DNA synthesis in detergent-treated mouse ascites sarcoma cells.

Mouse ascites sarcoma cells (SR-C3H/He cells) were made permeable to nucleoside triphosphates by treatment with nonionic detergents in a nearly isotonic condition. The permeable cells synthesized DNA in the presence of the four deoxyribonucleoside triphosphates, ATP, Mg2+, and the proper ionic environment. The optimum detergent concentration for DNA synthesis was 0.015--0.020% with Triton X-100, 0.020% with Nonidet P-40, and about 0.0025% with Brij 58. Higher concentrations of detergents were rather inhibitory to DNA synthesis. DNA synthesis in Triton-permeabilized cells was thought to be replicative, and the activity in the optimum conditions was much higher than that measured in hypotonic permeable cells or in isolated nuclei. These studies show the potential usefulness of detergent treatment for examining DNA replication in mammalian cells in vitro.     

DNA replication in isolated HeLa cell nuclei

DNA replication was investigated in HeLa cell nuclei isolated from different phases of the cell cycle. DNA synthesis occurred only in S-phase nuclei and was dependent on the presence of the four deoxynucleoside triphosphates, Mg⁺⁺, ATP and S-phase cytoplasm. G₁-phase cytoplasm was unable to support such DNA synthesis. A purified preparation of calf thymus DNA polymerase, however, was able to replace S-phase cytoplasm in supporting ATP dependent DNA synthesis, which suggests that the S-phase cytoplasmic factor is a DNA polymerase. G₁-phase nuclei could under no conditions be stimulated to initiate DNA replication prematurely.     

Inhibition of DNA replication in Escherichia coli by dibromophenol and other uncouplers.

DNA replication in Escherichia coli is inhibited by uncouplers such as 2,4-dibromophenol and 3,3'4',5-tetrachlorosalicylanilide. Inhibition occurs in either aerobically or anaerobically growing cells or in cells made permeable by toluene. With anaerobically growing cells, inhibition by dibromophenol is reversible and occurs under conditions in which there is no change in pools of ATP or deoxynucleoside triphosphates. With toluenized cells, inhibition is not due to breakdown of deoxynucleoside triphosphates. The rates of protein and RNA synthesis are not inhibited either in vivo or in toluenized cells by concentrations of dibromophenol or tetrachlorosalicylanilide which inhibit replication. It is generally believed that uncouplers inhibit many other cellular processes by collapsing a proton gradient across a membrane. However, the relative effectiveness of eight uncouplers and related compounds to inhibit replication did not parallel their ability to transport protons into E. coli cells. Therefore, the inhibition by uncouplers does not suggest that replication depends on a chemiosmotic process. A possible explanation for the uncoupler sensitivity is provided by the finding that many of the purified enzymes tested, including DNA polymerases II and III, are inhibited by dibromophenol and tetrachlorosalicylanilide.     

Chromatin replication in isolated nuclei from bovine lymphocytes

DNA replication in isolated nuclei from Concanavalin A-stimulated and resting bovine lymphocytes has been studied. Nuclei from S phase lymphocytes incorporate 4–7 times more (³H)dTTP than nuclei from resting cells. The DNA synthesis was dependent on ATP, Mg²⁺ and all four deoxynucleoside triphosphates and was linear for about 60 min. The newly synthesized DNA is nuclear and DNase-sensitive and is the product of discontinuous and semiconservative replication. After limited digestion with micrococcal nuclease the $\text{in vitro}$ replicated DNA was found to occur in nucleosomes prior to joining of primary DNA pieces. Addition of a protein extract from replicating cells stimulated the DNA synthesizing capacity of nuclei from resting lymphocytes. A preliminary characterization of this extract is given.     

Synthesis of herpes simplex virus DNA in a soluble nuclear extract.

A soluble extract prepared from nuclei of HeLa cells infected with herpes simplex virus type 1 has been found to synthesize herpes DNA in a process comparable to that observed in intact nuclei. The incorporation of [³H]dTTP has an absolute requirement for Mg⁺⁺ and for the other three deoxyribonucleoside triphosphates, and is relatively independent of added ATP. The reaction product, although of relatively short chain length, bands in CsCl density gradients at the density of herpes DNA and is essentially free of labeled cell DNA. Incorporation of BrdUTP results in a density shift suggesting extensive replication of endogenous DNA sequences.     

2'-Deoxy-2'-azidocytidine, a new inhibitor of DNA replication in mammalian cells.

Cell growth is reversibly inhibited by the nucleoside analogue, 2'-deoxy-2'-azidocytidine and the inhibition is a result of interference with DNA replication. The 5'-diphosphate of the analogue was earlier shown to specifically inactivate the enzyme ribonucleotide reductase in vitro. However, measurements of the pools of deoxyribonucleoside triphosphates in cells incubated in azidocytidine showed only minor changes which appeared to result from and not to be the cause of the inhibition of DNA replication. The DNA synthesized in polyoma-infected cells after incubation in azidocytidine showed a sedimentation pattern quite different from that seen after inhibition of DNA synthesis with arabinosyl cytosine or hydroxyurea. Experiments with nuclei isolated from azidocytidine-inhibited, polyoma-infected cells indicated (a) that the number of replicating molecules is decreased during the inhibition and (b) that upon incubation of the nuclei there is a rapid synthesis of DNA occurring in a new class of DNA molecules which are at a very early state of replication. Neither the 5'-triphosphate of azidocytidine nor the nucleoside itself inhibit DNA synthesis in vitro in isolated nuclei from polyoma-infected cells and at present the nature of the DNA-synthesis-inhibiting compound acting in the cells after addition of azidocytidine is unknown. Taken together the results suggest that azidocytidine inhibits DNA synthesis at an early stage, possible by blocking the initiation of DNA synthesis at the origin or by interfering with the elongation of newly initiated DNA molecules.     

Nucleotide requirements for DNA replication of SV40 chromatin in digitonin-treated and saponin-treated permeable cells

Permeable cell systems have been developed by treatment with digitonin or saponin for studying in vitro DNA replication of chromatin. DNA replication of SV40 nucleoprotein complexes (SV40 chromatin) in these systems requires the addition of ATP, dCTP, dGTP, dTTP, and Mg2+. Further addition of three other ribonucleoside triphosphates slightly increased DNA synthesis. Omission of ATP greatly reduced DNA synthesis. In the presence of ATP, however, omission of dATP did not significantly alter or rather slightly enhanced DNA synthesis. Analysis of replicated SV40 DNA by gel electrophoresis and autoradiography indicated that complete replication of SV40 DNA and chromatin occurred in these systems     

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.     

Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis.

Isolated nuclei from adenovirus type 2-infected HeLa cells catalyze the incorporation of all four deoxyribonucleoside triphosphates into viral DNA. The observed DNA synthesis occurs via a transient formation of DNA fragments with a sedimentation coefficient of 10S. The fragments are precursors to unit-length viral DNA, they are self-complementary to an extent of at least 70%, and they are distributed along most of the viral chromosome. In addition, accumulation of 10S DNA fragments is observed either in intact, virus-infected HeLa cells under conditions where viral DNA synthesis is inhibited by hydroxyurea or in isolated nuclei from virus-infected HeLa cells at low concentrations of deoxyribonucleotides. Under these suboptimal conditions for DNA synthesis in isolated nuclei, ribonucleoside triphosphates determine the size distribution of DNA intermediates. The evidence presented suggests that a ribonucleoside-dependent initiation step as well at two DNA polymerase catalyzed reactions are involved in the discontinuous replication of adenovirus type 2 DNA.     

The role of ATP in the transport of rapidly-labeled RNA from isolated nuclei of rat liver in vitro.

To understand the mechanism of the action of ATP on the in vitro transport of the rapidly-labeled RNA from isolated nuclei, the fate of ATP during the incubation as well as the effect of ATP, its analogues and other ribonucleoside triphosphates on the transport was examined and the following results were obtained. (1) More than 97% of added ATP remained acid soluble. No polyadenylation of the rapidly-labeled RNA in the released fraction by added ATP occurred although new polyadenylate segments smaller than 10 S were synthesized. (2) The addition of an ATP-generating system to the reaction mixture restored the initial rate of the release of the rapidly-labeled RNA from isolated nuclei. (3) Among the ribonucleoside triphosphates tested, ATP was most effective in stimulating the release. GTP was about 2/3 as effective as ATP. UTP showed some effect, but CTP showed no effect. EDTA was also non-effective. (4) When no ATP-generating system was added to the reaction mixture, AMP failed to mimic the effect of ATP. However, the combination of AMP and pyrophosphate could take the place of ATP. (5) Both AMP-CPP and AMP-PCP, the ATP analogues, showed the equal degree of their effect on the release, regardless of the position of the methylene bond. From these results, the principal role of ATP in the in vitro transport systems seemed to be its interaction with isolated nuclei to dissociate a structure which retains the rapidly-labeled RNA in the nucleus.     

RNA metabolism in isolated nuclei: processing and transport of immunoglobulin light chain sequences

Transport of prelabeled RNA from isolated myeloma nuclei is studied using conditions that permit RNA synthesis. Cytosol and spermidine are not required to maintain nuclear stability and inhibited RNA release. Omission of ATP or GTP decreased release 25 to 40%. The stimulatory effect of ATP or GTP is not due to hydrolysis of the triphosphates by the nuclear envelope NTPase, since addition of quercetin (an inhibitor of this NTPase) has no effect on the quantity of RNA released. The size distribution and percentage of poly A-containing species released from nuclei incubated with or without ATP or the other rNTPs are identical. Hybridization analysis of nuclear RNA before the transport assay revealed mature and precursor k light chain mRNA sequences. Following the transport assay, a significant fraction of k mRNA precursors is chased into mature k mRNA which is found both in nuclear-retained and released RNA.     

The effect of preincubation of HeLa cell nuclei with ATP on the degradation of mononucleosomal DNA by micrococcal nuclease.

HeLa cell nuclei with DNA labeled with [3H] thymidine have been preincubated under varying conditions and then incubated with micrococcal nuclease. Aliquots, removed at increasing times, were analyzed for mononucleosomal size DNA and for acid-soluble DNA, the ratios were plotted and a slope was determined. Preincubation with ATP and a regenerating system increased the slope 2 fold. Optimum ATP concentrations were above 0.25 mM. The ATP effect was reversed by novobiocin. No inhibition of the ATP effect was observed with nalidixic acid, coumermycin, oxolinic acid, VM-26, aphidicolin, or 3 amino-benzamide. NAD or cAMP or cGMP had no effect with or without ATP. Other nucleoside triphosphates could substitute to varying degrees for ATP as could ATP analogues. Nuclei from log phase cells showed no ATP effect, but log phase cells, partially depleted of ATP by incubation with deoxyglucose, showed the effect. The effect was lost in nuclei on long-term storage. No evidence was found for differential degradation of core histones, histone H-1 or DNA, and there was no evidence of nucleosome sliding.     

Effects of inhibition of basement membrane collagen deposition on rat mammary gland development

DNA biosynthesis by a system containing giant nuclei isolated from rat trophoblast cells at Day 13 of pregnancy has been studied. A method for the isolation of giant nuclei in good yield has been described. These nuclei were capable of incorporating [³H]dTTP into DNA for 2 hr and the incorporation was proportional to the amount of DNA template (nuclei). The system was highly dependent on the four deoxyribonucleoside triphosphates, ATP, and Mg²⁺ and was stimulated by monovalent ions such as K⁺. The optimum pH was 8.6. The product of the reaction was insensitive to RNase, sensitive to DNase, and banded at 1.710 g/ml in neutral CsCl together with bulk rat trophoblast DNA. Pulse-chase and density labeling experiments utilizing bromodeoxyuridine have indicated that replicative, discontinuous synthesis was taking place at sites previously active in vivo. DNA polymerases α, β, and γ were shown to be present in the nuclei. Experiments utilizing selective inhibitors of polymerases have demonstrated that DNA replication by trophoblast nuclei in vitro was insensitive to the specific α-polymerase inhibitor, aphidicolin, but almost completely inhibited by 2′, 3′-dideoxythymidine 5′-triphosphate as well as by N-ethylmaleimide suggesting that DNA replication observed in these trophoblast nuclei in vitro may be carried out by DNA polymerase γ.     

Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase

ATP hydrolysis was used to power the enzymatic release of clathrin from coated vesicles. The 70,000-mol-wt protein, purified on the basis of its ATP-dependent ability to disassemble clathrin cages, was found to possess a clathrin-dependent ATPase activity. Hydrolysis was specific for ATP; neither dATP nor other ribonucleotide triphosphates would either substitute for ATP or inhibit the hydrolysis of ATP in the presence of clathrin cages. The ATPase activity is elicited by clathrin in the form of assembled cages, but not by clathrin trimers, the product of cage disassembly. The 70,000-mol-wt polypeptide, but not clathrin, was labeled by ATP in photochemical cross-linking, indicating that the hydrolytic site for ATP resides on the uncoating protein. Conditions of low pH or high magnesium concentration uncouple ATP hydrolysis from clathrin release, as ATP is hydrolyzed although essentially no clathrin is released. This suggests that the recognition event triggering clathrin-dependent ATP hydrolysis occurs in the absence of clathrin release, and presumably precedes such release.     

Cellular integrity is required for inhibition of initiation of cellular DNA synthesis by reovirus type 3.

Synchronized HeLa cells, primed for entry into the synthesis phase by amethopterin, were prevented from initiating DNA synthesis 9 h after infection with reovirus type 3. However, nuclei isolated from synchronized cells infected with reovirus for 9 or 16 h demonstrated a restored ability to synthesize DNA. The addition of enucleated cytoplasmic extracts from infected or uninfected cells did not affect this restored capacity for synthesis. The addition of ribonucleotide triphosphates to nuclei isolated from infected cells stimulated additional DNA synthesis, suggesting that these nuclei were competent to initiate new rounds of DNA replication. Permeabilization of infected cells did not restore the ability of these cells to synthesize DNA. Nucleoids isolated from intact or permeabilized cells, infected for 9 or 16 h displayed an increased rate of sedimentation when compared with nucleoids isolated from uninfected cells. Nucleoids isolated from the nuclei of infected cells demonstrated a rate of sedimentation similar to that of nucleoids isolated from the nuclei of uninfected cells. The inhibition of initiation of cellular DNA synthesis by reovirus type 3 appears not to have been due to a permanent alteration of the replication complex, but this inhibition could be reversed by the removal of that complex from factors unique to the structural or metabolic integrity of the infected cell.     

Replication of a low-copy-number plasmid by a plasmid DNA-membrane complex extracted from minicells of Escherichia coli.

A DNA-membrane complex was extracted from minicells of an Escherichia coli mutant harboring a "miniplasmid" derivative (11.2 kilobases) of the low-copynumber plasmid RK2 (56 kilobases). The complex contained various species of supercoiled and intermediate forms of plasmid DNA, of which approximately 20% was bound firmly to the membrane after centrifugation in a CsCl density gradient. The plasmid DNA-membrane complex synthesized new plasmid DNA without the addition of exogenous template, enzymes, or other proteins. DNA synthesis appeared to proceed semi-conservatively, was dependent on the four deoxynucleoside triphosphates, partially dependent on ribonucleoside triphosphates, and was sensitive to rifampin, an antibiotic known to inhibit initiation of replication. Novobiocin and nalidixic acid also inhibited synthesis, as did the omission of ATP, N-Ethylmaleimide, an inhibitor of DNA polymerase II and III activity, but not DNA polymerase I activity, also partially inhibited the synthetic reaction, as did chloramphenicol. The plasmid DNA synthetic product was analyzed by alkaline sucrose and dye-CsCl gradient centrifugation, as well as by agarose gel electrophoresis. In each case, the product consisted of parental and intermediate forms of plasmid DNA. Some chromosomal DNA was also synthesized by a contaminating bacterial DNA-membrane complex, but this synthesis was rifampin insensitive and could be separated from plasmid DNA synthesis.     

DNA synthesis in isolated HeLa cell nuclei. Optimalization of the system and characterization of the product.

DNA replication in isolated nuclei from synchronized HeLa cells has been studied in an effort to optimalize the system and characterize the product. The synthesis was highly dependent on the four deoxyribonucleoside triphosphates, ATP, and Mg2+. Optimum pH was about 7.8. The system was further stimulated by monovalent ions with NH4Cl and Tris-HCl (each 65 mM) being the most effective. The four ribonucleoside triphosphates and glycerol gave a slight but very reproducible and additive stimulation. Low concentrations of spermine and spermidine (0.2-1.5 X 10(-4) M) were also slightly stimulatory (10-15%) whereas higher concentrations were inhibitory. The reaction product was DNase sensitive, and banded at 1.699 g/ml in neutral CsCl together with bulk HeLa nuclear DNA. When studied by neutral CsCl and alkaline Cs2SO4 gradients, the incorporation of [3H]TTP was mainly (more than 85%) due to further elongation of strands initiated in vivo as evidenced by BrdUrd labeling.     

ATP utilization by rep protein in the catalytic separation of DNA strands at a replicating fork.

Hydrolysis of ATP by rep protein proceeds in the presence of a single-stranded region of DNA 4 residues long, but the true effector for rep ATPase appears to be a replicating fork rather than a random coil. At or near a fork in duplex DNA, rep ATPase action is different from what it is on DNA lacking secondary structure (single-stranded): (i) Km for ATP is lower, (ii) specificity is for ATP and dATP with no action on other nucleoside triphosphates, (iii) sensitivity to certain ATP analogs is reduced, (iv) presence of a DNA-nicking enzyme (e.g. cistron A protein induced by phiX174) is required, and (v) Escherichia coli DNA binding protein facilitates rather than inhibits. During the separation of strands accompanying replication, 2 molecules of nucleoside triphosphate (ATP or dATP) are hydrolyzed for every nucleotide polymerized. Utilization of ATP by rep protein may provide energy for catalytic strand separation at a fork in advance of replication.     

RNA transport from isolated nuclei of cultured Djungarian hamster cells

The in vitro system of RNA transport containing isolated nuclei of Djungarian hamster cells transformed by SV-40 virus was studied. A functional test for cytoplasmic contaminations of the nuclei was proposed. The release of the newly synthesized RNA was found to be dependent on the duration of incubation, temperature and pH of the incubation medium as well as on the presence of spermine, spermidine, dithiothreitol, Mg2+, EDTA, exogenous RNA, nucleoside triphosphates and cytosol. The results obtained indicate that RNA release is an active process with activation energy of 12 kcal/mol. ATP, GTP, CTP and UTP have equal ability to serve as energy sources for the release of RNA. The nucleoside triphosphatase activity of the nuclei was the same in the presence of these four nucleoside triphosphates.