Intracellular Localization of DNA Polymerases in the Oocyte of Starfish, Asterina pectinifera

Oocytes of the starfish, Asterina pectinifera , were separated into karyoplasts and cytoplasts by centrifugation in a sucrose density gradient in the presence of cytochalasin B. More than 90% of the DNA polymerase activity of whole oocytes was recovered in the karyoplasts, and less than 10% in the cytoplasts. DNA polymerase α was specifically localized in karyoplasts. Of the DNA polymerase β activity of whole oocytes, 70–80% was found in the karyoplasts, and about 15% in the cytoplasts. The problem of whether DNA polymerases are localized within germinal vesicles in starfish oocytes is discussed.     

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.     

Synthesis of DNA in permeabilized cells of Kluyveromyces lactis.

Kluyveromyces lactis cells permeabilized with nystatin, though no longer viable, were able to incorporate 3H-dATP into DNA. Maximum rate of synthesis was obtained when all four deoxyribonucleoside triphosphates were present. For prolonged incorporation of 3H-dATP into DNA rATP or phosphoenolpyruvate were of absolute requirement. DNA synthesis was inhibited by p-chloromercuribenzoate, N-ethylmaleimide, nalidixate, ethidium bromide and distamycin A. The density of DNA synthesized in permeabilized cells grown on non-fermentable and fermentable carbon sources was analyzed on CsCl gradients in the presence or absence of distamycin A. The DNA synthesized by permeabilized cells previously grown on glycerol was essentially mitochondrial DNA; nuclear DNA (30% of total) was also synthesized by cells previously grown on glucose.     

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.     

Programmed macromolecular synthesis in regenerating karyoplasts.

Conditions for the preparation, purification, and maintenance of karyoplasts which could regenerate to reform whole viable cells were defined. Results of biochemical analyses of such karyoplasts at various times during regeneration indicated that a reproducible biosynthetic program was followed. Thus, an examination of the polypeptides made during regeneration by two-dimensional gel electrophoresis showed that the pattern of radiolabeled polypeptides synthesized at each time studied was specific and was significantly different from that observed at other times during regeneration. Polypeptides associated with three major cellular fractions--nuclear, cytoskeletal-microtrabecular, and soluble--were among the most dramatically regulated molecules. Other polypeptides, such as the major components of microfilaments and intermediate filaments, were synthesized at relatively constant rates and were assembled into structures throughout regeneration. Likewise, microtubules appeared to be reformed throughout regeneration, even in the absence of identifiable centriole-associated organizing centers. Finally, analysis of DNA synthesis by autoradiography showed that, even when prepared from whole cells synchronized at the G1/S interface, karyoplasts could not begin making DNA until they had regenerated an almost complete complement of cytoplasm.     

DNA fragmentation in permeabilized cells and nuclei. The role of (Ca2+ + Mg2+)-dependent endodeoxyribonuclease.

Permeabilized mammalian cells and isolated nuclei were used to study various aspects of DNA replication and repair. The present paper describes a progressive fragmentation of parental DNA in human lymphoblastoid cells that were permeabilized with L-alpha-lysophosphatidylcholine or with saponin and incubated at 37 degrees C in a DNA-synthesis mixture. The formation of DNA single-strand breaks (measured by alkaline elution) was linear with the time of incubation and was temperature-dependent. It was prevented by deleting Mg2+ or both Mg2+ and Ca2+ from the incubation mixture, or by the addition of EDTA. It was increased by deleting the components necessary for DNA synthesis, and by substituting Mn2+ for Mg2+ and Ca2+. DNA strand breaks also accumulated in isolated nuclei incubated in a DNA synthesis mixture, but not when Mg2+ was omitted. These results suggest that DNA fragmentation in permeabilized cells and nuclei was due to an activation of (Ca2+ + Mg2+)-dependent endodeoxyribonucleases. The integrity of template DNA needs to be ascertained when the conditions for measuring DNA synthesis in permeabilized cells or in nuclei are formulated.     

Identification of mammalian DNA repair factors using a reconstituted subcellular system : Partial characterization and subcellular location of a DNA repair-stimulating protein in hamster cells

By reconstituting lysolecithin-permeabilized hamster cells with endogenous proteins, a protein(s) which stimulated bleomycin-induced DNA repair synthesis was identified. The repair protein was inactivated by proteinase K and had an apparent molecular weight of 12000–15000 D. The following enzymatic activities were not detected in the partially purified DNA repair protein: general endonuclease, apurinic endonuclease, exonuclease, DNA polymerase or DNA polymerase β-stimulating activity. The subcellular location of the DNA repair-stimulating activity was investigated by cytochalasin B enucleation; approx. 80% of the activity was associated with karyoplasts, suggesting a nuclear location. Neither the activity nor subcellular location of the repair protein fluctuated appreciably during the cell cycle, consistent with a physiological role in DNA repair. Although the function of the DNA repair protein is not yet known, this approach should be useful in identifying and characterizing mammalian DNA repair proteins.     

Intracellular Localization and Sedimentation Coefficient of DNA Ligase in Oocytes of the Starfish, Asterina pectinifera

When immature oocytes of Asterina pectinifera were separated into karyoplasts (nuclear fragments) and cytoplasts (anuclear fragments) by cytochalasin B treatment and centrifugation in a sucrose gradient, almost all their DNA ligase activity was recovered in the karyoplasts. Thus, DNA ligase seems to be localized in the germinal vesicles or their vicinity in starfish oocytes. No ontogenic change in the activity of DNA ligase per oocyte, egg or embryo, or in its sedimentation coefficient (4.1 S) was observed during oocyte maturation, fertilization, or early development.     

Persistence of Interferon Action in Mouse Cells Permeabilized with Lysolecithin

DNA synthesis, as measured by incorporation of [(3)H]TTP, was inhibited in Swiss mouse 3T3 cells treated with interferon and subsequently permeabilized with lysolecithin. The degree of inhibition observed was similar in intact or permeabilized cells. The interferon-induced antiviral state was retained in permeabilized cells.     

Novobiocin—A specific inhibitor of semiconservative DNA replication in permeabilized Escherichia coli cells

The effect of novobiocin on macromolecule synthesis was investigated in Escherichia coli cells permeabilized by treatment with toluene or 2 m-sucrose. It was found that (1) semiconservative DNA replication is strongly and immediately inhibited and (2) ATP-independent DNA repair as well as RNA and protein synthesis are not affected.     

Effect of anti-cruciform DNA monoclonal antibodies on DNA replication.

To study the possible involvement of DNA cruciforms in the initiation of DNA replication, we used two monoclonal antibodies, 2D3 and 4B4, with anti-cruciform DNA specificity. Synchronized CV-1 cells were released into S phase for hourly intervals up to 6 h and permeabilized in the presence of monoclonal antibodies, under conditions that allow limited DNA replication. Exposure of the permeabilized cells to 2D3 or 4B4 resulted in a 2- to 6-fold enhancement of incorporation of labeled precursor nucleotide over the 6 h period. Approximately 50% of the enhanced synthesis was sensitive to aphidicolin, and the enhancing effect of 2D3 was abolished by absorption with immunobead anti-mouse immunoglobulin. Dot-blot hybridization analyses of DNA isolated from anti-cruciform antibody treatment groups showed a similar 2- to 11-fold increase in the relative copy number of low copy probes. In contrast, exposure of the permeabilized cells to a monoclonal antibody directed against Z-DNA and B-DNA had no significant effect on DNA synthesis. The results suggest that cruciforms are present in replicating DNA and that they are recognized and stabilized by the monoclonal antibodies.     

Excision repair of UV damage in human fibroblasts reversibly permeabilized by lysolecithin

We have examined nucleotide excision repair synthesis in confluent human diploid fibroblasts permeabilized with lysolecithin. Following a UV dose of 12 J/m2, maximal incorporation of [alpha 35S]dNTPs occurred at a lysolecithin concentration (approximately 80 micrograms/ml) where slightly more than 90% of the cells were initially permeable to trypan blue. However, autoradiography of cells, permeabilized at this lysolecithin concentration, demonstrated that only about 20% of the total cell population incorporated significant levels of 35S into DNA. This result presumably reflected the fact that approximately 20% of the total cell population remained permeable for much longer periods of time (up to 2 h) than the remaining cell population (less than 20 min). The incorporation of dNTPs by UV-irradiated, permeabilized cells appeared to be bona fide excision repair synthesis since: (1) Incorporation was completely absent in unirradiated, permeabilized cells and in irradiated, permeabilized repair-deficient cells. (2) Nucleotides incorporated in the presence of BrdUTP were associated with normal density DNA. (3) The apparent Km for all 4 dNTPs was 50-100 nM, in agreement with past reports on human fibroblasts irreversibly permeabilized by cell lysis. (4) DNA associated with the newly incorporated dNTPs underwent ligation and rearrangements in chromatin structure analogous to what is observed in intact human cells. Repair incorporation of dNTPs was rapid and linear during the first 2 h after UV irradiation and permeabilization. After this time, incorporation ceased or continued at a much slower rate. Cell viability experiments and autoradiography demonstrated that the cells permeabilized to [3H]dNTPs were capable of carrying out DNA replication and cell division. Thus, confluent human diploid fibroblasts can be reversibly permeabilized to labeled dNTPs by lysolecithin for the study of excision repair following physiologic doses of UV radiation. However, under these conditions, only a fraction of the cells remain permeable for an extended period of time.     

Inhibition of DNA synthesis in permeabilized L cells by novobiocin

Novobiocin was equipotent in inhibiting DNA and RNA synthesis in cultured mouse L cells. It also suppressed in vitro DNA and RNA synthesis in permeabilized L cells and nuclei; 50 percent inhibition of DNA and RNA synthesis was obtained by 1 mM and 20 mM novobiocin, respectively. ATP antagonized the effect of novobiocin. Nalidixic acid had a weak inhibitory effect on in vitro DNA synthesis; 10 mM nalidixic acid showed 60 percent inhibition. ATP did not antagonize nalidixic acid. The inhibitory effect of novobiocin exceeded that of aphidicolin. These findings suggest a participation of a gyrase- and/or type II topoisomerase-like enzyme in the DNA replication machinery in L cells.     

Synthesis of phage 2C-DNA in permeabilized B. subtilis.

Summary Bacillus subtilis cells, infected with bacteriophage 2C and then permeabilized, (plasmolysis, protoplast conversion, or treatment with organic solvents) incorporate dATP into DNA through a polymerization reaction requiring the 4 deoxyribonucleosidetri-phosphates dATP, dGTP, dCTP and dTTP. While uracil is an in vivo precursor of phage 2C DNA (in which hydroxymethyluracil completely replaces thymine), neither uracil, or dUTP, nor dUMP are incorporated into viral DNA by phage-infected permeabilized cells. Although the amount of dTTP incorporated under these conditions is small, this compound greatly enhances the incorporation of dATP into viral DNA.Synthesis of 2C-DNA in permeabilized cells is discontinuous; however, the Okazaki fragments (2x10⁶ daltons) which accumulate under these conditions, show no tendency to join and to form full strands, as they do in intact host cells. Finally, density shift experiments suggest that, in addition to repair synthesis, semiconservative duplication takes place within the permeabilized cells.When phage-infected bacteria are permeabilized at different moments of the viral cycle, labeled precursors are mainly incorporated into cell DNA during the eclipse phase, and into viral DNA during the maturation phase. Moreover, viral DNA formation is prevented when cells are infected with virions previously irradiated with ultraviolet light.Since most metabolic pathways and gene regulation patterns are not altered by the permeabilization process, allowing the use of direct DNA precursors, the systems of virus-infected permeabilized cells prove exceptional tools for a study of virus-host relationship.     

Suppression of dexamethasone-stimulated DNA synthesis in an oncogene construct containing rat cell line by a DNA site-oriented ligand of poly-ADP-ribose polymerase: 6-amino-1,2-benzopyrone

The cellular inhibitory effects of 6-amino-1,2-benzopyrone (6-ABP), a DNA site-specific ligand of adenosine diphosphoribosyl transferase (ADPRT), were determined in a dexamethasone-sensitive EJ-ras gene construct containing cell line (14C cells). Dexamethasone in vitro transforms these cells to a tumorigenic phenotype and also stimulates cell replication. At a non-toxic concentrations (0.2 mM) 6-ABP treatment of intact cells for 4 days inhibits the dexamethasone-stimulated increment of cellular DNA content, depresses replicative DNA synthesis as assayed by thymidine incorporation to the level of cells that were not exposed to dexamethasone, and in permeabilized cells reduces the dexamethasone-stimulated increase of deoxyribonucleotide incorporation into DNA to the level of untreated cells. In situ pulse labeling of cells pretreated with 6-ABP indicated an inhibition of DNA synthesis at a stage prior to the formation of the 10-kb intermediate species. The drug had no direct effect on cellular DNA polymerases as tested in vitro, and the inhibition of DNA synthesis in permeabilized cells following drug treatment for 4 days was abolished by externally added DNA templates. Neither dexamethasone nor the drug influenced the cellular quantity of ADPRT molecules, tested immunochemically.     

The control of DNA replication in hybrids between neutrophils and fibroblasts

Abstract. DNA synthesis regulation in heterokaryons between mouse neutrophils and cultured cells of various proliferative potentials has been studied. The following features have been found. Both immortalized and non-immortalized cells can reactivate DNA synthesis in neutrophil nuclei. The reactivation ability of cultured cells increases after immortalization and is not changed by further transformation. Neutrophils inhibit the entry of cultured cell nuclei into S phase and have no effect on ongoing DNA synthesis. Malignant cells are much less sensitive to the inhibitory action of neutrophils than non-malignant ones. Non-malignant immortalized cells are as sensitive to this effect as non-immortalized cells. Neutrophil karyoplasts do not influence DNA synthesis in partner cultured cell nuclei. Cycloheximide pretreatment of neutrophils drastically diminishes their inhibitory effect.     

Subcellular localization of phosphatidylinositol synthesis

It is well-established that the endoplasmic reticulum is the major site of phosphatidylinositol (PtdIns) synthesis. The PtdIns synthetic ability of other organelles, such as plasma membrane and nucleus, remains controversial. In the present study, we re-examine this question by comparing PtdIns synthesis in isolated cytoplasts (enucleated cells) with that in corresponding karyoplasts (nuclei surrounded by plasma membrane but lacking most cytoplasmic components). We report that cytoplasts are competent to carry out both basal and stimulated PtdIns synthesis as well as polyphosphoinositide hydrolysis, while karyoplasts can neither synthesize PtdIns nor hydrolyze phosphoinositides in response to agonists. The karyoplasts are, however, capable of synthesizing phosphatidylcholine (PtdCho), as previously reported. From these data, we conclude that PtdIns synthesis is limited to cytoplasmic components, and cannot be sustained by either plasma membrane or nucleus under conditions that permit robust PtdCho synthesis.     

Does butylphenyl-deoxyguanosine triphosphate differentially inhibit DNA polymerase alpha and delta activities in permeabilized HeLa cells?

In eukaryotic cells, two enzymes, DNA polymerases alpha and delta, are thought to play major roles in DNA synthesis. I have used butylphenyl dGTP (BuPdGTP), a potent inhibitor of purified DNA polymerase alpha, to assess the relative activities of these enzymes in two permeabilized cell systems. In both instances BuPdGTP eliminated all of the activity which was sensitive to aphidicolin. However, no conditions were found where BuPdGTP preferentially inhibited the synthesis of Okazaki fragments--the presumed products of DNA polymerase alpha activity. This implies that DNA polymerase activities on the two sides of the replication fork are unable to operate independently, being just two elements of the integrated replication machinery that undertakes DNA synthesis in permeabilized cells.     

In situ nucleoprotein structure involving origin-proximal SV40 DNA control elements.

Nucleoprotein structures at the SV40 GC-box and adjacent AT-rich region have been probed by nucleases in permeabilized cells at nucleotide level resolution. The patterns of nuclease protection and hypersensitivity in these permeabilized cells that allow initiation of RNA and DNA synthesis are quite different from those observed in isolated nuclei that are inactive. Whereas simple DNA protection by factors is found in nuclei, the pattern in permeabilized cells includes very strong nuclease hypersensitive sites. Their arrangement suggests that the region exists as a higher order nucleoprotein complex in vivo, which is disturbed during the preparation of nuclei. The pattern is also found to be disturbed in permeabilized cells when T-antigen is inactivated by temperature-sensitive mutation. Since T-antigen origin binding sites and the GC-box region have been shown previously to interact functionally, the existence of a higher order structure involving both components provides a likely physical basis for the functional interaction of separate control elements.