DNA synthesis in vivo and in vitro in Escherichia coli irradiated with ultraviolet light

DNA synthesis was followed in vivo and in permeable Escherichia coli after ultraviolet light irradiation, irradiation and incubation in a growth medium containing chloramphenicol and in unirradiated cells. In vitro, replicative type DNA synthesis was partially restored after incubation of cells in medium containing chloramphenicol, but not in vivo. The DNA was pulse-labeled in permeable cells in the presence of deoxyribonucleoside triphosphates and ribonucleoside triphosphates. dCTP was replaced by 5-Hg-dCTP as a substrate for DNA synthesis. Hg-DNA was separated from cellular nucleic acids on thiol-agarose affinity columns. The 5' termini of newly synthesized DNA were analyzed after treatment with alkaline phosphatase and rephosphorylation with polynucleotide kinase and [gamma-32P]ATP. DNA synthesis in unirradiated permeable E. coli represents a replicative process dependent on ATP and inhibited by novobiocin. About 70% of the nascent DNA carried terminally labeled RNA moiety at its 5' end. In vitro DNA synthesis in irradiated cells was suppressed and hardly influenced by the presence of ATP or novobiocin. The 5'-RNA content of this cell population was less than 5%.     

DNA synthesis and repair in permeable cells of Micrococcus radiodurans.

Cells permeable to deoxyribonucleoside triphosphate were prepared from Micrococcus radiodurans, and DNA synthesis and rejoining of strand scissions induced by gamma-rays were investigated. DNA synthesis was stimulated by ATP at an optimal concentration of 1mM. This reaction requires four deoxyribonucleoside triphosphates and MgCl2. NAD inhibited the reaction, but no rejoining of primer DNA was observed. Even in the presence of NAD, DNA which was synthesized in the unirradiated permeable cells had a peak molecular weight of only 1.3 - 10(6). DNA synthesis was stimulated by irradiation of the permeable cells with gamma-rays, but this stimulatory effect was eliminated by the addition of NAD. Both primer and synthesized DNA in the irradiated permeable cells were rejoined in vitro in the presence of NAD and deoxyribonucleoside triphosphates, while those in the unirradiated permeable cells were not rejoined.     

Macromolecular synthesis in permeable liver cells

Macromolecular synthesis was studied in individual liver cells rendered permeable to macromolecules and charged molecules by treatment with toluene. Toluene-treated cells were compared to intact cells with regard to their ability to synthesize protein, RNA, and DNA. The permeable cells catalyzed the incorporation of amino acids into protein in a system which was sensitive to cycloheximide. Maximal incorporation required the addition of tRNA, ATP, GTP, an energy source and various cations. RNA synthesis also took place in these cells and was inhibited by actinomycin D. Maximal incorporation required all four ribonucleoside triphosphates, an energy-generating system, and Mn²⁺, K⁺, and F^?. The toluene-treated cells also were active for DNA synthesis when Ca²⁺ was present to induce endonucleolytic cleavage of the endogenous DNA. For maximal synthesis, all four deoxyribonucleoside triphosphates, ATP, K⁺, Mg²⁺, polyamines, and mercaptoethanol were required. These studies serve to emphasize the potential usefulness of toluene treatment for studying biosynthetic processes in mammalian cells.     

DNA Replication in Permeabilized Cells of Sea Urchin Embryos

For study of the regulation of DNA replication in sea urchin embryos during the early stages of development, an embryonic cell system that was permeable to exogenously supplied nucleotides was established. Embryos were permeabilized by incubating them in hypotonic buffer containing 0.3 M glucose. The permeabilized embryonic cells maintained their morphological integrity, and synthesized DNA when supplied with exogenous dNTPs.
DNA synthesis in these permeabilized embryonic cells required the presence of ATP and three other deoxyribonucleoside triphosphates in addition to labeled dTTP. DNA synthesis was almost completely inhibited by N-ethylmaleimide, and proceeded in a discontinuous fashion. Only cells permeabilized during the S phase could incorporate nucleoside triphosphates into DNA: cells permeabilized during other phases did not synthesize DNA. During a 60 min-incubation period, over 10% of the genomic DNA was replicated under the experimental conditions used.     

Nucleoside triphosphate dependence of repair replication in toluenized Escherichia coli

Ultraviolet irradiation of Escherichia coli stimulates non-conservative DNA synthesis in cells rendered permeable to nucleoside triphosphates by treatment with toluene. This synthesis, like semi-conservative replication, proceeds in the presence of millimolar concentrations of ATP. Unlike semi-conservative replication, the ultraviolet-stimulated DNA synthesis can proceed if other nucleoside triphosphates are substituted for ATP. The selective dependence of semi-conservative replication upon ATP has been used to study the repair mode of synthesis in the absence of the semi-conservative mode and to demonstrate the dependence of ultraviolet-stimulated synthesis upon the uvrA gene product. Studies with recB mutants show that the nucleoside triphosphate-dependent ultravioletstimulated DNA synthesis occurs in strains deficient in the RecBC deoxyribonuclease.     

Separation of DNA-dependent DNA polymerase activities in Micrococcus radiodurans.

DNA polymerase activities in Micrococcus radiodurans were separated into two fractions after purification more than 2000 fold. They differ in pH optimum and residual activities in the absence of a full deoxyribonucleoside triphosphates complement. NAD partly inhibited one of the activities. Both activities were eluted as a single peak on gel filtration and sedimented at the same rate on glycerol gradient centrifugation. Molecular weight 140000 was calculated from Stokes radius and sedimentation constant. Deoxyribonuclease activity was detected on one of the polymerase activities which preferentially degraded double-stranded DNA. Priming activity of nicked DNA was reduced by gamma-irradiation. These results have been related to the possible rolls in repair synthesis in vivo or DNA synthesis in permeable cells of M. radiodurans.     

Semi-in vitro Repair of DNA in Germinating Spores of Bacillus subtilis Irradiated with γ-Ray

DNA repair synthesis was studied in germinating spores of Bacillus subtilis made permeable to deoxyribonucleoside triphosphates by treatment with Brij 58. The synthesis is dependent on the presence of all four deoxyribonucleoside triphosphates, but does not require adenosine triphosphate. Repair synthesis in the γ-ray irradiated and Brij 58 treated germinating spores was observed in wild type strain 168Tt, but not in DNA polymerase I-deficient mutant strain D22. Furthermore, the single-strand breaks of DNA in the germinating spores of strain 168Tt induced by γ -ray irradiation were rejoined during postirradiation incubation in the presence of four deoxyribonucleoside triphosphates, nicotinamide adenine dinucleotide and magnesium ion. In the case of a mutant D22, the γ-ray induced DNA single-strand breaks were not rejoined.     

Bacteriophage T4-Directed DNA Synthesis in Toluene-Treated Cells

DNA synthesis has been studied in T4-infected Escherichia coli cells made permeable to nucleotides by treatment with toluene. The rate of incorporation of labeled deoxyribonucleoside triphosphates into DNA at various times after infection is proportional to the in vivo rate. This in vitro incorporation is dependent on all four deoxyribonucleoside triphosphates (5-hydroxymethyldeoxy-cytidine triphosphate can substitute for dCTP) and Mg(2+). It is stimulated by rATP, partially inhibited by pancreatic DNase, and abolished by N-ethylmalei-mide and 1-beta-d-arabinofuranosylcytosine triphosphate. T4 amber DO (DNA negative) and temperature-sensitive DO mutants under nonpermissive conditions of infection fail to induce DNA synthesis in vitro. The synthesizing activity is intracellular and the DNA product is exclusively T4 DNA. The in vitro synthesis proceeds in a discontinuous manner involving synthesis and subsequent joining of small DNA fragments (about 10S in alkaline sucrose gradients) into larger molecules predominantly one-half the length of mature T4 DNA. No restriction of C-containing or nonglucosylated HMC-containing T4 DNA product is observed in this system.     

Organellar DNA synthesis in permeabilized soybean cells

Summary Cultured cells of Glycine max (L.) Merr. v. Corsoy were permeabilized by treatment with L--lysophosphatidylcholine (LPC). The permeabilized cells were capable of uptake and incorporation of deoxynucleoside triphosphates into DNA. Incorporation of exogenous nucleotides into DNA was linear for at least 90 minutes and the initial rate of incorporation approached 50% of the theoretical in vivo rate of DNA synthesis. However, DNA synthesis in the permeabilized cells was unaffected by the potent DNA polymerase inhibitor, aphidicolin. Analysis of newly synthesized DNA by molecular hybridization revealed that only organellar DNA was synthesized by the permeabilized cells. The LPC treated cells were also permeable to a protein as large as DNase I. The permeabilized cells were capable of RNA and protein synthesis as indicated by incorporation of radiolabeled UTP and leucine, respectively, into acid-precipitable material.     

DNA polymerase in nucleoli isolated from Ehrlich ascites tumor cells

DNA replication was investigated in nucleoli isolated from Ehrlich ascites tumor cells. DNA synthesis was dependent on the presence of the four deoxynucleoside triphosphates and magnesium, but was reduced in the presence of ATP. The pH optimum for DNA replication was 8.5 to 9.0 N-Ethyl-maleimide reduced the reaction significantly. DNA synthesis occurred on nucleolar chromatin and was stimulated by treatment of the nucleoli with a small amount of DNase I. Addition of exogenous DNA to the reaction mixture significantly stimulated [³H]dTMP incorporation.     

Reversibly permeable hepatoma cells in culture

A brief treatment of H35 hepatoma cells with lysolecithin resulted in a cell population which is permeable to low-molecular weight charged molecules that cannot normally cross the plasma membrane. These include deoxynucleotide and nucleotide triphosphates, folyl and methotrexate polyglutamates, and trypan blue. As a result dTTP can be incorporated into the DNA of the permeable cells, providing the required nucleotides and deoxynucleotides are added to the medium. This result, combined with only a slight observed loss (20–25%) in total cell protein, lactate dehydrogenase (EC 1.1.1.27) activity and tyrosine aminotransferase (EC 2.6.1.5) activity, demonstrated that permeation of the cells does not extensively disrupt membrane integrity. Further support for this view comes from the fact that the permeable cells could seal when placed in enriched medium. The process of sealing was inhibited by cycloheximide and tunicamycin. The sealed cells, whose surfaces appeared identical to those of untreated cells by scanning electron microscopy, were fully capable of cell division when exposed to serum. Values for several other parameters, including dexamethasone-dependent tyrosine aminotransferase induction, thymidine incorporation into DNA, leucine incorporation into protein and folate coenzyme transport, supported the conclusion that sealed cells and untreated H35 cells have identical properties. Based on the characteristics of the permeable and sealed H35 cells, a discussion of the experimental potential of these preparations for studying macromolecular synthesis, investigating enzymes in situ and depleting cells of folate coenzymes is presented.     

DNA synthesis in permeabilized mouse L cells.

Mouse L cells are rendered permeable to nucleoside triphosphates by a cold shock with a near isotonic buffer. These cells retain their morphologic integrity and use exogenously supplied nucleotides and deoxynucleotides to synthesize RNA and DNA. The newly synthesized DNA is nuclear and is the product of semiconservative replication. Incorporation of deoxynucleotides into DNA by thymidine kinase-deficient cells were used to conform rigorously that the exogenously supplied deoxynucleotides were incorporated into DNA without intermediate processing through nucleosides. DNA synthesis requires the presence of Na+, ATP, all 4 deoxynucleotides, and Mg2+. The reaction is inhibited by N-ethylmaleimide, p-hydroxymercuribenzoate and actinomycin D. Hydroxy-urea and arabinosylcytosine do not inhibit the reaction whereas cytosine arabinoside triphosphate shows competitive inhibition with the deoxynucleotides. These findings indicate that the permeable cell system can be used for in situ evaluations of the replicative DNA polymerase using the endogenous DNA template.     

The ATP dependence of the incision and resynthesis steps of excision repair.

Escherichia coli made permeable by treatment with toluene can perform a mode of DNA synthesis that is stimulated by ultraviolet radiation and closely resembles the resynthesis step of excision repair. If ultraviolet-irradiated toulene-treated cells are incubated in an assay mixture with ATP but without the four deoxyribonucleoside triphosphates (dNTPs) or NAD, accumulations of single-strand breaks in the DNA are detected by alkaline sucrose gradient analysis. A second incubation with the dNTP'S and NAD but without ATP produces nonconservative DNA synthesis in strains with normal levels of DNA polymerase I. However, in PolA strains, ATP must be present during the second incubation in order to produce measurable amounts of ultraviolet-stimulated DNA synthesis. These results suggest that in strains deficient in DNA polymerase I there may be two ATP-dependent steps in this repair pathway, one required for incision and one associated with resynthesis.     

The mode of action of 1-beta-D-arabinofuranosylcytosine in inhibiting DNA repair; new evidence using a sensitive assay for repair DNA synthesis and ligation in permeable cells

Mammalian cells permeabilised by treatment with saponin are capable of UV excision repair. We have developed an assay system which permits measurement of the later stages of repair, i.e. repair synthesis and ligation. Incomplete repair sites are accumulated in UV-irradiated cells by incubating them with DNA synthesis inhibitors hydroxyurea and aphidicolin. On removal of the inhibitors at the time of permeabilisation, these incomplete sites, detected as DNA breaks, are rapidly ligated in a reaction that requires deoxyribonucleoside triphosphates and is blocked by aphidicolin. Thus ligation is possible only after a significant amount of DNA synthesis. We have used the assay to clarify the mode of inhibition of DNA repair by 1-beta-D-arabinofuranosylcytosine (ara C), another DNA polymerase inhibitor. It is well known that incomplete repair sites accumulated in whole cells with ara C are ligated at a slow rate, if at all. The hypothesis that ara C blocks or reduces further polymerisation after its incorporation into repair patches is disproved by our demonstration that, in permeable cells, the accumulated DNA breaks are ligated very rapidly. The likely explanation of the action of ara C is that, once phosphorylated, it remains in the cell as ara CTP and continues to inhibit polymerisation through competition with dCTP; in permeable cells, it readily leaks out.     

Comparison of the bile salts and sodium dodecyl sulfate stress responses in Enterococcus faecalis.

The resistance to detergents and detergent-induced tolerance of a gastrointestinal organism, Enterococcus faecalis ATCC 19433, were examined. The most remarkable observation was the rapid response of cells in contact with bile salts and sodium dodecyl sulfate (SDS). The killing by high concentrations of detergents was nearly instantaneous. A 5-s adaptation with moderate sublethal concentrations of bile salts or SDS (0.08 or 0.01%, respectively) was sufficient to induce significant adaptation against homologous lethal conditions (0.3% bile salts or 0.017% SDS). However, resistance to a subsequent lethal challenge progressively increased further to a maximum reached after 30 min of adaptation. Furthermore, extremely strong cross-resistances were observed with bile salts- and SDS-adapted cells. However, no relationship seems to exist between levels of tolerance and de novo-synthesized proteins, since blockage of protein synthesis during adaptation had no effect on induction of resistance to bile salts and SDS. We conclude that this induced tolerance to detergent stress is independent of protein synthesis. Nevertheless, the stress-induced protein patterns of E. faecalis ATCC 19433 showed significant modifications. The rates of synthesis of 45 and 34 proteins were enhanced after treatments with bile salts and SDS, respectively. In spite of the overlap of 12 polypeptides, the protein profiles induced by the two detergents were different, suggesting that these detergents trigger different responses in E. faecalis. Therefore, bile salts cannot be substituted for SDS in biochemical detergent shock experiments with bacteria.     

Characterization of a CMP-sialic acid:lactosylceramide sialyltransferase activity in cultured hamster cells

Previous studies have shown a strong correlation between reduced levels of GM3 ganglioside and an increase in the oncogenic transformation of cultured cells. CMP-sialic acid:lactosylceramide sialyltransferase, which catalyzes GM3 synthesis, was characterized in cultured hamster fibroblasts (NIL-8) with respect to substrate binding, pH optimum, detergent requirements, metal ion requirements, activity during cell cycle phases and activity during cell growth phases. The apparent Km values for CMP-sialic acid and lactosylceramide were 0.16 and 0.11 mM, respectively. The enzyme required Mn2+ (15 mM) for maximal, but Mg2+ and Ca2+ were able to substitute to a lesser extent. Triton CF-54 (0.3%, w/v) compared to other nonionic detergents gave the greatest enzyme activation, while ionic detergents inhibited the enzyme. A broad pH optimum (4.5-8.0) was obtained, with maximum activity at pH 6.5 in cacodylate-HCl buffer. No buffer effects on enzyme activity were seen. Sialyltransferase activity was found to be highest in the M and G1 phases of the cell cycle and in the contact-inhibited phase of cell growth.     

Introduction of deoxyribonucleoside triphosphates into intact cells by electroporation

A simple method, employing high-voltage electric discharge (electroporation), was developed to introduce phosphorylated nucleosides into the cytoplasm of viable cells. HL-60 leukemia cells permeabilized by this technique remained viable and incorporated deoxyribonucleoside triphosphates into nuclear DNA. Furthermore, DNA synthesis was depressed for at least 24 h in HL-60 cells made permeable to 1-beta-D-arabinosylcytosine 5'-triphosphate by this methodology. Electroporation was found to be applicable to the permeabilization of a wide variety of cell lines in culture to nucleotides, suggesting that this methodology may be useful for the introduction into intact cells of a wide variety of molecules that are not normally transported effectively.