Differential effect of neomycin on DNA dependent -DNA and RNA synthesis in vitro

Neomycin inhibits $\text{in vitro}$ DNA dependent DNA and RNA synthesis catalyzed by DNA polymerase I and RNA polymerase from $\text{E. coli}$. The effect of the antibiotic is more pronounced towards DNA synthesis. The inhibition of DNA synthesis is competitive with template DNA, does not reverse with excess deoxynucleoside triphosphate, Mg²⁺ or enzyme $\text{E. coli}$ DNA polymerase I. Neomycin does not reduce the number of potential 3′ -OH end or primer. It seems to shorten the size of the newly formed polynucleotide.     

Studies on invitro DNA synthesis: II. Isolation of a protein which stimulates deoxynucleotide incorporation catalyzed by DNA polymerases of E.coli

Purified RNA polymerase, DNA polymerase III and unwinding protein of $\text{Escherichia}\text{coli}$ catalyze limited rifampicin sensitive fd or ØX 174 DNA-dependent DNA synthesis. A protein has been partially purified from $\text{E.}\text{coli}$ which stimulates rifampicin sensitive dXMP incorporation in this system 20 to 30 fold. This protein also stimulates DNA synthesis catalyzed by DNA polymerases I and II; the stimulation occurs in reactions primed with natural and synthetic DNAs as well as RNA-DNA hybrids. The protein is not a product of the known dna genes. In contrast to the above system of purified enzymes, rifampicin sensitive dXMP incorporation in crude extracts of $\text{E.}\text{coli}$ is specifically dependent on fd but not ØX 174 DNA. An additional factor has been isolated from extracts of $\text{E.}\text{coli}$ which restores specificity to the purified rifampicin sensitive system by preventing ØX 174 DNA from serving as a template.     

Further characterization of a ribonucleotide-polymerizing enzyme from Histoplasma capsulatum. II. Possible role in cellular metabolism

An enzyme, ribonucleotide polymerase, isolated from the yeast phase of a fungus, $\text{Histoplasma capsulatum}$ has been found to stimulate the incorporation of dTMP in the reaction catalysed by DNA polymerase from $\text{H. capsulatum}$ and $\text{E. coli}$. The stimulation is dependent on the amount of ribonucleotide polymerase added. The data indicate that protein-protein interaction is responsible for the increase in DNA synthesis. It is suggested that ribonucleotide polymerase may be involved in supplying short RNA primers for DNA polymerase.     

1,10-Phenanthroline-cuprous ion complex, a potent inhibitor of DNA and RNA polymerases.

The inhibition by 1,10-phenanthroline of $\text{E. coli}$ DNA polymerase I has recently been attributed to the formation in the assay mixtures of a unique and effective inhibitor, the 2:1 1,10-phenanthroline-cuprous ion complex (1). We have now found that this coordination complex is also an effective inhibitor of $\text{E. coli}$ DNA dependent RNA polymerase, Micrococcus luteus DNA dependent DNA polymerase, and T-4 DNA dependent DNA polymerase. This conclusion is based either on the requirement of a thiol for 1,10-phenanthroline inhibition or on the reversal of 1,10-phenanthroline inhibition by the non-inhibitory cuprous ion specific chelating agent 2,9-dimethyl-1,10-phenanthroline. 2,2′,2″-Terpyridine is also very effective at relieving 1,10-phenanthroline inhibition. The reversal of 1,10-phenanthroline inhibition should be attempted before it is claimed that 1,10-phenanthroline inhibits any polymerases by coordinating a zinc ion at the active site.     

A new eukaryotic RNA polymerase factor: a factor from chicken myeloblastosis cells which stimulates transcription of denatured DNA

A heat-stable protein factor, capable of stimulating RNA synthesis by nuclear RNA polymerase II, was found in isolated nuclei of chicken myeloblastosis cells. It is adsorbed to a DEAE-Sephadex column used for RNA polymerase purification and then is eluted with 0.1 M ammonium sulfate. This factor appears to differ from previously reported eukaryotic RNA polymerase factors in its property of stimulating the activity of denatured (or single-stranded) DNA template. When heated, this factor contains no detectable endonuclease or exonuclease activity. The degree of stimulation is greater with chicken myeloblastosis RNA polymerase IIb than IIa and is most efficient when homologous DNA is used as template. This factor causes no stimulation of $\text{E. coli}$ RNA polymerase.     

Pyridoxal 5' phosphate: a selective inhibitor of oncornaviral DNA polymerases.

Pyridoxal 5′ phosphate at concentrations < 0.5 mM inhibits $\text{polymerization}$ of deoxynucleoside triphosphate catalysed by variety of DNA polymerases isolated from type C RNA tumor viruses, as well as $\text{E.}\text{coli}$, but $\text{does}\text{not}$ affect the polymerase associated RNase H activity. Both phosphate and aldehyde groups of pyridoxal phosphate are essential for the inhibition which appears to be mediated through the reversible Schiff base.     

Translation of AKR-murine leukemia viral RNA in an E. coli cell-free system

High molecular weight RNA isolated from the oncogenic type C murine leukemia virus, AKR-MuLV, stimulates the incorporation of radioactive amino acids into protein in an $\text{E. coli}$ cell-free system. Analysis of the translational products by SDS polyacrylamide gel electrophoresis demonstrated the synthesis of at least three proteins corresponding in molecular weight to several authentic viral proteins. Positive immunoprecipitation tests also confirm the translational product as AKR-MuLV related. Although at least 18 proteins were found on analysis of disrupted murine leukemia virions, only three were synthesized $\text{in vitro}$ in response to AKR-MuLV RNA in the $\text{E. coli}$ cell-free system.     

Evidence for two RNA polymerases in Arthrobacter, a morphogenetic bacterium

Two DNA-dependent RNA polymerases have been isolated from $\text{Arthrobacter crystallopoietes}$, a bacterium with a distinct morphological life cycle. The two enzymes are different with respect to chromatographic and electrophoretic behavior, divalent cation requirement, and template activity with different DNA species. One appears to be similar in its properties and structure to $\text{E. coli}$ polymerase, the other is different, but the nature of the difference is not yet clear. The possible relationship of the two enzymes in a regulatory role in the life cycle has yet to be investigated.     

Localization of the structural gene for the ' subunit of RNA polymerase in Escherichia coli

A minicell-producing strain of $\text{E.}$$\text{coli}$ carrying an F′ factor, KLF10-1, forms minicells that contain plasmid but not chromosomal DNA. These minicells were found to synthesize two polypeptides corresponding precisely to the β and β′ subunits of RNA polymerase in SDS-polyacrylamide gel electrophoresis. In contrast, minicells obtained from an isogenic strain carrying F13-1 do not synthesize these proteins under similar conditions. These results indicate that the structural genes for the β′ as well as β subunits of the polymerase are located on the chromosomal segment (78 to 81 min on the standard genetic map of $\text{E.}$$\text{coli}$) carried by KLF10-1.     

The effect of ionising radiation and chemical methylation upon the activity and accuracy of E. COLI DNA polymerase I

The activity of $\text{E. coli}$ DNA polymerase I decreases on treatment with γ-rays, methylnitrosourea or dimethyl sulphate. In the case of the first two agents the decrease in activity is accompanied by a decrease in the accuracy of the enzyme in an $\text{in vitro}$ assay. There is no detectable change in the ratio of DNA polymerase activity to 3′→5′ exonuclease activity on treatment.     

Electron microscopic studies of the binding of Escherichia coli RNA polymerase to DNA: II. Formation of multiple promoter-like complexes at non-promoter sites

The interactions between Escherichia coli RNA polymerase holoenzyme and a 3800 base-pair restriction fragment of bacteriophage T7 DNA (Mbo-IC) have been examined by electron microscopy. In addition to exhibiting weak, non-specific interactions (K_a ～ 10⁴ M^?1), RNA polymerase is able to form up to 15 to 20 relatively stable complexes with this template (K_a est 10⁹ M^?1). Only one of these complexes is formed at the T7 promoter E, that maps at 92.2 ± 1% on the conventionalgenome. The remaining complexes seem to be situated non-randomly on this fragment and possibly involve interactions with specific DNA sequences. The association kinetics of formation have been examined and give rise to a second-order rate constant of ～ 10⁵ M^?1s^?1. Formation of these complexes is markedly reduced at low temperatures. Under standard binding conditions (50 mM-NaCl) the dissociation rate of these complexes is slow ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; ~\; 30\; </mtext><mtext>min</mtext>}}$), but increases rapidly with increasing salt concentration and at reduced temperatures. It is unaffected by the presence of heparin up to 5 μg/ml. Thus it appears that E. coli RNA polymerase can form complexes with promoter-like properties at many different sites on T7 DNA.     

Activation of the template activity of isolated rat liver nuclei for DNA synthesis and its inhibition by NAD

The template activity of isolated rat liver nuclei for DNA synthesis assayed with $\text{E.}$$\text{coli}$ DNA polymerase was found to be dependent upon the presence of Ca²⁺ or Mg²⁺ in the incubation medium. DNA was prepared from isolated nuclei subjected to conditions which activated the template and centrifuged in an alkaline sucrose gradient. The distribution profile showed that smaller fragments were formed, suggesting enhancement of endonucleolytic activity. When isolated nuclei were incubated with NAD to induce poly(adenosine diphosphate ribose) formation and were subjected to the activation conditions, the template for DNA synthesis remained unchanged. The distribution profile in an alkaline sucrose gradient of DNA prepared from these nuclei and control nuclei was identical. The present findings suggest that the template-activating system for DNA synthesis was blocked when isolated nuclei were treated with NAD $\text{in}$$\text{vitro}$.