whereas the RNA polymerase I activity appears at a further stage of germination.