Replication of colicinogenic factor E1 DNA in plasmolysed Escherichia coli cells. Coupling of DNA replication and RNA synthesis.

Plasmolysed chloramphenicol-treated Escherichia coli cells carrying the colicinogenic factor E1 utilize deoxynucleoside triphosphates for the semi-conservative synthesis of Col E1 DNA. Col E1 DNA replication in plasmolysed cells can be dissociated into two temporally separated processes: (a) a rifampicin-sensitive RNA synthesis, which is stimulated by adenosine 3':5'-monophosphate (cyclic AMP) and requires all four ribonucleoside triphosphates and (b) an ATP-dependent DNA synthesis, which is inhibited by arabinosylnucleoside triphosphates and sulfhydryl-blocking reagents. Thes two processes exhibit different sensitivities to inhibition by polyamines and actinomycin D.     

Energy coupling in the transport of beta-galactosides by Escherichia coli: effect of proton conductors

Escherichia coli accumulates thiomethyl-beta-d-galactoside against a concentration gradient under anaerobic conditions. The accumulation was abolished by carbonylcyanide m-chlorophenylhydrazone, tetrachlorosalicylanilide, 2,4 dinitrophenol, and other uncouplers of oxidative phosphorylation even though oxidative phosphorylation would not be expected to occur anaerobically. In the presence of the uncouplers, the beta-galactoside carrier remained functional and catalyzed equilibration of thiomethylgalactoside across the membrane. The uncouplers did not inhibit the generation of adenosine triphosphate or protein turnover, or the accumulation of alpha-methylglucoside and glycerol by phosphorylation. We conclude that, at least anaerobically, uncouplers of oxidative phosphorylation do not interfere with energy metabolism in general, but prevent the utilization of metabolic energy for the active transport of galactosides. The uncouplers also facilitate passage of protons across the membrane. Various hypotheses are considered to explain why a proton-impermeable membrane may be required for active transport of galactosides and other substrates.     

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 mode of inhibitory action by aphidicolin on eukaryotic DNA polymerase alpha.

The mode of action by aphidicolin on DNA polymerase alpha from the nuclear fraction of sea-urchin blastulae was studied. The inhibition of DNA polymerase alpha by aphidicolin was uncompetive with activated DNA and competitive with the four deoxynucleoside triphosphates using activated DNA as a template-primer. For truncated (residual or limited) DNA synthesis with only three deoxynucleoside triphosphates, aphidicolin inhibited the residual synthesis more strongly in the absence of dCTP than in the absence of each of the other three deoxynucleoside triphosphates. The inhibition was reversed with excess dCTP but not with the other three deoxynucleoside triphosphates. That is, aphidicolin inhibited DNA polymerase alpha by competing with dCTP with a Ki value of 0.5 microgram/ml and by not competing with the other three deoxynucleoside triphosphates. dTMP incorporation with the activated DNA was more sensitive to aphidicolin than dGMP or dTMP incorporation with poly(dC). (dG)12-18 or poly(dA) . (dT)12-18. Similar results were obtained for DNA polymerase alpha (B form) from mouse myeloma MOPC 104E.     

Specific inhibition of DNA biosynthesis induced by 3'-amino-2',3'-dideoxycytidine

3'-Amino-2',3'-dideoxycytidine (3'-NH2-dCyd) produced an S-phase-specific block in exponentially growing L1210 leukemia cells. The monophosphate and triphosphate forms of the drug were detected within a few hours of 3'-NH2-dCyd treatment of intact cells. No significant change in the deoxynucleoside triphosphate levels was observed during the early stages of treatment. However, by 24 h a 2-fold increase in the amount of the deoxynucleoside triphosphates was seen. The triphosphate form of the drug competitively inhibited dCTP incorporation into calf thymus DNA using highly purified DNA polymerase alpha. The Ki was determined to be 9.6 microM with respect to dCTP. Incorporation of the analogue into DNA was not detected. On the other hand, sucrose gradient analysis suggested that incorporation of the analogue into actively synthesized DNA may account for the biological activity of this compound. Treatment with 3'-NH2-dCyd induced single-strand breaks in actively synthesized DNA, but no double-strand breaks were observed in the presence of the analogue. The data indicate that 3'-amino-2',3'-dideoxycytidine specifically interferes with DNA replication at the level of DNA polymerase by inhibiting chain elongation.     

Inhibition of DNA polymerase activity by methyl methanesulfonate

Methyl methanesulfonate (MMS) inhibits both thymidine incorporation into DNA in mitogen-activated human lymphocytes and deoxythymidine triphosphate incorporation into template DNA by DNA polymerase-alpha in a cell-free system. When MMS-modified DNA was used as the template for DNA synthesis utilizing unmodified DNA polymerase-alpha, nucleotide incorporation into template DNA was not inhibited. When unmodified DNA was used as the template for DNA synthesis utilizing MMS-modified DNA polymerase-alpha, nucleotide incorporation was differentially inhibited dependent on the MMS concentration. An analysis of the kinetics of DNA polymerase-alpha inhibition showed that incorporation of all 4 deoxynucleoside triphosphates into DNA template was noncompetitively inhibited by MMS, which is consistent with nonspecific MMS modification of the enzyme. These data indicate that MMS modification of DNA polymerase-alpha alone is sufficient to inhibit the incorporation of deoxynucleoside triphosphates into template DNA in vitro. The data further indicate that alkylation of both DNA polymerase-alpha and DNA template synergistically increases inhibition of DNA synthesis.     

Search for a DNA gyrase in mammalian mitochondria.

Incorporation of labeled deoxynucleoside triphosphates into mtDNA by isolated rat liver mitochondria has been shown previously to reflect DNA replication. We have used this system to seek evidence for a mtDNA gyrase. Coumermycin, novobiocin, nalidixic acid, and oxolinic acid are known to be inhibitors of Escherichia coli gyrase, to inhibit E. coli DNA replication, to abolish colicin E1 replication, and to depress the supercoiling of phage lambda DNA, the last two via inhibition of the DNA gyrase. Our results show that these agents inhibit [3H]dATP incorporation into bulk mtDNA at concentrations similar to those used for E. coli. Analysis by sucrose gradient sedimentation confirms the inhibition and shows further that the synthesis of the highly supercoiled form of mtDNA (i.e. 39 S DNA) is depressed relative to other mtDNA forms (i.e. 27 S DNA), suggesting an inhibition of the supercoiling process. Analysis of the DNA by CsCl/propidium diiodide centrifugation shows, in addition, that incubation with coumermycin results in the appearance of a mtDNA form shown to be relaxed mtDNA. The results are consistent with the occurrence of a mtDNA gyrase and its operation in mtDNA replication.     

Relations between synthesis of deoxyribonucleotides and DNA replication in 3T6 fibroblasts

In exponentially growing 3T6 cells, the synthesis of deoxythymidine triphosphate (dTTP) is balanced by its utilization for DNA replication, with a turnover of the dTTP pool of around 5 min. We now investigate the effects of two inhibitors of DNA synthesis (aphidicolin and hydroxyurea) on the synthesis and degradation of pyrimidine deoxynucleoside triphosphates (dNTPs). Complete inhibition of DNA replication with aphidicolin did not decrease the turnover of pyrimidine dNTP pools labeled from the corresponding [3H]deoxynucleosides, only partially inhibited the in situ activity of thymidylate synthetase and resulted in excretion into the medium of thymidine derived from breakdown of dTTP synthesized de novo. These data demonstrate continued synthesis of dTTP in the absence of DNA replication. In contrast, hydroxyurea decreased the turnover of pyrimidine dNTP pools 5-50-fold. Hydroxyurea is an inhibitor of ribonucleotide reductase and stops DNA synthesis by depleting cells of purine dNTPs but not pyrimidine dNTPs. Our results suggest that degradation of dNTPs is turned off by an unknown mechanism when de novo synthesis is blocked.     

Inhibition of erythrocyte plasma membrane NADH dehydrogenase by nucleotides and uncouplers

Erythrocyte ghost NADH dehydrogenase is inhibited in a competitive fashion by ATP and ADP whereas other nucleoside di- and triphosphates, cyclic nucleosides, as well as non-phosphorylating ATP analogs are relatively ineffective. In addition, this enzyme, measured with ferricyanide as electron acceptor, is inhibited by uncouplers of oxidative phosphorylation (proton-conducting reagents), the inhibition being competitive in character (i.e., the uncouplers were without influence upon maximum velocity). The effectiveness of the uncouplers was in the order of their hydrophobic character with the presence of the alkyl side chain rendering nonyl-dinitrophenol much more active than 2,6-dinitrophenol itself. Hydrophobic compounds that are not protonophores (e.g., eosin, proflavin or valinomycin) were not inhibitory. Whereas adenine nucleotides probably inhibit NADH oxidation competitively through structural similarity with the substrate, it appears unlikely that uncouplers compete at the NADH site directly. Rather, the apparently-competitive inhibition in the latter case may reflect competition for proton transfer to an acceptor residing in a hydrophobic region of the enzyme complex.     

Inhibition of erythrocyte plasma membrane NADH dehydrogenase by nucleotides and uncouplers

Erythrocyte ghost NADH dehydrogenase is inhibited in a competitive fashion by ATP and ADP whereas other nucleoside di- and triphosphates, cyclic nucleosides, as well as non-phosphorylating ATP analogs are relatively ineffective. In addition, this enzyme, measured with ferricyanide as electron acceptor, is inhibited by uncouplers of oxidative phosphorylation (proton-conducting reagents), the inhibition being competitive in character (i.e., the uncouplers were without influence upon maximum velocity). The effectiveness of the uncouplers was in the order of their hydrophobic character with the presence of the alkyl side chain rendering nonyl-dinitrophenol much more active than 2,6-dinitrophenol itself. Hydrophobic compounds that are not protonophores (e.g., eosin, proflavin or valinomycin) were not inhibitory. Whereas adenine nucleotides probably inhibit NADH oxidation competitively through structural similarity with the substrate, it appears unlikely that uncouplers compete at the NADH site directly. Rather, the apparently-competitive inhibition in the latter case may reflect competition for proton transfer to an acceptor residing in a hydrophobic region of the enzyme complex.     

Replication of T4 DNA in Escherichia coli Treated with Toluene

Deoxyribonucleoside triphosphates are incorporated into T4 DNA in infected cells treated with toluene. Under the proper conditions the incorporation is controlled by the known T4 DNA polymerase and proceeds by a semiconservative mechanism. Both strands of the phage DNA are replicated into a high molecular weight progeny molecule. The replication system is accessible to extracellular pancreatic DNase added to the reaction mixture. At early times after infection a second replication system, not under control of the gene 43 polymerase, has been detected which synthesizes T4 DNA in toluenized cells.     

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.     

Replication of bacteriophage M13 DNA in plasmolysed Escherichia coli cells

Plasmolysed M13 infected E. coli cells utilize deoxynucleoside triphosphates to synthesize phage-specific DNA in an ATP-dependent, nalidixic acid sensitive, semi-conservative replication process. Whereas the major fraction of the reaction product consists of replicative form I molecules (RF) labeled asymmetrically in the viral strand, a minor fraction of the label is found in mature viral single strands. We therefore conclude that the system is capable of initiating second rounds of replication, for which ring closure seems to be a precondition.     

Dissociation of the DNA replicase system of bovine lymphocyte nuclei.

Exposure of S-phase nuclei or subnuclear preparations from phytohemagglutinin-stimulated bovine lymphocytes to 0.02 M ATP caused an immediate and almost total loss of their ability to replicate DNA in vitro. Other ribonucleoside and deoxyribonucleoside triphosphates caused a similar inhibition of DNA replication. Levels of ATP which inhibit replication cause the release of DNA polymerases alpha and beta and small pieces of DNA from these nuclei. This release occurs both at 4 and 37 degrees C. The data support the conclusion that high levels of ATP or other nucleoside triphosphates inhibit DNA replication in nuclei by dissolution of the DNA replication complex. The limited success in reconstitution of the DNA replicase complexes is discussed.     

Development of an in vitro bacteriophage N4 DNA replication system

An in vitro DNA replication system from bacteriophage N4-infected Escherichia coli has been developed. It requires MgCl2, all four deoxyribonucleoside triphosphates, and exogenously added N4 phage DNA; other DNAs are used inefficiently or not at all. Ribonucleoside triphosphates are not required, although they stimulate DNA synthesis. In vitro replication starts at the ends of the N4 genome and moves progressively inward. Initiation occurs through hairpin priming at the 3' ends of the genome, but shows a strong preference for the right end. Three N4 gene products (dnp, dbp, and exo) required in vivo for N4 DNA synthesis are absolutely required in the in vitro system. These findings are discussed with respect to the mode of N4 DNA replication.     

Source of Energy for Gliding Motility in Flexibacter polymorphus: Effects of Metabolic and Respiratory Inhibitors on Gliding Movement

The effects of selected metabolic and respiratory inhibitors on the gliding motility of Flexibacter polymorphus were examined. Motility and oxygen consumption were quantitatively inhibited in a reversible manner by specific respiratory poisons, suggesting that gliding velocity was linked to electron transport activity. Arsenate had little influence on the number or rate of gliding filaments, despite a 95% decrease in the concentration of intracellular adenosine 5′-triphosphate (ATP). At concentrations of cyanide or azide that abolished gliding movement, cells possessed a level of ATP that should have been sufficient to allow motility. Proton-conducting uncouplers of oxidative phosphorylation, such as carbonylcyanide m-chlorophenylhydrazone (CCCP) and tetrachlorosalicylanilide, strongly inhibited locomotion yet did not suppress respiratory activity or intracellular ATP sufficiently to account for their effect on movement. Inhibition of motility by CCCP (but not by tetrachlorosalicylanilide) was partially reversed by sulfhydryl compounds. However, unlike CCCP, inhibition of motility by p-chloromercuribenzoate, a known sulfhydryl-blocking reagent, was associated with a corresponding reduction in respiratory activity and ATP content of cells. Protein synthesis was not blocked by concentrations of CCCP inhibitory for motility, indicating that utilization of existing ATP in this energy-requiring process was not impaired. These data suggest (but do not unequivocally prove) that ATP may not function as the sole energy donor for the gliding mechanism, but that some additional product of electron transport is required (e.g., the intermediate of oxidative phosphorylation).     

Inhibition of membrane transport in Streptococcus faecalis by uncouplers of oxidative phosphorylation and its relationship to proton conduction

We studied the effect of compounds that uncouple oxidative phosphorylation on membrane function in Streptoccocus faecalis, an organism which relies upon glycolysis for the generation of metabolic energy. At low concentrations (ranging from 10(-7) to 10(-4)m), tetrachlorosalicylanilide, tetramethyldipicrylamine, carbonylcyanide m-chlorophenylhydrazone, pentachlorophenol, and dicoumarol strongly inhibited energy-dependent transport of rubidium, phosphate, and certain amino acids. However, these compounds had little effect on the generation of adenosine triphosphate via glycolysis or on its utilization for the synthesis of macromolecules. They also did not seriously inhibit uptake of those monosaccharides and amino acids which do not require concurrent metabolism. It is proposed that the uncouplers interfere with the utilization of metabolic energy for membrane transport. The uncouplers accelerated the translocation of protons across the cytoplasmic membrane. It appears that a proton-impermeable membrane is required for transport, perhaps, because a proton gradient is involved in the coupling of metabolic energy to the translocation of substrates across the membrane.     

Tests for Deoxyribonucleic Acid Synthesis in Toluenized Bacillus subtilis Cells

Several tests were devised to further characterize deoxyribonucleic acid (DNA) synthesis in toluenized Bacillus subtilis cells. Vigorous agitation of toluenized cells (localization test) demonstrated that the DNA replication is exclusively a cell-associated process. A DNA "repair" condition was also applied to toluenized cells and shown to be distinct from DNA replication in its DNA polymerase I dependency and its ability to synthesize DNA on template which is either cell associated or free, outside the cell. This repair condition was used in conjunction with the localization test to demonstrate the penetration of deoxyribonuclease I and possibly DNA polymerase I into toluenized cells. Therefore, we suggest that the localization test can be used to test the penetration of proteins into toluenized cells for both the DNA repair and replication processes.