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.     

Ruthenium red as a carrier of electrons between external NADH and cytochrome c in rat liver mitochondria.

We have determined that Co²⁺, Ni²⁺ or Zn²⁺ may substitute for Mg²⁺ during DNA synthesis with $\text{E.}\text{coli}$ DNA polymerase I, sea urchin nuclear DNA polymerase and the DNA polymerase from avian myeloblastosis virus (AMV). In addition, the frequency of non-complementary nucleotide incorporation using AMV DNA polymerase was increased using Co²⁺ or Mn²⁺ as the metal activator. These results suggest that the fidelity of DNA synthesis may be influenced by the metal activator used during catalysis.     

Inhibition of exonuclease V after infection of E. coli by bacteriophage T7

Exonuclease V ($\text{recBC}$ DNase) is inactivated in $\text{E. coli}$ between 4 and 7 min after infection by T7. This process requires protein sythesis. The inactivation does not occur when T7 is deficient for its RNA polymerase and thus does not express the genes involved in DNA replication and phage maturation. Some implications of this new function of T7 are discussed with respect to the processes of infection and DNA replication.     

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.     

E. gracilis RNA polymerase I: a zinc metalloenzyme.

$\text{E. gracilis}$ DNA dependent RNA polymerase I has been purified to homogeneity. α-amanitin, over the concentration range 0.05 to 200 μg/ml, does not affect its activity, consistent with its being classified as an RNA polymerase I. Based on a molecular weight of 624,000 daltons the enzyme contains 2.2 g atom of Zn but no Mn, Cu, Fe, as determined by microwave excitation emission spectrometry. Zinc is essential for activity since the chelating agent, 1,10-phenanthroline, inhibits enzymatic function but its non-chelating analogue, 4,7-phenanthroline is ineffective. Thus, like the RNA polymerase II, zinc is a catalytically essential component of $\text{E. gracilis}$ RNA polymerase I (1).     

Excision of thymine dimers by proteolytic and amber fragments of E. coli DNA polymerase I

Excision of thymine dimers from specifically incised ultraviolet irradiated DNA by $\text{E. coli}$ DNA polymerase I is stimulated by concurrent DNA synthesis. The 36,000 molecular-weight “small fragment” obtained by limited proteolysis of DNA polymerase I, which retains only the 5′ → 3′ exonuclease activity, also excises thymine dimers, but at one-tenth the rate of the intact enzyme. However, the rate of excision is increased by addition of the “large” 76,000-molecular weight fragment. With the further addition of the 4 deoxynucleoside triphosphates, permitting DNA synthesis to occur, excision approaches rates observed with the intact enzyme. The same result was obtained with a fragment of DNA polymerase I with 5′ → 3′ exonuclease activity that is present uniquely in polymerase I $\text{amber}$ mutants.     

Carbon monoxide and hydroxymercuribenzoate sensitivity of a fatty acid (omega-2) hydroxylase from Bacillus megaterium.

DNA-free minicells of $\text{Escherichia coli}$ will not allow growth of phage T-7, nor is RNA synthesis stimulated by phage infection. Thus, these miniature cells seem not to contain $\text{in vivo}$ RNA polymerase activity. However, DNA-dependent RNA polymerase activity can be unmasked in extracts with poly(dA-T) and Mn²⁺. This activity may represent a cytoplasmic pool of inactive RNA polymerase in normal cells.     

Evidence for template-specific sites in DNA polymerases

Using rabbit hemoglobin messenger RNA as template, $\text{E. coli}$ polymerase I produces poly (dT), poly (dA)·(dT) and antimessenger DNA products. Mild heating of the enzyme causes a differential loss in activity as indicated by three rates of inactivation for the three types of synthesis. Heat inactivation studies have also been carried out with DNA polymerases from oncogenic RNA viruses and mammalian sources using various homopolymer-oligomer pairs as primertemplates. In general, for any given enzyme these synthetic primer-templates reveal different extents of inactivation of the polymerase. These findings may be interpreted to suggest a) that the binding of DNA polymerase to various primer-templates produces conformational changes in the enzyme which are dependent on the type of template bound, or b) that many, if not all, DNA polymerases have different subsites for different templates.     

The template specificities of DNA polymerase in cell free lysates of Xenopus laevis eggs, larvae and immature ovaries

DNA polymerase activities in cell-free lysates of unfertilized eggs, larvae and immature ovaries of $\text{Xenopus}\text{laevis}$ were compared to purified $\text{E.}\text{coli}$ DNA polymerase I using several natural and synthetic templates. The templates were tested as the native and denatured forms of normal and DNase I treated molecules. Although the $\text{Xenopus}$ polymerases tended to prefer DNase I treated Xenopus DNA over the other templates tested, so did the $\text{E.}\text{coli}$ polymerase I. In general, the template preferences of the polymerases studied depended in complex ways on both the form and the species of origin of the template.     

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}$.     

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.     

Synthesis and properties of a new fluorescent analog of ATP: Adenosine-5′-triphosphoro-γ-1-(5-sulfonic acid) napthylamidate

An analog of ATP has been synthesized which contains the fluorophore, 1-aminonapthalene-5-sulfonate attached via a γ-phosphoamidate bond. This analog is strongly fluorescent (quantum yield = 0.63) with an emission maximum at 460 nm; the excited state lifetime is 20 nsec. It is a substrate for DNA-dependent RNA polymerase of $\text{E. coli}$ and wheat germ RNA polymerase II. It is also a substrate for $\text{E. coli}$ valyl t-RNA synthetase, venom phosphodiesterase, and potato apyrase. Cleavage of the α-β phosphoryl bond as a result of RNA synthesis or by venom phosphodiesterase produces a 15 nm red shift in the fluorescence emission spectrum. This property should make this nucleotide useful for studies of the mechanisms of enzymatic reactions involving cleavage of the α-β phosphoryl bond.     

Inhibition of RNA chain initiation in E. coli core RNA polymerase with 2-hydroxy-5-nitrobenzyl bromide

The modification of $\text{E. coli}$ core RNA polymerase with 2-hydroxy-5-nitrobenzyl bromide (Koshland's Reagent) resulted in the benzylation of 6 out of 13 cysteines, and 10 out of 20 tryptophans in the polymerase, and occurred with an 8% decrease in its [θ]₂₂₀. The modification resulted in a maximal inhibition of 60% of the RNA chains on both calf thymus and micrococcal DNA templates. γ-³²P-ATP studies showed the inhibition occurred at RNA chain initiation. This study raises the possibility that the modified core polymerase may synthesize specific RNA(s).