Effects of Ribonucleosides on Thymidine Incorporation: Selective Reversal of the Inhibition of Deoxyribonucleic Acid Synthesis in Thymineless Auxotrophs of Escherichia coli

Addition of certain ribonucleosides to exponentially growing cultures of Escherichia coli increased the extent of thymidine incorporation. The prolonged uptake of thymidine was correlative with the ability of these ribonucleosides to prevent the degradation of thymidine. In addition to protecting thymidine, uridine reversed partially (70 to 80%) the inhibition of deoxyribonucleic acid (DNA) synthesis in thymineless auxotrophs by cytosine arabinoside, hydroxyurea, and nalidixic acid. This reversal was selective for auxotrophic strains since no reversal of inhibition by uridine was observed in any of the prototrophic strains examined. In the presence of uridine, the rapid assimilation of thymidine by prototrophic and auxotrophic strains was prevented and the rate of DNA synthesis became a function of the available exogenous thymidine. Under these conditions, prototrophic strains accumulated equivalent amounts of thymidine into the acid-soluble (pool) and acid-insoluble (DNA) cell fractions. In contrast, 95 to 98% of the thymidine taken up by auxotrophs was found in the acid-insoluble (DNA) cell fraction. The results suggest that different mechanisms for DNA synthesis exist in auxotrophs and prototrophs. Based on these observed differences, some possible mechanisms for the selective reversal of the inhibition of DNA synthesis in auxotrophs are discussed.     

Induction of Excessive Deoxyribonucleic Acid Synthesis in Escherichia coli by Nalidixic Acid

Prior treatment of Escherichia coli with nalidixic acid in nutritionally complete medium altered the subsequent pattern of deoxyribonucleic acid (DNA) synthesis normally observed in nutritionally deficient medium. Transfer of E. coli 15 TAU to an amino acid- and pyrimidine-deficient medium usually resulted in a 40 to 50% increase in DNA content. Previous treatment with nalidixic acid caused a 200 to 300% increase in DNA content under these conditions. The extent of this DNA synthesis depended on the duration of prior exposure to nalidixic acid. The maximal rate of synthesis was obtained after a 40- to 60-min exposure to nalidixic acid and was two to three times that of the control. The induction of this excessive DNA synthesis was prevented by chloramphenicol or phenethyl alcohol, but the synthesis of this DNA was only partially sensitive to these agents. With E. coli TAU-bar, the rate of DNA synthesis, after removal of nalidixic acid, was similar to that of E. coli 15 TAU, but the maximal amount of DNA synthesized was 180 to 185% of that initially present. Cesium chloride density gradient analysis demonstrated that DNA synthesis after removal of nalidixic acid occurs by a semiconservative mode of replication. The density distribution of this DNA was similar to that obtained after thymine starvation. These results suggest that nalidixic acid treatment may induce additional sites for DNA synthesis in E.coli.     

Effect of Putative Deoxyribonucleic Acid Inhibitors on Macromolecular Synthesis in Saccharomyces cerevisiae

The effects of inhibitors of bacterial deoxyribonucleic acid (DNA) synthesis upon logarithmically growing cultures of Saccharomyces cerevisiae were investigated. Cell division, ribonucleic acid (RNA) synthesis, and DNA synthesis were measured after addition of nalidixic acid, fluorodeoxyuridine, or phenethyl alcohol to cultures of yeast growing in defined and complex media. Both nalidixic acid and fluorodeoxyuridine had only temporary effects on nucleic acid synthesis in cultures growing in defined medium, and little or no observable effect on cultures growing in complex medium. Neither compound inhibited colony formation on complex solid medium, although growth was slow on defined solid medium. Phenethyl alcohol caused complete inhibition of DNA synthesis, RNA synthesis, and cell division in cultures growing in defined medium. In cultures growing in complex medium, RNA synthesis and cell division were inhibited to a lesser extent. A slight increase in DNA was observed in the presence of the inhibitor.     

Reduced DNA repair during differentiation of a myogenic cell line

Repair synthesis induced by 4-nitroquinoline-1-oxide (4NQO) in L6 myoblasts before and after cellular fusion was measured by [3H] thymidine incorporation into unreplicated DNA. The level of repair synthesis was reuced after the cells had fused into myotubes. The terminal addition of radioactive nucleotides into DNA strands occurred only to a minor extent, and the dilution of [3H] thymidine by intracellular nucleotide pools was shown not to be responsible for the observed difference in repair synthesis, Both the initial rate and the overall incorporation of [3H] thymidine were found to be 50% lower in the myotubes. 4NQO treatment of myoblasts and myotubes induced modifications in the DNA which were observed as single-strand breaks during alkaline sucrose sedimentation. After the myoblasts were allowed a post-treatment incubation, most of the single-strand breaks were not longer apparent. In contrast, a post-treatment incubation of myotubes did not change the extent of single-strand breakage seen. Both myoblasts and myotubes were equally effective in repairing single- strand breaks induced by X radiation. It would appear that when myoblasts fuse, a repair enzyme activity is lost, probably an endonuclease that recognizes one of the 4 NQO modifications of DNA. The result observed is a partial loss of repair synthetic ability and a complete loss of ability to remove the modification that appears as a single-strand break in alkali.     

Single-strand breakage in DNA of Escherichia coli exposed to Cd2+.

When a growing culture of Escherichia coli was exposed to 3 X 10(-6) M Cd2+, 85 to 95% of the cells lost their ability to form colonies on agar plates. Loss of viability was accompanied by considerable single-strand breakage in the DNA, with no detectable increase in double-strand breaks. A direct correlation appeared to exist between the number of single-strand breaks and the concentrations of Cd2+ to which the cells were exposed. Exposure of DNA in vitro to a Cd2+ concentration of 3 X 10(-6) M or higher, followed by sedimentation in alkaline sucrose gradients, demonstrated no single-strand breaks. Cadmium-exposed cells recovered viability when incubated in Cd2+-free liquid medium containing 10 mM hydroxyurea. During the early period of recovery, there was a lag in the incorporation of labeled thymidine, but cellular DNA, at least in part, appeared to be repaired.     

Pleiotropic Effect of the recA Gene of Escherichia coli: Uncoupling of Cell Division from Deoxyribonucleic Acid Replication

A defective recA gene, which is involved in recombination, is shown in this article to permit limited cell division, when deoxyribonucleic acid (DNA) synthesis is blocked. Thymidine starvation or nalidixic acid blocked DNA synthesis, and stopped cell division of a rec(+)thy(-) strain of Escherichia coli. However, with the same treatments, a recAthy(-) strain could continue to divide for at least 5 hr, and cell numbers increased 2.5- to 4-fold. After several hours of thymidine starvation, the culture contained very long cells (snakes) and small (normal-sized) cells. The short cells contained very little, if any, DNA. Cells of all ages divided in the absence of thymidine. Specific differences in membrane proteins were observed between thymidine-starved rec(+) and recA cells, as expected from previous experiments in which these proteins were associated with cell division and DNA synthesis. It is proposed that septum formation is controlled negatively by the recA(+) gene.     

Deoxyribonucleic Acid Breaks in Heated Salmonella typhimurium LT-2 After Exposure to Nutritionally Complex Media

Minimal medium recovery of heat-treated Salmonella typhimurium LT-2 has been expressed as the reduced viability on trypticase soy agar supplemented with 0.5% yeast extract (TSY) relative to a glucose-salts (M-9) agar. Incubation of S. typhimurium LT-2 in water at 50 C for 15 min did not change the sedimentation patterns of deoxyribonucleic acid (DNA) in alkaline sucrose gradients. The same pattern was obtained if the heated cells were further incubated for 15 min at 37 C in M-9 broth. However, a marked increase in DNA single-strand breakage accompanied by a loss of viability was observed after a similar incubation of heated bacteria in TSY broth. If heated bacteria were incubated in M-9 broth before TSY broth, there was a decrease in the single-strand breakage occurring in the TSY broth. This decrease is believed to be the result of repair of heat-induced damage. We conclude that minimal medium recovery after heat treatment is due to DNA damage caused by sequential exposure to heat and TSY medium, such damage not occurring after sequential exposure to heat and M-9 medium.     

Influence of Thymine Starvation on the Integrity of Deoxyribonucleic Acid in Escherichia coli

The influence of thymine starvation on the single-strand molecular weight of deoxyribonucleic acid (DNA) from Escherichia coli was determined by sedimentation through gradients of alkaline sucrose. Growth of cells for as long as 150 min in thymineless medium did not significantly reduce the molecular weight below the control value of 2.4 +/- 0.3 x 10(8) daltons. Incubation of cells in thymineless medium after exposure to 500 ergs/mm(2) of ultraviolet light or 20 krad of (137)Cs gamma rays did not appear to block the rejoining of single-strand breaks associated with irradiation. Thus, DNA repair enzymes, presumably including DNA ligase, are not significantly inhibited by thymine starvation.     

Deoxyribonucleic Acid Synthesis and Cell Division in a lon− Mutant of Escherichia coli

The lon(-) mutants of Escherichia coli form long filamentous cells after temporary inhibition of deoxyribonucleic acid (DNA) synthesis by ultraviolet irradiation, treatment with nalidixic acid, or thymine starvation. The kinetics of DNA synthesis and cell division after a period of thymine starvation have been compared in lon(+) and lon(-) cells. After this treatment, both kinds of cells recover their normal DNA to mass ratio with the same kinetics. In contrast to previous reports, cell division is found to recommence in both lon(+) and in lon(-) cells after such a temporary period of inhibition of DNA synthesis. However, the delay separating the recommencement of DNA synthesis and of cell division is approximately three times as long in lon(-) as in lon(+) cells. Low concentrations of penicillin inhibit cell division in both lon(+) and lon(-) cells. In this case, cell division recommences with the same kinetics in both strains after the removal of penicillin. This suggests that different steps in the cell division process are blocked by inhibition of DNA synthesis and by penicillin treatment. The lon(-) mutation appears to affect the former of these steps.     

Effect of Nalidixic Acid on Deoxyribonucleic Acid Synthesis in Bacteriophage SPO1-infected Bacillus subtilis

The effect of nalidixic acid on deoxyribonucleic acid (DNA) synthesis in Bacillus subtilis cells infected with bacteriophage SPO1 was studied. Nalidixic acid had little inhibitory effect on SPO1 DNA synthesis at concentrations that drastically inhibited B. subtilis DNA synthesis. Inhibition of DNA synthesis, appropriate to the concentration used, was imposed within 1 min after addition of nalidixic acid, suggesting that it acts directly on DNA synthesis in both infected and uninfected cells. The SPO1 DNA synthesized in the presence of high concentrations of nalidixic acid had a density characteristic of normal SPO1 DNA and was packaged into viable progeny phage particles, but its rate of synthesis was reduced and bacterial lysis was delayed.     

Inhibition of Ribonucleic Acid Synthesis by Nalidixic Acid in Escherichia coli

The effect of low concentrations of nalidixic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was examined. It was observed that RNA synthesis in exponentially growing cells was not significantly affected, in harmony with previous studies. However, RNA synthesis was markedly depressed by nalidixic acid during starvation for an amino acid or during chloramphenicol treatment. This effect was not caused by increased killing or inhibition of nucleoside triphosphate synthesis by nalidixic acid. The pattern of radioactive uracil incorporation into transfer RNA or ribosomes was not changed by the drug. The sensitivity of RNA synthesis to nalidixic acid in the absence of protein production may be useful in probing the amino acid control of RNA synthesis.     

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Effect of Nalidixic Acid

During the conjugal transfer of the R64-11 plasmid at 42 C from donor cells thermosensitive for vegetative deoxyribonucleic acid (DNA) synthesis to recipient minicells, the plasmids are conjugally replicated in the donor cells. This conjugal replication is inhibited by nalidixic acid, and the degree of inhibition is comparable to the reduction in the amount of plasmid DNA transferred to the recipient minicells in the presence of the drug. In addition, the size of DNA transferred to the minicells and the fraction of conjugally replicated DNA in the donor cells that can be isolated as closed-circular plasmid DNA under alkaline conditions are both reduced by nalidixic acid. When the drug is added to a mating that is underway, the rate of conjugal replication is immediately reduced. This change is accompanied by a reduction in the amount of conjugally replicated DNA in the donor cells that can be isolated as closed-circular plasmid DNA. Furthermore, conjugally replicated plasmid DNA that is not associated with the donor cell membrane becomes membrane bound after the addition of nalidixic acid.     

The repair of methyl methanesulphonate-induced lesions in the DNA of a murine lymphoma cell

The introduction of single-strand breaks into the DNA of a murine lymphoma (L5178Y) cell treated in vivo with methyl methanesulphonate (MMS) and the behaviour of these breaks on post-treatment incubation were studied. A large proportion of single-strand breaks present after MMS treatment could be repaired as shown by sedimentation in alkaline sucrose. Two inhibitors of DNA synthesis, hydroxyurea and cytosine arabinoside affected the repair process differently-hydroxyurea had only a small effect while cytosine arabinoside blocked repair and at some doses allowed further degradation of the DNA. It was also found that the level of ‘repair replication’ in the presence of cytosine arabinoside was lower than that found in the presence of hydroxyurea.     

Formation of chromatid-type aberrations in G2 stage of the cell cycle

CHO cells were treated in G₁ stage of the cell cycle with chromosome-breaking agents that act in an S-dependent manner. The cells were challenged in G₂ stage, before fixation, with various inhibitors of DNA synthesis or repair. Short-wave UV, mitomycin C, decarbomyl mitomycin and 4-nitroquinoline oxide (4NQO) were used as chromosome-breaking agents. The inhibitors of DNA repair or synthesis used were hydroxyurea, aphidicolin and caffeine. Permeabilization of cells followed by a treatment with Neurospora endonuclease (a treatment to convert DNA single-strand breaks into double-strand breaks) did not have any influence on the frequencies of chromatid aberrations induced by the chemicals used, whereas with the inhibitors the extent of potentiation varied depending on the mutagen and the inhibitor used.     

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.     

The increases in the rates of synthesis of ribosomal proteins and ribosomal RNA during refeeding of starved Tetrahymena cells are not dependent on DNA replication

We have analysed the effects of an inhibition of DNA replication by hydroxyurea on the synthesis of ribosomal proteins (r-proteins) and ribosomal RNA (rRNA) in Tetrahymena cells resuming growth after long-term starvation. The coordinate regulation of the synthesis of individual r-proteins and their increased rate of synthesis during refeeding are not impaired by inhibition of DNA replication. Moreover, the presence of hydroxyurea does not prevent an increase in the rate of synthesis of rRNA around 70-80 min after refeeding. Previously, this increase was claimed to be gene dose-dependent. Up to 180 min after refeeding, the synthesis of r-proteins appears to be closely coupled with that of rRNA and proceed in stoichiometric balance, irrespective of whether hydroxyurea is present or not. After 180 min of refeeding in the presence of hydroxyurea, this stoichiometric balance breaks down, and the rate of synthesis of r-proteins clearly exceeds that of the rRNA synthesis.     

Damage and Repair of DNA in 5-Bromodeoxyuridine-Labeled Chinese Hamster Cells Exposed to Fluorescent Light

Illumination of Chinese hamster cells with fluorescent light after 5-bromodeoxyuridine incorporation leads to extensive single-strand breakage in the DNA of the exposed cells. The rate of production of single-strand breaks is dependent on the extent to which thymine is replaced by 5-bromouracil. At least some of the breaks observed with alkaline gradients are probably produced in vivo and are probably not contingent upon alkaline hydrolysis since breakage can be demonstrated with neutral gradients also. Cells are able to rejoin most of the single-strand breaks within 60 min; however, damage to the DNA-containing material (the “complex”) initially released from cells is repaired more slowly. Cysteamine protects against single-strand breakage with a dose-modifying factor of 2.8. A comparison is made between the production of single-strand breaks by fluorescent light and X-rays, and the significance of such breaks relative to cell survival is discussed.     

Deoxyribonucleic Acid Degradation and the Lethal Effect by Myxin in Escherichia coli

Exposure of Escherichia coli 15T(-) cells to the antibiotic myxin results in the inhibition of deoxyribonucleic acid (DNA) biosynthesis, degradation of intracellular DNA, and death of the cells. Each of these effects was markedly enhanced when protein synthesis was simultaneously inhibited by chloramphenicol. In the continued presence of chloramphenicol, a brief (1 min) exposure to myxin resulted in a rate of DNA degradation and cell death equivalent to that found in the continued presence of myxin alone. Single-strand breaks were present in the DNA of cells exposed to myxin, but when chloramphenicol was also present the breaks were found much earlier. Degradation of DNA in cells exposed to myxin was found to be distributed randomly in both strands and extended over the genome with no restriction to the vicinity of the replication point. There was no release of DNA from its attachment to the cellular membrane in myxin-exposed cells. The possibility that the chloramphenicol effect is due to the inhibition of repair enzyme synthesis which is stimulated by exposure of the cells to myxin is discussed. These data indicate that the extent of the lethal and metabolic damage to the cells by an exposure to myxin represents the result of competition between damage to and repair of cellular DNA.     

Increased DNA polymerase and ATP-dependent deoxyribonuclease activities following DNA damage in Mycobacterium smegmatis

Summary Treatment of growing cultures of Mycobacterium smegmatis with alkylating agents (methyl methaneusulphonate, ethyl methanesulphonate, nitrogen mustard, or mitomycin C) or with ultraviolet light resulted in enhanced specific activities of a DNA polymerase and of an ATP-dependent deoxyribonuclease. Similar results had previously been obtained with hydroxyurea and with iron limitation. The three of these treatments which were tested (methyl methane-sulphonate, mitomycin C and hydroxyurea) produced strand breaks or alkali-labile regions in the DNA of this organism. The increased enzyme activities could be prevented by simultaneous treatment with inhibitors of protein synthesis.In contrast, treatment of the cultures with intercalating agents (ethidium bromide, acridine orange, or proflavine), 5-fluorouracil, caffeine, or nalidixic acid, inhibited DNA synthesis without increasing the enzyme activities. These treatments did not produce strand breaks in the DNA of this organism.The results support the hypothesis that, in M. smegmatis, damage to DNA induces increased synthesis of enzymes associated with DNA repair.     

Survival of CHO cells that replicated DNA in the presence of hydroxyurea

Synchronized CHO cells in S phase were treated with different concentrations of hydroxyurea for various time intervals. In the presence of 2 mM hydroxyurea DNA replication was inhibited by more than 95% and S phase cells were killed within 20 h. With 0.1 mM hydroxyurea, however, when DNA replication was inhibited by about 70%, more than 90% of S phase cells survived a 40 h treatment. DNA replication in the presence of hydroxyurea had normal characteristics for up to 5 h except that the average rate of DNA chain elongation (fork displacement) was reduced. Fluorodeoxyuridine, excess thymidine, and cycloheximide caused a similar loss of reproductive viability as hydroxyurea, if DNA replication was inhibited to the same extent. The results suggest that killing of S phase cells might be induced by inhibition of DNA replication itself, i.e. by completely blocking displacement of forks.