Properties of a Bacillus subtilis strain lacking DNA polymerase I

We have isolated a mutant of Bacillussubtilis deficient in DNA polymerase I, denominated polA42, which shows a reduced ability to repair the damage to DNA by UV radiation, MMS and mitomycin C;the ability to perform recombination is not appreciably impaired.DEAE cellulose chromatography allows the separation of polymerases I and II from the parental strain;a simple procedure is also described which allows to separate rapidly the polymerases II and III of the mutant strain. The three separated polymerases have similar catalytic properties but they can be distinguished for their sensitivity to inhibitors: PCMB inhibits polymerases II and III but not polymerase I; HPUra inhibits only polymerase III. All three enzymes are unaffected by nalidixate. The DNA synthesis occurring in cells of the polA42 strain permeabilized with toluene is inhibited by nalidixate, whereas the synthesis occurring in polA⁺ toluenized cells is unaffected by the drug. The polA gene has been mapped by transduction and localized between the phe₁₂ and argA₃ genes.     

The effect of antimycin A on cytochromes b561, b566, and their relationship to ubiquinone and the iron-sulfer centers S-1 (+N-2) and S-3.

Three nuclear RNA polymerases and one poly(A) polymerase were isolated from the yeast, Saccharomyces cerevisiae. The ability of cordycepin triphosphate to inhibit each was determined. RNA polymerase II was significantly more sensitive to this compound than the other polymerases. RNA polymerase I was relatively insensitive, being inhibited less than 20% by 40 μm cordycepin triphosphate. The calculated apparent K_i values of RNA polymerases II and III and poly(A) polymerase were, respectively, 0.3, 3.0, and 4.6 μm. Inhibition was competitive with regard to ATP. These data do not support the idea that, in yeast, poly(A) addition to preformed RNA in vivo is the primary site of cordycepin action.     

The hyperthermophilic archaeon Pyrodictium occultum has two alpha-like DNA polymerases.

We cloned two genes encoding DNA polymerases from the hyperthermophilic archaeon Pyrodictium occultum. The deduced primary structures of the two gene products have several amino acid sequences which are conserved in the alpha-like (family B) DNA polymerases. Both genes were expressed in Escherichia coli, and highly purified gene products, DNA polymerases I and II (pol I and pol II), were biochemically characterized. Both DNA polymerase activities were heat stable, but only pol II was sensitive to aphidicolin. Both pol I and pol II have associated 5'-->3' and 3'-->5' exonuclease activities. In addition, these DNA polymerases have higher affinity to single-primed single-stranded DNA than to activated DNA; even their primer extension abilities by themselves were very weak. A comparison of the complete amino acid sequences of pol I and pol II with two alpha-like DNA polymerases from yeast cells showed that both pol I and pol II were more similar to yeast DNA polymerase III (ypol III) than to yeast DNA polymerase II (ypol II), in particular in the regions from exo II to exo III and from motif A to motif C. However, comparisons region by region of each polymerase showed that pol I was similar to ypol II and pol II was similar to ypol III from motif C to the C terminus. In contrast, pol I and pol II were similar to ypol III and ypol II, respectively, in the region from exo III to motif A. These findings suggest that both enzymes from P. occultum play a role in the replication of the genomic DNA of this organism and, furthermore, that the study of DNA replication in this thermophilic archaeon may lead to an understanding of the prototypical mechanism of eukaryotic DNA replication.     

A comparative study of nuclear and chloroplast DNA dependent RNA polymerases from wheat leaves

• 1.1. Three DNA dependent RNA polymerases have been purified from chromatin and chloroplast fractions of wheat leaves.
• 2.2. The purified enzymes were completely dependent on exogenous DNA after purification by glycerol gradient, DEAE-Sephadex and phosphocellulose chromatography.
• 3.3. The nuclear enzymes, I and II, showed a strong preference for denatured nuclear DNA, whereas the chloroplast enzyme preferred denatured chloroplast DNA.
• 4.4. The three enzymes require either Mg²⁺ or Mn²⁺ for activity.
• 5.5. α-amanitin specifically inhibited RNA polymerase II but has no effect on polymerase I and chloroplast polymerase.
• 6.6. Enzyme I is most active at very low ionic strength (0.10 mM KC1), whereas enzyme II and chloroplast enzyme show maximum activity at 150mM and 50 mM KC1 respectively.

     

DNA polymerases from bakers' yeast.

Two DNA polymerases are present in extracts of commercial bakers' yeast and wild type Saccharomyces cerevisiae grown aerobically to late log phase. Yeast DNA polymerase I and yeast DNA polymerase II can be separated by DEAE-cellulose, hydroxylapatite, and denatured DNA-cellulose chromatography from the postmitochondrial supernatants of yeast lysates. The yeast polymerases are both of high molecular weight (greater than 100,000) but are clearly separate species by the lack of immunological cross-reactivity. Analysis of associated enzyme activities and other reaction properties of yeast DNA polymerases provides additional evidence for distinguishing the two species. Enzyme I has no associated nuclease activity but does carry out pyrophosphate exchange and pyrophosphorolysis reactions, and has an associated 3'-exonuclease activity. Enzyme I does not degrade deoxynucleoside triphosphates and cannot utilize a mismatched template. Enzyme II does carry out a template-dependent deoxynucleoside triphosphate degradation reaction and can excise mismatched 3'-nucleotides from suitable template systems. Earlier studies have shown that both Enzyme I and Enzyme II are inhibited by N-ethylmaleimide. The yeast enzymes are not identical to any known eukaryotic or prokaryotic DNA polymerases. In general, Enzyme I appears to be most similar to eukaryotic DNA polymerase alpha and Ezyme II exhibits properties of prokaryotic DNA polymerases II and III.     

Separation and partial characterization of two ribonucleic Acid polymerases from pea seedlings

Two DNA-dependent RNA polymerases (ribonucleoside triphosphate:RNA nucleotidyl transferase, EC 2.7.7.6) have been isolated from pea (Pisum sativum) seedlings. The enzymes were solubilized by sonication in high salt buffer and were separated by chromatography on diethylaminoethyl cellulose using a linear salt gradient. Polymerase I eluted at 0.10 m (NH₄)₂SO₄, accounted for about 10% of the recovered activity and was completely insensitive to α-amanitin. Polymerase II eluted at 0.14 m (NH₄)₂SO₄, accounted for the remaining 90% of recovered activity and was strongly inhibited by α-amanitin. Both enzymes preferred denatured to native DNA as template, both showed an absolute requirement of divalent cation, and both were sensitive to the ionic strength of the assay medium. The developing pea seedling seems a promising system for studies of possible changes in relative activities and roles of multiple RNA polymerases during eukaryotic development.     

Peptidases of yeast and filamentous forms ofCandida albicans

A study of the peptidases of the yeast and filamentous forms ofCandida albicans H-317 was carried out. At least three distinct peptidase activities in cell extracts of the yeast and filamentous phases were separated by gel filtration column chromatography or native polyacrylamide gel electrophoresis and measured by an enzyme-coupled colorimetric assay. The first peak of hydrolyzing activity to elute from a Sephadex G-200 column was designated as peak I, an aminopeptidase(s) with broad substrate specificity, and was characterized as follows: It had a molecular weight of 550,000, pH optima 7.5 to 8.0, andK_m of 1 × 10⁻⁴M with trimethionine as substrate, and it was inhibited by EDTA. The activity of peak I showed stereospecificity since it hydrolyzedd-methionyl-l-methionyl-l-methionine andl-methionyl-l-methionyl-d-methionine but notl-methionyl-d-methionyl-l-methionine. The second peak to elute from the gel filtration column was designated peak II. Its molecular weight was estimated to be 120,000, it had a pH optimum of 7.0, and it was inhibited by EDTA. TheK_m with trimethionine as substrate for the yeast and filamentous phase peak II was 3.2 × 10⁻⁴ and 7.5 × 10⁻⁵M, respectively. In addition to peak I and peak II peptidase(s), a dipeptidase activity with a molecular weight of 150,000 was detected in both the yeast and filamentous phases. Thus, a multiplicity of peptidases was present in cell extracts ofC. albicans and no major differences were noted in the partially purified peptidases from either the yeast or filamentous phase.     

Chloroplast and mitochondrial DNA polymerases from cultured soybean cells

DNA polymerases were purified from chloroplasts and mitochondria of cultured Glycine max cells. The chloroplast enzyme exists in two forms which are indistinguishable from each other biochemically. All three organellar enzymes have an estimated molecular weight of 85,000 to 90,000 and prefer poly(rA)dT_12-18 over activated DNA as a template in vitro. Maximum activity of the chloroplast and mitochondrial DNA polymerases requires KCl and a reducing agent, and the enzymes are completely resistant to inhibitors of DNA polymerase α. Taken together, these properties classify the soybean organellar enzymes as DNA polymerases γ. A unique feature that distinguishes the plant enzymes from their animal counterparts is their resistance to dideoxyribonucleotides.     

DNA-dependent RNA-polymerases from Artemia embryos. characterization of polymerases I and II from nauplius larvae.

Partial purification and characterization of DNA-dependent RNA-polymerases from nauplius larvae of the brine shrimp, Artemia salina, are described. Fractionation of solubilized RNA-polymerases on columns of DEAE-cellulose yielded partially purified preparations of RNA polymerases I and II. The properties of these enzymes were found to be similar to properties of corresponding enzymes from other animal sources. A significant change in the relative amounts of polymerases I and II occurs between 36 and 72 hr of development. Polymerase activity obtained from 36-hr nauplii consisted of approximately equal amounts of polymerases I and II, whereas polymerase II accounted for more than 80% of the activity recovered from 72-hr nauplii. Total polymerase activity was lower at 72 than at 36 hr. The significance of these changes in relation to the decrease in RNA synthesis in vivo that occurs after 36 hr is discussed.     

Nuclear Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerases from Saccharomyces cerevisiae

Two deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerases (I, II) have been solubilized from isolated Saccharomyces cerevisiae nuclei. The enzymes can be separated by chromatography on O-diethylaminoethyl Sephadex. Both enzymes are active with high-molecular-weight nuclear yeast DNA, although RNA polymerase I has a higher affinity for polydeoxy-adenylic-thymidylic acid and RNA polymerase II for denatured DNA. RNA polymerase I is active only with manganese. alpha-Amanitin inhibits only the activity of RNA polymerase II.     

Inactivation of DNA Polymerases of Murine Leukaemia Viruses by Calcium Elenolate

THE monoterpene elenolic acid can be isolated, after mild acid hydrolysis, from aqueous extracts of the olive plant (Olea europa)^1,2. The hydrolysate yields crystalline calcium elenolate [(C₁₁H₁₃O₆)₂Ca] which is virucidal in vitro for a number of both DNA and RNA viruses³. It also reduces virus yields from hamsters infected with parainfluenza 3 virus⁴ and has minimal toxicity when administered to animals⁵. I report the inhibition of RNA-dependent DNA polymerases of murine leukaemia viruses by calcium elenolate.     

Phosphorylation of yeast DNA-dependent RNA polymerases in vivo and in vitro. Isolation of enzymes and identification of phosphorylated subunits.

Yeast DNA-dependent RNA polymerases I, II, and III are phosphorylated in vivo. Yeast cells were grown continuously in 32Pi and the RNA polymerases were isolated by a new procedure which allows the simultaneous purification of these enzymes from small quantities (35 to 60 g) of cells. Each of the RNA polymerases was phosphorylated. The following phosphorylated polymerase polypeptides were identified: polymerase I subunits of 185,000, 44,000, 36,000, 24,000, and 20,000 daltons; a polymerase II subunit of 24,000 daltons; and polymerase III subunits of 24,000 and 20,000 daltons. The incorporated 32P was acid-stable but base-labile. Phosphoserine and phosphothreonine were identified after partial acid hydrolysis of purified [32P]polymerase I. A yeast protein kinase that co-purifies with polymerase I during part of the isolation procedure was partially purified and characterized. This protein kinase phosphorylates the subunits of the purified polymerases that are phosphorylated in vivo and, in addition, a polymerase I subunit of 48,000 daltons and a polymerase II subunit of 33,500 daltons. Phosphorylation of the purified enzymes with this protein kinase had no substantial effect on polymerase activity in simple assays using native yeast DNA as a template. Preincubation of purified polymerase I with acid or alkaline phosphatase also had no detectable effect on polymerase activity.     

Purification and characterization of two extracellular polyhydroxyalkanoate depolymerases from Pseudomonas mendocina

Two polyhydroxyalkanoate depolymerases, PHAase I and PHAase II, were purified to homogeneity from the culture supernatant of an effective PHA-degrading bacterium, Pseudomonas mendocina DS04-T. The molecular masses of PHAase I and PHAase II were determined by SDS-PAGE as 59.4 and 33.8 kDa, respectively. Their optimum pH values were 8.5 and 8, respectively. Enzymatic activity was optimal at 50 °C. Both purified enzymes could degrade PHB, PHBV, and P(3HB-co-4HB). Addition of Na⁺ and K⁺ slightly increased the rate of PHAase II. EDTA significantly inhibited PHAase II but not PHAase I. Mercaptoethanol and H₂O₂ also inhibited the activities of both enzymes.     

DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene

Three nuclear DNA polymerases have been described in yeast: DNA polymerases I, II, and III. DNA polymerase I is encoded by the POL1 gene and is essential for DNA replication. Since the S. cerevisiae CDC2 gene has recently been shown to have DNA sequence similarity to the active site regions of other known DNA polymerases, but to nevertheless be different from DNA polymerase I, we examined cdc2 mutants for the presence of DNA polymerases II and III. DNA polymerase II was not affected by the cdc2 mutation. DNA polymerase III activity was significantly reduced in the cdc2-1 cell extracts. We conclude that the CDC2 gene encodes yeast DNA polymerase III and that DNA polymerase III, therefore, represents a second essential DNA polymerase in yeast.