A template independent, rifampicin sensitive poly (A).poly (U) synthesizing activity present in Bacillus subtilis.

A template independent poly (A)·poly (U) synthesizing activity has been isolated from $\text{Bacillus subtilis}$. This activity is eluted from a DNA-cellulose column along with DNA-dependent RNA polymerase. The column fractions which exhibit this activity contain RNA polymerase holoenzyme plus a polypeptide which is slightly larger than sigma factor; pure RNA polymerase holoenzyme did not synthesize poly (A)·poly (U). The activity was dependent on the presence of ATP, UTP, and Mn⁺⁺ (Mg⁺⁺ could not substitute), and was inhibited by rifampicin, streptolydigin, and Cibacron Blue. The incorporation of nucleotides was not linear with time, but appeared after a lag period. The results suggest that a modified form of DNA-dependent RNA polymerase analogous to $\text{Escherichia coli}$ holoenzyme II is catalyzing the synthesis of poly (A)·poly (U).     

Thyrotropin releasing hormone receptors in regulation of prolactin gene expression in GH cells

Calf thymus DNA polymerase β and mammalian type C retroviral DNA polymerases are strongly inhibited by low concentrations (1–2mM) of inorganic phosphate (Pi). A detailed analysis of this phenomenon revealed that Pi-mediated inhibition: a) requires the presence of Mn²⁺ (Mg²⁺ neither supports nor relieves this inhibition; b) is strongly affected by the stoichiometric relationship between Mn²⁺ and Pi concentrations; c) is competitive with respect to deoxynucleoside triphosphate (dNTP) concentration, and d) increasing the concentration of substrate or non-substrate dNTPs in reaction mixtures raised the concentration of Mn²⁺ at which significant inhibition by a fixed concentration of Pi was first seen. These findings suggested that Mn²⁺, dNTPs, and Pi may interact in Pi-mediated inhibition. Thin-layer chromatographic analysis revealed the formation of an Mn-dNTP-Pi complex, while Mg²⁺ did not participate in such complex formation. We propose that it is this tripartite complex which is responsible for the Pi-mediated inhibition of sensitive DNA polymerases.     

Characterization of an alpha-like DNA polymerase from Bombyx mori silkglands.

A soluble DNA polymerase has been purified near to homogeneity from Bombyx mori silkglands. The following characteristics were observed: high molecular weight (about 150 000 - 220 00); optimum pH about 8; inhibition by high salt concentrations, sulfhydryl-group blocking agents and polyamines; absence of nuclease activity; preference for magnesium as required divalent cation with all the efficient template-primers tested; and clear template-primer specificity, the purified enzyme being able to copy primed - polydeoxyribonucleotide templates [activated DNA, poly(dA).oligo(dT), poly(dA).oligo(rU)] but not polyribonucleotide chains [poly(rA).oligo(dT), poly(rA).oligo(rU)] in the presence of either Mg++ or MN++. Believed to represent the bulk of silkgland DNA polymerase activity, the purified soluble enzyme most resembles vertebrate DNA polymerases alpha when it is compared to other eukaryotic DNA polymerases as yet characterized.     

DNA polymerases β and λ as potential participants of TLS during genomic DNA replication on the lagging strand

The main strategy used by pro-and eukaryotic cells for replication of damaged DNA is translesion synthesis (TLS). Here, we investigate the TLS process catalyzed by DNA polymerases β and λ on DNA substrates using mono-or dinucleotide gaps opposite damage located in the template strand. An analog of a natural apurinic/apyrimidinic site, the 3-hydroxy-2-hydroxymetylthetrahydrofuran residue (THF), was used as damage. DNA was synthesized in the presence of either Mg²⁺ or Mn²⁺. DNA polymerases β and λ were able to catalyze DNA synthesis across THF only in the presence of Mn²⁺. Moreover, strand displacement synthesis was not observed. The primer was elongated by only one nucleotide. Another unusual aspect of the synthesis is that dTTP could not serve as a substrate in all cases. dATP was a preferential substrate for synthesis catalyzed by DNA polymerase β. As for DNA polymerase λ, dGMP was the only incorporated nucleotide out of four investigated. Modified on heterocyclic base photoreactive analogs of dCTP and dUTP showed substrate specificity for DNA polymerase β. In contrast, the dCTP analog modified on the exocyclic amino group was a substrate for DNA polymerase λ. We also observed that human replication protein A inhibited polymerase incorporation by both DNA polymerases β and λ on DNA templates containing damage.     

Three Dimensional Association of Double-Stranded Helices Are Produced in Conditions for Z-DNA Formation

Abstract

The Z form of alternating poly(dG-dC)·poly(dG-dC) can be induced when the concentration of NaCl, MgCl₂ or ethanol are increased. In order to obtain more information concerning this Z structure, the B?Z transition is analyzed on the same sample, both by UV spectrophotometry and electron microscopy. The procedures used in this work provide high resolution images with minimal alterations of the molecules. It is shown that at high vlaues of cations or ethanol, the polymer makes complex associations of numerous molecules stuck together parallelly. By decreasing the salt or ethanol concentrations, a progressive decondensation of the molecules is obtained. At low concentrations of Mg⁺⁺ (2.10^?2 M), alterations of the linear secondary structure of the molecules are observed, although the UV spectrum is of the B-type. In the presence of that low concentration of Mg⁺⁺, natural DNAs (øX174 and yeast mitochondrial DNA fragment inserted in pBR) exhibit structural modifications similar to those observed with the poly(dG-dC)·poly(dG-dC). These structures mainly consist in four-stranded hairpins and loops built up by the sticking of two segments of DNA. The correlation between these intertwining of short DNA segments and the presence of potentially Z-forming sequences is discussed.     

Nuclear DNA-dependent RNA polymerases of Ehrlich ascites tumor cells: two discrete -amanitin-sensitive forms

DNA-dependent RNA polymerase was solubilized from nuclei of ascites tumor cells by the standard techniques of ultrasonic treatment in 0.3 M ammonium sulfate, salt fractionation, and dialysis. Three discrete forms of RNA polymerase (I, II, III) were separated on DEAE-Sephadex A-25. Forms II and III were inhibited by α-amanitin, but no form was sensitive to rifampicin. Each form was more active with Mn⁺⁺ than with Mg⁺⁺ ions, more active with denatured than with native calf thymus DNA, and differed from the others with respect to optimal concentrations of (NH₄)₂SO₄, Mn⁺⁺ ions and DNA.     

Electronmicroscopy and Circular Dichroism of the Dynamics of the Formation and Dissolution of Supramolecular Forms of Z-DNA

Abstract

Previous electronmicroscopic studies had shown that N-acetylaminofluorene (AAF)- substituted poly(dG-dC)·poly(dG-dC) in the Z conformation, in lOmM Mg^{+ +}, condensed into periodically banded, branched structures. We now show that similar structures are seen when poly(dG-dC) ·poly(dG-dC) is converted to the Z conformation by heating to 60°C in ImM Mn⁺⁺ or to 65°C in the presence of 0.5mM Mn^{+ +}. We demonstrate that these banded structures form in solution, i.e. they are not artifacts of the preparative procedures used for electronmicroscopy, by crosslinking the Z conformers in solution with DL-diepoxybutane (DEB), and then restoring the solution to conditions that favor return to the B conformation. Circular dichroism (CD) and immunochemical studies showed that the Z conformation was maintained and the banded supramolecular structures were still seen by electronmicroscopy. Electronmicroscopy and CD were also used to follow the dissolution of the supramolecular structures by controlled scission of the crosslinks with the eventual return to the short double stranded molecules typical of the B conformers. During this process, supercoiled structures, both toroidal and interwound, were observed. The relationship of the toroids to the banded structure is discussed in the context of two possible structures for the condensed polynucleotide. We conclude that DNA, whether in the B or Z conformation, is extremely flexible in the presence of appropriate counter ions, and we present evidence that earlier estimates of their persistence lengths are too high. The inherent tendency to form condensed, highly organized structures is a property of DNA that could play an important role in its “packaging,” and in its functions, and might have been critical for the evolution and replication of early life forms.     

DNA Polymerases of Myeloma: Template and Primer Specificities of Two Enzymes

INTEREST in the viral RNA-dependent DNA polymerases^1,2 has led to an examination of mammalian DNA polymerases and to the characterization in certain systems of DNA polymerases which can transcribe ribopolymer chains of appropriate ribopolymer-oligodeoxyribopolymer hybrids^3,4. The nature of these enzymes and their function in cells are unclear. We have reported the separation, by chromatography on DEAE-cellulose, of the DNA polymerases of the murine myeloma tumour into two separate fractions which we have designated as fractions D-I and D-II (ref. 5). Each of these fractions contains an activity capable of utilizing activated DNA and of transcribing the ribopolymer strand of poly rA oligo dT. We describe here the separation of two DNA polymerase enzymes in fraction D-I.     

Acid phosphatase fractions from various growth zones of broad bean root revealed by disc electrophoresis

Fractions of acid phosphate (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) were studied in extracts of segments from three growth zones of broad bean roots by means of electrophoresis in acrylamide gel. The azocoupling reaction with α-naphtyl phosphate was used for detection. The phosphatase activity was investigated in the range of pH 3·6–7·2. Altogether nine fractions moving towards the anode were revealed. Some fractions differed slightly in their pH optimum. The presence of Mg⁺⁺ in the incubation medium resulted in the activation of two fractions, Mn⁺⁺ showed activation of three fractions and inhibition of the rest of the fractions; the presence of Zn⁺⁺ resulted in a slight inhibition of all fractions. Between electrophoreograms of extracts of segments from the division zone and electrophoreograms of extracts of segments from the enlargement zone and from the maturation zone considerable quantitative differences were found with one fraction; proportions of the other fractions were approximately identical in electrophoreograms of all three growth zones. The response to the presence of Mg⁺⁺, Mn⁺⁺ and Zn⁺⁺ in the incubation medium as well as the pH optima of the individual fractions were identical for all three growth zones.     

Chelation of divalent cations by lomofungin: role in inhibition of nucleic acid synthesis

Lomofungin inhibition of yeast growth and RNA synthesis is prevented by Cu⁺⁺ or Zn⁺⁺ ions which chelate with the antibiotic and prevent its uptake by the cells. EDTA potentiates the inhibition. Mg⁺⁺ ions do not protect $\text{in vivo}$ or against the inhibition of purified bacterial RNA and DNA polymerases. Lomofungin prevents formation of the RNA polymerase. DNA initiation complex, probably by chelation with the firmly bound Zn⁺⁺ of the enzyme.     

Cation dependence of guinea pig epidermal cell spreading

Summary To understand the earliest phases of epidermal cell spreading we have sought a defined in vitro system. We studied the divalent cation dependence of guinea pig epidermal cell spreading in media containing varying concentrations of cations. No spreading occurred in calcium-magnesium-free Dulbecco's modified Eagle's medium (CMF-DME) in the presence of cation-free fetal bovine serum; however, significant spreading occurred if the medium was supplemented with Mg⁺⁺ plus Ca⁺⁺ or Mg⁺⁺ alone. Supplementing with Ca⁺⁺ alone led to much less spreading. These cations in CMF-DME did not support spreading in the absence of serum or the presence of serum albumin. Assaying cell spreading in a simple salt solution consisting of NaCl, KCl, Tris buffer, pH 7.4 plus dialyzed serum and a series of divalent cation supplements (Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, Co⁺⁺, Zn⁺⁺, Ni⁺⁺), showed that only Mg⁺⁺ and Mn⁺⁺, and to a lesser extent, Ca⁺⁺, supported cell spreading. In contrast to Mg⁺⁺, however, Mn⁺⁺ could support spreading in the absence of whole serum if serum albumin were present. Although Mn⁺⁺ plus serum albumin supported more rapid spreading at lower cation concentrations than Mg⁺⁺ plus serum, equal concentrations of Ca⁺⁺ completely blocked the Mn⁺⁺ effect. In contrast to the increasing cell spreading, which occurred in Mg⁺⁺-containing medium with time, cell death occurred in Mn⁺⁺-containing medium by 24 h. Consonant with studies from other laboratories, human foreskin fibroblasts spread in Mn⁺⁺-containing salt solution in the absence of protein supplements. These experiments indicate for epidermal cell spreading that Mg⁺⁺ is the important cation in tissue culture media, that under proper cation conditions epidermal cells do not need a specific spreading protein (i.e. a protein that has been demonstrated to support cell spreading), that Mn⁺⁺ and Mg⁺⁺-induced spreading seem to represent different mechanisms, that fibroblastic and epidermal cells have different cation requirements for in vitro spreading, and that the crucial role cations play in cell spreading remains to be elucidated. This work was supported in part by Public Health Service grant CA34470-01 (KSS) awarded by the National Cancer Institute, Bethesda, Md.     

DNA polymerases in midgestation mouse embryo, trophoblast, and decidua

The DNA polymerases of midgestation mouse embryo, trophoblast, and decidua have been examined. A low molecular weight, nuclear. DNA-dependent polymerase (D-DNA polymerase) and a higher molecular weight cytoplasmic enzyme were found in all three cell types. A DNA polymerase which utilized the poly(A) strand of oligo(dT) · poly(A) as template (R-DNA polymerase) was also found in the three cell types. This enzyme was present both in the nucleus and the cytoplasm. All enzyme levels were highest in the rapidly dividing embryonic cells, substantially lower in the DNA replicating but nondividing trophoblast cells, and lowest in the nonreplicating, nondividing decidual cells. Our observations are consistent with the idea that the nuclear and cytoplasmic D-DNA polymerases are under coordinate control. The relationship of these enzymes to DNA synthesis in vivo is discussed.     

Solubilization and characterization of RNA polymerase from a higher plant

RNA polymerase has been solubilized from sugar beet chromatin. With calf thmus or sugar beet DNA as template enzyme activity was linear with respect to protein concentration and required the presence of all four nucleoside triphospahates, added DNA and divalent metal ions. The enzyme exhibited a sharp Mn²⁺ optimum of 1·25 mM and a Mg²⁺ optimum at 10mM. The Mn²⁺/Mg²⁺ activity ratio (activity at optimum concentrations) was 2·0 with an optimum salt concentration of 50 mM. Based on data including inhibition with α-amanitin (0·025 μg/ml), the majority of the total activity appeared to be RNA polymerase I. Subsequent fractionation by DEAE-Sephadex column chromatography resulted in one peak of activity eluted with 0·18 M (NH₄)₂SO₄.