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.     

Nascent DNA chains synthesized in reversibly permeable cells of mouse thymocytes

Freshly prepared thymocytes continue to synthesize DNA under hypotonic conditions in the presence of 4.5% dextran T-150, the four deoxyribonucleoside triphosphates and ATP. Permeable cells could seal the membrane in a serum-enriched medium within a few hours. 2'-Deoxycytidine 5'-triphosphate is effectively substituted by 5-mercuri-2'-deoxycytidine 5'-triphosphate as a substrate. The newly synthesized mercurated DNA can be separated from cellular DNA and RNA on a thiol-agarose affinity matrix. The rate of incorporation of [3H]thymidine triphosphate into permeable cells is the same as that of the incorporation of [3H]thymidine into intact cells, corresponding to approximately 30% of the rate in vivo. Synthesis in permeable cells reflects DNA replication shown by inhibitors such as 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (aCTP), nalidixic acid and novobiocin and by density shift experiments. More than 80% of the newly synthesized low-molecular-mass DNA, 8-60 nucleotides in length, consists of RNA-linked DNA. This conclusion is based on phosphorylation with [gamma-32]ATP and polynucleotide kinase and rephosphorylation after alkaline hydrolysis. The 5' end of RNA consists of adenylate, guanylate, cytidylate and uridylate residues in a ratio of 4:3:1.5:1.5.     

Escherichia coli DNA synthesis in vitro: insensitivity of ATP-dependent DNA repair to inhibition by novobiocin.

Novobiocin, an effective inhibitor of DNA replicaion in Escherichia coli, is shown to have no effect on the ATP-dependent DNA repair carried out by toluenized cells after ultraviolet irradiation. Therefore novobiocin can be considered a selective inhibitor of replicative DNA synthesis in vitro.     

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.     

Triton X-100 activates nucleoside triphosphate-dependent, recBC-dependent DNA synthesis in toluene-treated Escherichia coli.

Escherichia coli cells whose chromosome replication has been terminated in vivo, either by growth into stationary phase or by incubation of a mutant carrying a temperature-sensitive initiation mutation under restrictive conditions, are inactive in in vitro DNA synthesis as measured in toluene-treated cells. Addition of the non-ionic detergent Triton X-100 to such inactive systems results in a marked stimulation of ATP-dependent in vitro DNA synthesis. This Triton-stimulated DNA synthesis appears to proceed by a semi-conservative mechanism, in that DNA synthesized in vitro in the presence of a density labeled precursor bands in CsCl equilibrium centrifugation at a hybrid density. Neutral sucrose gradient centrifugation demonstrates that most of this hybrid material exhibits a molecular weight in excess of 1 X 10(7). Triton-stimulated synthesis requires the presence of DNA polymerase III, as does normal in vivo replication. We show here, however, several anomalous properties of the DNA synthesis in the Triton/toluene system. In particular, Triton-stimulated synthesis is absent in cells harboring a recB mutation which lack the ATP-dependent exonuclease V, an enzyme implicated in recombinational repair synthesis in vivo. Furthermore, the ATP requirement for Triton-stimulated synthesis is relatively non-sepcific, and a variety of nucleoside triphosphates can effectively substitute for ATP. Finally, despite their high molecular weight in neutral sucrose gradient centrifugation, Triton-stimulated DNA synthesis generates DNA molecules of low molecular weight (less than 500 000) as determined by alkaline sucrose gradient centrifugation. In contrast, DNA synthesis in the normal toluene-treated cell system is not dependent on recB activity, shows a nearly absolute requirement for ATP which cannot be replaced by other nucleoside triphosphates, and produces molecules of far greater molecular weight as measured on alkaline sucrose gradients. Taken altogether the data strongly suggest that Triton activates an unusual form of DNA synthesis in toluene-treated cells which shows both repair and replicative aspects. These results caution against the use of Triton-activated toluene-treated cells system, for studying simple replicative DNA synthesis.     

In Vitro Polyoma DNA Synthesis: Characterization of a System from Infected 3T3 Cells

A lysate from hypotonically swollen polyoma-infected BALB/3T3 cells incorporated labeled deoxynucleotide triphosphates into both viral and cellular DNAs. The incorporation was stimulated by the presence of ATP, deoxynucleotide triphosphates, thiols, and magnesium ions. Strong inhibition of incorporation was observed with thiol reagents and arabinosyl nucleotide triphosphates. The rate of in vitro synthesis increased with the temperature of incubation as expected. Incorporation into cellular DNA for up to 2 h was observed in lysates from virus-infected and serum-stimulated cells but not from resting cells. Synthesis in the system, therefore, appeared to reflect the physiological state of the cells before preparation of the lysate. Incorporation into viral DNA stopped far sooner than that into cellular DNA. During the initial phase of the in vitro incubation, incorporation occurred into viral replicative intermediates (RI). These RIs had identical properties to those isolated after in vivo pulse labeling and a substantial proportion of them was matured to form I DNA at later times in the incubation through all the stages known to occur in vivo. Density labeling of the in vitro product showed that practically all of the RIs pre-existing in the infected cell took part in the in vitro reaction. Analysis of DNA labeled in vitro in the presence of 5-bromodeoxyuridine triphosphate showed that synthesis occurred on RIs at all stages of replication and that the progeny strands were elongated by up to 80% of unit viral DNA length. Pre-existing RIs, pulse labeled in vivo, showed evidence of a pool at a late stage of replication which required elongation of their progeny strands by approximately 25% during conversion to form I molecules. From density-labeling experiments, we were also able to show that viral DNA synthesis in vitro was semiconservative. The major reason for cessation of viral DNA synthesis in vitro was the very limited ability of the lysate to initiate new rounds of viral DNA synthesis.     

Mechanism of gap-filling during postreplication repair of ultraviolet damage in Haemophilus influenzae.

Deoxyribonucleic acid (DNA), pulse labeled after ultraviolet irradiation of excision-defective mutants of Haemophilus influenzae, is of lower single strand molecular weight than that of unirradiated cells but approaches the size of DNA from unirradiated cells upon further incubation in growth medium. This gap-filling process is controlled by the rec-1 gene. Gap-filling occurs normally in a temperature-sensitive DNA synthesis mutant at the restrictive temperature showing that normal semiconservative DNA synthesis is not necessary for gap-filling. To test for recombinational events after irradiation, the DNA synthesized after irradiation was radioactively labeled for a short time in medium containing 5-bromodeoxyuridine followed by incubation for various times in non-radioactive, 5-bromodeoxyuridine-containing medium. The DNA was denatured and analyzed isopycnically. The labeled DNA was initially "heavy," but later shifted toward lighter densities. This shift occurred in the temperature-sensitive DNA synthesis mutant at the restrictive temperature and in the recombination-defective mutant rec-2, but was not seen in the rec-1 mutant. The density shift can be interpreted as evidence that rather extensive exchanges occurred between parental DNA and the DNA made after irradiation. These results suggest that such exchanges are important for gap-filling in H. influenzae.     

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.     

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.     

Requirement for Protein Synthesis in rec-Dependent Repair of Deoxyribonucleic Acid in Escherichia coli after Ultraviolet or X Irradiation

Deprivation of amino acids required for growth or treatment with chloramphenicol or puromycin after irradiation reduced the survival of Rec(+) cells of Escherichia coli K-12 which had been exposed to either ultraviolet (UV) or X radiation. In contrast, these treatments caused little or no reduction in the survival of irradiated recA or recB mutants. The effect of chloramphenicol on the survival of X-irradiated cells was correlated with an inhibition of repair of single-strand breaks in irradiated deoxyribonucleic acid (DNA), previously shown to be controlled by recA and recB. In UV-irradiated cells no effect of chloramphenicol was detected on the repair of single-strand discontinuities in DNA replicated from UV-damaged templates, a process controlled by recA but not by recB. From this we concluded that inhibiting protein synthesis in UV or X-irradiated cells may interfere with some biochemical step in repair dependent upon the recB gene. When irradiated Rec(+) cells were cultured for a sufficient period of time in minimal growth medium before chloramphenicol treatment their survival was no longer decreased by the drug. After X irradiation this occurred in less than one generation time of the unirradiated control cells. After UV irradiation it occurred more slowly and was only complete after several generation times of the unirradiated controls. These observations indicated that replication of the entire irradiated genome was probably not required for rec-dependent repair of X-irradiated cells, although it might be required for rec-dependent repair of UV-irradiated cells.     

Methylation-dependent DNA synthesis in Escherichia coli mediated by DNA polymerase I.

An in vitro system was used to study DNA synthesis in lysates of Escherichia coli cells which had been grown in the presence of ethionine. Such lysates showed a reduced capacity to incorporate [3H]TTP into high-molecular-weight material. Activity could be restored by incubation with S-adenosyl methionine and ATP. S-adenosyl methionine-reactivated TTP incorporation required the presence of DNA polymerase I, ATP, and all four deoxyribonucleotide triphosphates. DNA polymerase III was not required.     

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.     

Novobiocin; an inhibitor of the repair of UV-induced but not X-ray-induced damage in mammalian cells.

In addition to inhibiting replicative DNA synthesis in HeLa cells, novobiocin has a severe effect on the cellular response to UV irradiation, reducing the number of breaks made in pre-existing DNA by the excision repair process. The inhibition of UV repair by novobiocin is reflected in enhanced UV-killing of these cells. Rejoining of DNA after X irradiation is not impaired by novobiocin. The recognition and removal of UV damage may require unwinding of the DNA by gyrase, which--in bacteria--is the target for novobiocin.     

Physiological aspects of modification and restoration of chromosomal synthesis in bacteria after x-irradiation

Billen, Daniel (The University of Texas, Houston), and Roger Hewitt. Physiological aspects of modification and restoration of chromosomal synthesis in bacteria after X irradiation. J. Bacteriol. 90:1218-1225. 1965.-A study was made of the effect of amino acid deprivation or chloramphenicol on the character of postirradiation deoxyribonucleic acid (DNA) replication in bacteria with the use of radioisotopes and 5-bromouracil as a density label. CsCl density-gradient studies of DNA showed that postirradiation incubation of amino acid-requiring Escherichia coli in an amino acid-free medium interfered with continued linear chromosomal replication. In the presence of the required amino acids, linear chromosomal replication was shown to resume. Addition of chloramphenicol was found to prevent this resumption. Deletion of the required amino acids or the presence of chloramphenicol in a fully supplemented medium allowed the detection of altered DNA synthesis in bacteria at X-ray doses as low as 500 r. The character of the limited DNA made in the presence of the density label after irradiation is described. The results are interpreted as showing that the synthesis of a protein(s) is required for restoration of linear chromosomal replication in the irradiated cells.     

Effects of 2',3'-dideoxythymidine triphosphate on replicative DNA synthesis and unscheduled DNA synthesis in permeable mouse sarcoma cells

2',3'-Dideoxythymidine triphosphate differentially inhibited replicative DNA synthesis in permeable mouse ascites sarcoma cells and unscheduled DNA synthesis in bleomycin-treated permeable cells or in isolated rat liver nuclei. The mode of inhibition of 2',3'-dideoxythymidine triphosphate was competitive with respect to deoxythymidine triphosphate. 2',3'-Dideoxythymidine triphosphate inhibited replicative DNA synthesis with a Ki of 8 microM, whereas unscheduled DNA synthesis was more sensitive, the Ki being 0.5 microM. Referring to the differential sensitivity of DNA polymerases alpha and beta to 2',3'-dideoxythymidine triphosphate and to other related information reported previously, the present results suggested that DNA polymerase alpha is playing a major role in replicative DNA synthesis, and DNA polymerase beta in unscheduled DNA synthesis.     

An evaluation of the evidence for H+ pumping by reconstituted cytochrome c oxidase in the light of recent criticism

Caffeine inhibited DNA synthesis in toluene-treated Escherichia coli K12 strains to the same extent as in intact cells using the incorporation of [3H]thymidine as a measure of DNA synthesis. The inhibition was found to be competitive with ATP, and it was not influenced by the concentrations of deoxynucleoside triphosphates to any extent. When caffeine was added together with other DNA synthesis inhibitors such as novobiocin, nalidixic acid or actinomycin D, the inhibition in all cases was non-additive. It is suggested that caffeine inhibits one of the ATP-requiring enzymes in the DNA replication machinery, possibly DNA polymerase III or one of the DNA helicases.     

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.     

Two modes of excision repair in toluene-treated Escherichia coli.

In toluene-treated Escherichia coli incision breaks accumulate during post-irradiation incubation in the presence of adenosine 5'-triphosphate (ATP). It is shown that incised deoxyribonucleic acid (DNA) is converted to high-molecular-weight DNA during reincubation in the presence of the four deoxyribonucleoside triphosphates (dNTP's) and nicotinamide adenine dinucleotide (NAD). This restitution process is ATP independent and N-ethylmaleimide insensitive and takes place only in polA+ strains. It is defective in strains carrying a mutation in the 5' leads to 3' exonucleolytic activity associated with DNA polymerase I. Repair of accumulated incision breaks differs from repair in which all the steps of the excision repair process occur simultaneously or in rapid succession. The latter is observed if toluene-treated E. coli are incubated immediately after irradiation in the presence of the four dNTP's, NAD, and ATP. It is shown that under these conditions dimer excision occurs to a larger extent than during repair of accumulated incision breaks and that, except in strains defective in polynucleotide ligase, incision breaks do not accumulate. This consecutive mode of repair is detectable in polA+ strains and at low doses also in polA mutants.     

Dependence of mammalian DNA synthesis on DNA supercoiling. III. Characterization of the inhibition of replicative and repair-type DNA synthesis by novobiocin and nalidixic acid

Novobiocin and nalidixic acid, inhibitors of the bacterial enzyme DNA gyrase, inhibit DNA, RNA and protein synthesis in several human and rodent cell lines. The sensitivity of DNA synthesis (both replicative and repair) to inhibition by novobiocin and nalidixic acid is greater than that of protein synthesis. Novobiocin inhibits RNA synthesis about half as effectively as it does DNA synthesis, whereas nalidixic acid inhibits both equally well. Replicative DNA synthesis, as measured by incorporation of [3H]thymidine, is blocked by novobiocin in a number of cell strains; the inhibition is reversible with respect to both DNA synthesis and cell killing, and continues for as long as 20--30 h if the cells are kept in novobiocin-containing growth medium. Both novobiocin and nalidixic acid inhibit repair DNA synthesis (measured by BND-cellulose chromatography) induced by ultraviolet light or N-methyl-N'-nitro-N-nitrosoguanidine (but not that induced by methyl methanesulfonate) at lower concentration (as low as 5 micrograms/ml) than those required to inhibit replicative DNA synthesis (50 micrograms/ml or greater). Neither novobiocin nor nalidixic acid alone induces DNA repair synthesis. Incubation of ultraviolet-irradiated cells with 10--100 micrograms/ml novobiocin results in little, if any, further reduction of colony-forming ability (beyond that caused by the ultraviolet irradiation). Novobiocin at sufficiently low concentrations (200 micrograms/ml) apparently generates a quiescent state (in terms of cellular DNA metabolism) from which recovery is possible. Under more drastic conditions of time in contact with cells and concentration, however, novobiocin itself induces mammalian cell killing.