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Starting from 2-thiodeoxythymidine, the mono- and triphosphate of the nucleoside analog were prepared by enzymatic phosphorylation with thymidine kinase and thymidylate kinase, respectively. 2-Thiodeoxythymidine triphosphate was also obtained by chemical pyrophosphorylation of the monophosphate. The analog nucleoside-triphosphate is a substra@ for Escherichia coli DNA polymerase. A copolymer consisting of alternating deoxyadenosine- and 2-thiodeoxythyunidine nucleotides is synthesized extensively by the enzyme. The polymer was characterized by nearest neighbour analysis, sedimentation and buoyant density in the analytical ultracentrifuge, ultraviolet spectra and stability against thermal and alkaline denaturation. [ABSTRACT FROM AUTHOR]

We have studied the ribonucleosidediphosphate reductase of a deoxyuridine-requiring mutant LD195 of Escherichia coli K. This enzyme was purified 50 fold from the mutant and a control strain. In all steps of the purification the mutant strain yielded normal activity of the Bi subunit of the enzyme but approximately 10 % of the wild-type activity of the B2 subunit The activity is similarly reduced for all three substrates tested CDP, UDP, and GDP. The mutation appears to affect the physical structure of the B2 subunit, since addition of high concentrations of sodium acetate can restore enzymatic activity to the partially purified B2 subunit from the mutant strain. In contrast, the same concentrations strongly inhibit the wild- type B2 subunit. Although the mutant has a requirement of deoxyuridine at 42 °C but not at 30 °C (as shown in an accompanying paper), mutant protein B2 is not more thermo-labile than wild-type protein B2 This requirement therefore probably reflects an increased demand of deoxyribonucleotides at higher temperatures. Hydroxyurea inhibits mutant and wild-type ribonucleosidediphosphate reductase to the same extent in vitro. In spite of this, hydroxyurea inhibits growth of the mutant strain at a 20 fold lower concentration than that required for comparable inhibition of growth of the parent. This is explained by the greatly decreased protein B2 activity of the mutant. [ABSTRACT FROM AUTHOR]

A procedure to isolate Escherichia coli mutants defective in ribonucleotide reduction is described. Using a parental strain defective in dCTP deaminase (paxA) and thus unable to synthesize dUMP from deoxycytidine nucleotides, we selected for cells unable to reduce UDP at a rate sufficient to satisfy the cell's need for thymidine nucleotides. This was accomplished by selecting for cells resistant to 2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine (trimethoprim) in the presence of thymidine and screening these cells for a thymidine requirement that could be satisfied by deoxyuridine. Genetic analysis as well as enzyme assays of four such mutants chosen for study indicated that three had a defective thymidylate synthetase. One, LD195, which had normal thymidylate synthetase activity, was found to have a conditional deoxyuridine requirement that is only expressed under favourable growth conditions. Since restoration of a functional dCTP deaminase to derivatives of LD195 eliminated the deoxyuridine requirement, it was concluded that a combination of two mutations is responsible for the expression of this phenotype. Analysis of nucleotide pools of LD195 grown exponentially under permissive conditions revealed that both the dTMP and dTTP pools were 6-fold lower than in the parent indicating a deficiency in the production of a thymidine nucleotide precursor. Enzyme assays of crude extracts of LD195 indicated that this deficiency was due to a defective ribonucleosidediphosphate reductase. This conclusion is proven by an extensive enzymatic analysis presented in an accompanying paper. [ABSTRACT FROM AUTHOR]

Infection of Escherichia coli with bacteriophage T5 caused a more than 20-fold increase in ribonucleotide reductase activity in crude cell extracts. The phage induced the formation of a new ribonucleotide reductase and a new thioredoxin. The T5-specific ribonucleotide reductase was defined by its ability to accept the T5-induced thioredoxin as hydrogen donor. However, the enzyme could also accept E. coli thioredoxin as hydrogen donor and it was inhibited by antisera against subunit B2 of E. coli ribonucleotide reductase. The crude phage enzyme preparation studied preferred CTP to CDP and CMP as substrate and required magnesium and ATP for activity. Its behaviour towards allosteric effectors was different from that of the host enzyme. T5-induced thioredoxin chromatographed together with E. coli thioredoxin on DEAEcellulose but could be separated from the latter by immunoadsorbent chromatography. T5 thioredoxin was reduced by E. coli thioredoxin reductase but could not serve as hydrogen donor of E. coli ribonucleotide reductase. Bacteriophage T6 induced a new ribonucleotide reductase and a new thioredoxin upon infection. The T6-induced proteins cross-reacted functionally and immunologically with the corresponding T4-induced proteins. Infection of E. coli B with phage T7 or induction of the lysogens E. coli K-39 (λ+) and E. coli W-3350 (∼c I857) caused no increase in reductase activity and chromatography of cell extracts gave no evidence for the presence of new thioredoxins. [ABSTRACT FROM AUTHOR]

The aminoacylation of 15 fragments from yeast tRNAPhe and of various mixtures of these fragments was studied in detail. The aminoacylation conditions were systematically varied in order to obtain maximal incorporation of phenylalanine. No fragment by itself accepted phenylalanine. Fragment combinations in which parts of the anticodon loop or the dihydrouridine loop were missing, accepted phenylalanine to an extent of 20–75% compared to intact tRNAPhe. Modifications in both regions together inactivated the molecule. With the anticodon stem and/or the manilooop missing, low but significant activity was found. Splits in the dihydrouridine loop and in the T-ψ-C loop together did not abolish the phenylalanine acceptance. Combinations in which parts of the dihydrouridine stem or of the T-ψ-C stem were missing could not be aminoacylated. Complete association of complementary fragments was observed in all cases. The results of charging and heating experiments, however, indicate the coexistence of different flagment combinations in a fragment mixture. The incomplete aminoaeylation of fragment mixtures is explained by the presence of fragment combinations with an unsuitable three-dimensional structure. The rate of aminoacylation was lower for fragment combinations than for tRNAPhe. The Michaelis constants of 3 fragment combinations were identical to the one of t RNAPne. Two fragment mixtures inhibited the rate of charging of tRNAPhe. Phenylalanine was the only amino acid which was accepted by the fragment mixtures with synthetases from yeast or Escherichia coli. No miseharging was observed. The results together with those of related publications are discussed with respect to recognition sites of tRNAPhe for the cognate synthetase. [ABSTRACT FROM AUTHOR]

Cells made permeable to nucleotides by treatment with ether synthesize DNA from exogenous deoxynucleoside triphosphates in a semi-conservative fashion, The reaction proceeds from sites of previous synthesis on the chromosome, and not detectably from new sites. Short chains appear as precursors of long units and have a mean sedimentation coefficient of about 10 S in alkali, like Okazaki-pieces when synthesized in the presence of 10 to 20 μM deoxynucleoside tri- phosphates, and of only 5 S when synthesized in the presence of 1μM dNTP, Under the former conditions short chains contain not more than half of the newly synthesized DNA; under the latter conditions they can contain more than half, the remainder of the newly synthesized DNA being in long units, Nascent short chains labeled after several minutes of synthesis in the presence of 5-bromodeoxyuridine 5'-triphosphate at high deoxynucleoside triphosphate concentrations show bimodal density distribution in an alkaline Cs2SO4 equilibrium gradient with peaks of material corresponding to the density of heavy single strands and almost light single strands, respectively. The implications of these findings are discussed. Judged from controls, repair synthesis was not a source of error in these experiments. In contrast with results reported for intact cells, ether-treated polA1- (Kornberg-polymerase deficient) cells join nascent short chains as efficiently as do polA+ cells. [ABSTRACT FROM AUTHOR]

Glutamine synthetase adenylyltransferase was purified 300-fold from Escherichia coli B grown on minimal medium. The purified enzyme was found to be homogeneous in the ultracentrifuge, but by electrophoresis on polyacrylamide gel two enzymatically active protein components were separated, that differed from batch to batch in their relative proportions. The apparent Km values of the various substrates and activators of the adenylylation of glutamine synthetase were determined. The adenylylating enzyme requires Mg2+ or Mn2+ and ATP for activity. Several other divalent cations and nucleotides were tested and found to be ineffective. In the presence of glutamine the rate of the reaction decreases at ATP concentrations greater than 1 mM. Addition of ADP, CTP, UTP and ITP to the standard assay mixture, containing ATP results likewise in a decrease of the rate of the reaction. Various intermediates of glycolytic pathway and Krebs cycle inhibit the adenylylation reaction. The optimum pH for adenylylation is about 7.6. The activation energy for the adenylylation was estimated from rate measurements. It is 13 kcal/mole in the temperature range from 5° to 32°. Gel filtration on Sephadex G-150 indicates an average molecular weight of 145000 daltons. On storage of the enzyme in solution inactive aggregates of higher molecular weight are formed. Bovine serum albumin prevents inactivation and aggregation. [ABSTRACT FROM AUTHOR]

The effects of nucleotide analogues on DNA synthesis ere studied in nucleotide permeable Escherichia coli cells. 2′,3′,Dideoxyribosylthymine 5′-triphosphate inhibited replicative DNA synthesis far more strongly than endonuclease-induced DNA repair synthesis. In ϕX174-infected cells this analogue caused the formation of short pieces (7 S) of ϕX replicative form DNA in which newly synthesized DNA was found covalently linked to preformed primer. Deoxyuridine triphosphate led to even shorter pieces (3 S) of ϕX replicative form DNA. Nicotinamide mononucleotide, an inhibitor of E. coli DNA ligase, promoted accumulation of short (10 S) E. coil DNA pieces. Newly synthesized 5′-triphosphate ends were not detected in these nucleotide-permeable cells during the early stages of ϕX replicative form replication, nor was evidence obtained that nuoleoside monophosphates are immediate precursors of DNA. [ABSTRACT FROM AUTHOR]