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At least 19 recordsLinked to original sources
PubMed1977

Regulation of deoxyribonucleotide biosynthesis during in vivo bacteriophage T4 DNA replication. Intrinsic control of synthesis of thymine and 5-hydroxymethylcytosine deoxyribonucleotides at precise ratio found in DNA.

The kinetics of the de novo formation of pyrimidine deoxyribonucleotides is the same after infection by wild type bacteriophage T4, which generate very low steady state levels of deoxytibonucleotides, and by T4 DNA synthesis-negative mutatants (Dna-), which accumulate high levels, suggesting that the control is not by a feedback mechanism. In this study, the ratio of the de novo synthesis of dTMP to HmdCMP derivatives was measured by determining the total thymine and 5-hydroxylxytosine (HmCyt) deoxyribonucleotides synthesized by the reductive pathways from [6-3H]uracil including those in DNA and any degradation products excreted into the medium. The ratio of the de novo synthesis of Thy/HmCyt derivatives remained constant at 2.1 +/- 0.1 for at least 45 min after infection by wild type phage, i.e. precisely at the Thy/HmCyt ratio in T4 DNA. On infection by phage mutated in the Dna-genes 32, 41, 44, or 45, the ratio still remained close to 2 to 1 for at least 25 min. Only after the pyrimidine deoxyribonucleotide concentrations reached levels about 100-fold greater than the initial values did the ratio begin to increase. However, a mutant of the structural gene for T4 DNA polymerase showed some increase in ratio by 15 min. Mutants of gene 1 (HmdCMP kinase) were distinct in that the Thy/HmCyt ratio dropped to about 1.0 by 25 min, and then remained quite constant. Uniquely, in these mutants a significant quantity of 5-hydroxymethyluracil or a derivative was found, about 40% being in the medium. The product was shown to be derived by deamination of a 5-HmCyt derivative. All Dna- mutants tested excreted 35 to 50% of their thymine derivatives, mostly as thymine, into the medium. Neither thymine nor 5-hydroxymethyluracil derivates were excreted after wild type phage infection. We propose that pyrimidine deoxyribonucleotide synthesis is regulated at a Thy:HmCyt ratio of 2:1 as an intrinsic property of a complex of enzymes synthesizing and channeling deoxyribonucleotides for T4 DNA replication and not exclusively by effector-sensitive mechanisms.

Coliphages↗
PubMed1978

A ribo-deoxyribonucleotide primer synthesized by primase.

The 29-residue ribonucleotide primer formed by primase at the origin of phage G4 DNA replication (Bouché, J.-P, Rowen, L., and Kornberg, A. (1978) J. Biol. Chem. 253, 765-769) was shorter in the presence of deoxynucleoside triphosphates (dNTPs). At 50 micrometer dNTPs and 20 micrometer rNTPs, RNA trancripts no longer than 6 residues were synthesized and these were still effective in priming replication by the DNA polymerase III holoenzyme. Primer synthesis was initiated with ATP; adenosine 5'-O-(3-thiotriphosphate) (Appp(S)), adenosine 5'-tetraphosphate, adenylyl imidodiphosphate (App(NH)p), and ADP were able to substitute for ATP. dATP and GTP were ineffective in initiating replication. DNA replication was stimulated by GTP, suggesting that incorporation of this nucleotide into the second position of the primer trancript by primase produces a more efficient primer. Each of the dNTPs can be incorporated into a hybrid ribonucleotide-deoxyribonucleotide transcript, indicating that primase is able to add either a ribonucleotide or deoxyribonucleotide to the 3'-OH of either of ribo residue or a deoxy residue of the primer terminus. Incorporation of an individual dNTP was less efficient than that of the corresponding rNTP, and the presence of all four dNTPs profoundly depressed RNA synthesis by primase.

Adenosine Triphosphate↗
PubMed1975

Ribo- and deoxyribonucleotide triphosphate pools in synchronized populations of Tetrahymena pyriformis.

The content of ribo- and deoxyribonucleotide triphosphates has been measured in asynchronous and synchronized populations of Tetrahymena pyriformis. The concentration of ribonucleotide triphosphates does not vary through the cell cycle. It seems to be a rule that the ATP pool is reduced during division only in cell types which also reduce macromolecular syntheses and respiration. The content of deoxyribonucleotide triphosphates varies through the cell cycle. The pools are maximal during the S-period, but measurable amounts are present in all phases.

Adenosine Triphosphate↗
PubMed1976

[The interaction between DNase I and the constituent deoxyribonucleotides of DNA].

A study of the interaction between deoxyribonucleotides and DNase I (E.C. 3.1.4.5) has disclosed a greater affinity of puric deoxyribonucleotides for the enzyme. DNase I showed one binding site for dAMP with an affinity constant of 2-4 X 10(4) M(-1). MnCl2 decreases the fixation of dAMP more effectively than MgCl2.

Adenine Nucleotides↗
PubMed1978

Age-dependent DNA labeling and deoxyribonucleotide synthesis in wheat seeds.

Utilisation of ribonucleosides as precursors of DNA biosynthesis was studied in germinating wheat embryos because the reductive pathway leading to deoxyribonucleotides is very difficult to demonstrate in extracts of higher plants in vitro. [5-3H]Cytidine and [6-3H]uridine are incorporated into wheat DNA (RNA-free) via ribonucleotide reduction without intermediate scission of the glycosidic bond. This reaction is observed at 20-30 h after the onset of germination only in aged (2-4-year-old) seeds while the embryos isolated from fresh grains show very little cytidine incorporation; in contrast, thymidine incorporation into DNA between 10 and 18 h of germination is not age dependent. Fresh wheat contains a soluble, heat-stable inhibitor fraction, most probably a modified oligonucleotide, which efficiently prevents cytidine incorporation when added to old embryos together with the labeled nucleoside. This material also inhibits purified Escherichia coli ribonucleotide reductase and is thought to be part of the control system for ribonucleotide reduction in wheat; it may gradually decay during storage of the seeds. Dry wheat embryos do not contain deoxyribonucleoside triphosphates. Pool sizes of dATP and dTTP in germinating embryos were found to reach 1 pmol/microgram DNA at 10-15 h of germination (i.e. before ribonucleotide reduction) and were independent of the age of seeds. These data suggest that wheat contains other preformed dexoyribonucleoside derivatives which are phosphorylated at an early time and can initially sustain DNA synthesis. Induction of measurable ribonucleotide reductase activity in fresh winter wheat was for the first time accomplished by 15 days of vernalization of the seeds at +2 degrees C.

Cytidine↗
PubMed1976

Role of vitamin B12 in the reduction of ribonucleotides into deoxyribonucleotides in Drosophila cells grown in vitro.

Adenosine was found to inhibit growth of Drosophila melanogaster cells in culture. This toxic effect is prevented by the addition of uridine + deoxyuridine, or uridine + deoxycytidine. In the presence of vitamin B12, uridine alone is sufficient to sustain proliferation of Drosophila cells inhibited by adenosine. Moreover, vitamin B12 increases the incorporation of [3H] uridine into DNA, and decreases the incorporation of [3H] thymidine. No modification of the incorporation of [14C] adenine, [14C] adenonsine or [3H] cytosine into DNA could be found in the presence of vitamin B12. It is concluded that vitamin B12 is involved in an enhanced conversion of uridine ribonucleotides into deoxyribonucleotides derived from uridine.

Adenosine↗
PubMed1978

Interactions of 4-nitroquinoline 1-oxide with four deoxyribonucleotides.

The interactions of 4-nitroquinoline 1-oxide (NQO) with the four 5'-deoxyribonucleotides were probed using absorption spectra of the charge transfer bands and 1H and 13C nuclear magnetic resonance (NMR) spectra of nucleotide-NQO mixtures. Spectral data yielded equilibrium constants (K(dpG:NQO) = 16 M-1, K(dpA:NQO) = 12 M-1, K(dpT:NQO) = K(dpC:NQO) = 4 M-1) which suggest the preference of NQO for the guanine residue in a DNA. This is in agreement with the data of Okano, T., et al. [(1969) Gann 60, 295]. From 13C and 1H NMR data on nucleosides, a structure for the dpG:NQO complex is proposed.

4-Nitroquinoline-1-oxide↗
PubMed1975

Cyclopenta[f]isoquinoline derivatives designed to bind specifically to native deoxyribonucleic acid. 2. Synthesis of 6-carbamylmethyl-8-methyl-7(5)H-cyclopenta[f]isoquinolin-3(2H)-one and its interaction with deoxyribonucleic acids and poly(deoxyribonucleotides).

3-Ethoxy-8-methyl-5,6-dihydro-7H-cyclopenta[f]isoquinolin-5-one (2) was converted to 6-carbethoxymethyl-3-ethoxy-8-methyl-5,6-dihydro-7H-cyclopenta[f]isoquinolin-5-one (6) through an oxalyl derivative. Treatment of 6 with ammonia gave the corresponding amide 7 which on sodium borohydride reduction and subsequent dehydration yielded 6-carbamylmethyl-3-ethoxy-8-methyl-7(5)H-cyclopenta[f]isoquinoline (9). The analogous ester 10 was similarly obtained from 6. Numerous attempts to dealkylate the 3-ethoxy group of 9 or 10 failed. However, 6 coould easily be dealkylated on heating with 25% hydrochloric acid in a sealed tube.The ester, 6-carbethoxymethyl-8-methyl-5,6-dihydro-7H-cyclopenta[f]isoquinoline-3(2H),5-dione (11), so obtained was converted to the corresponding amide 12 which on reduction with sodium borohydride and subsequent dehydration afforded the desired compound, 6-car-bamylmethyl-8-methyl-7(5)H-cyclopental[f]isoquinolin-3-(2H)-one (1). 1 was found to be mildly cytotoxic againstL5178Y mouse leukemia cells in culture.1 was also found to bind to native calf thymus DNA. 1 inhibited RNA synthesis by a DNA-dependent RNA polymerase and a higher inhibition of RNA synthesis was observed when poly(dG-dC) was used as a template than when poly(dA-dT) was used. A significant increase of thermal transition temperature of calf thymus DNA and poly(dG)-poly(dC) was observed in the presence of 1. The accumulated evidence demonstrates that 1 interacts weakly with calf thymus DNA and interacts preferentially with poly(deoxyribonucleotides)-containing GC pairs.

Animals↗
PubMed1976

Simultaneous initiation of synthesis of bacteriophage T4 DNA and of deoxyribonucleotides.

In earlier reports we have suggested that bacteriophate T4 DNA replication occurs in a complex composed of the proteins required for polymerization and the system of enzymes synthesizing the deoxyribonucleoside triphosphate precursors of DNA. T4-induced dCMP hydroxymethylase and dTMP synthetase, though demonstrable in extracts soon after infection, are not active in vivo until about 5 min. The in vivo activities increase exponentially for approximately 15 min and then become constant. We have suggested that the exponential period represents the formation of the complexes. This paper shows that the initiation of DNA synthesis and of the two deoxyribonucleotide-synthesizing activities occurs simultaneously and with coinciding exponential kinetics. The in vivo activities of the two enzymes were tested after infection by a number of T4 amber Dna- mutants. Their activities were essentially unchanged compared to the wild-type phage, except on infection by mutants of gene 43 (T4 DNA nucleotidyltransferase or DNA polymerase). With these mutants the rate of increase of dTMP synthetase and dCMP hydroxymethylase activities was always substantially lower than after infection by wild-type phage. It is proposed that an intimate interaction occurs between T4-induced DNA polymerase and the complex of enzymes forming 5-hydroxymethyl-dCMP and dTMP.

Chloramphenicol↗
PubMed1978

Nuclear-magnetic-resonance studies of 5'-ribonucleotide and 5'-deoxyribonucleotide conformations in solution using the lanthanide probe method.

The conformations of the metal-bound 5'-ribonucleotides and 5'-deoxyribonucleotides in aqueous solution at different pH values have been studied using the lanthanide probe method. The conformational analysis, based on mixing different conformations in fast exchange within the nuclear magnetic resonance time scale, agrees well with the results from coupling constants, nuclear Over-hauser effects and spin-lattice relaxation times, obtained for the metal-fixed systems. The equilibrium between the two basic conformational combinations for the 5'-nucleotides, anti-(N in equalibrium S)-gg-g'g' and syn-(N in equalibrium S)-gt-g'g' depends on the nature of the furanose ring, the base and also on the state of base protonation and phosphate ionization. The effect of base protonation is particularly strong for the guanine nucleotides.

Adenosine Monophosphate↗
PubMed1979

Deoxyribonucleotide synthesis and DNA polymerase activity in plant cells (Vicia faba and Glycine max).

Enzymes of deoxyribonucleotide and DNA biosynthesis, which are little known in plants, were studied in root tips of germinating broad beans (Vicia faba) and in fast-growing cultures of soybean cells (Glycine max). The plant cells contain a ribonucleoside 5'-diphosphate reductase which is detected in vitro only during a limited period of growth, viz. 30--32 h after inhibition of Vicia seeds, and between the second and third day after inoculation of soybean cultures. In both species ribonucleotide reductase activity precedes maximum DNA synthesis. The reductases could be precipitated with ammonium sulfate but were not purified further due to the extremely low enzyme content of the plant extracts. Therefore the reductive pathway of deoxyribotide formation was also established in Vicia root tips by efficient labeling of the plant DNA with a ribonucleoside, [5-3H]cytidine, which reaches a maximum at the same time as the reductase activity measured in vitro. Cycloheximide inhibits this process, indicating the need for de novo enzyme induction. In contrast, DNA polymerase is present in the tissue throughout the entire development and rises only 2-fold in activity during the S phase. The soluble polymerases were partially characterized in both legume species and were found very similar to the DNA polymerase of pea seedlings. Ribonucleotide reductase is more likely a limiting component of DNA formation during the plant cell cycle than DNA polymerase.

DNA↗
PubMed1975

Deoxyribonucleotide metabolism in Herpes simplex virus infected HeLa cells.

The effect of Rolly No. 11 strain herpes simplex virus infection of HeLa cells in culture on deoxynucleotide metabolism and the level of various enzymes concerned with the biosynthesis of DNA has been investigated. Of 18 enzyme activities studied, thymidine kinase, DNA polymerase and deoxyribonuclease were markedly augmented, a finding in agreement with previous reports. Deoxycytidine kinase, ribonucleotide reductase, thymidylate kinase and deoxycytidylate deaminase activities, in contrast with previous reports, did not increase; the activities of the other enzymes studied, also did not increase. Whereas most of the radioactivity derived from [14-C] thymidine in the acid-soluble fraction of the uninfected cells was present as deoxythymidine triphosphate, that present in the infected cells was primarily in the form of deoxythymidine monophosphate. Thus, in the infected cell deoxythymidylate kinase is a rate-limiting enzyme in the biosynthesis of deoxythymidine triphosphate. A marked increase in the pools of the four naturally occurring deoxynucleoside triphosphates (dTTP, dCTP, dATP, dGTP) was found. The rate of formation of the virus-induced enzymes was determined, as were the various nucleoside triphosphate pools and the other phosphorylated derivatives of thymidine; a maximum was reached for all these csmponents between 6 to 8 h post infection. Although an apparent greater synthesis of DNA occurred in the uninefected cells, when the specific activity of the radioactive deoxythymidine triphosphate was taken into account, there was actually a greater rate of DNA synthesis in the infected cells, with the peak at 8 h post infection.

Biological Transport↗
PubMed1976

Deoxyribonucleotide pools in mouse-fibroblast cell lines with altered ribonucleotide reductase.

Mutant cells lines of 3T6 mouse fibroblasts, resistant to thymidine and deoxyadenosine, have an altered allosteric regulation of the enzyme ribonucleotide reductase (Meuth, M. and Green, H., Cell, 3, 367, 1974). Compared to 3T6, these lines contain larger pools of deoxynucleoside triphosphates, in particular deoxycytidine triphosphate, but show a normal rate of DNA synthesis. Addition of thymidine or deoxyadenosine to 3T6 cells results in large accumulations of the corresponding triphosphates and a dramatic decrease in the dCTP pool, concomitant with inhibition of DNA synthesis. Addition of thymidine to the mutant cell lines also leads to an increase in the dTTP pool but does not result in a depletion of dCTP or inhibition of DNA synthesis. Addition of deoxyadenosine only leads to a small increase of the dATP pool. In general the change in the allosteric regulation of bibonucleotide reductase is reflected in the deoxynucleotide pools.

Animals↗
PubMed1976

Altered deoxyribonucleotide pools in P2 eductants of Escherichia coli K-12 due to deletion of the dcd gene.

Deletion of the Escherichia coli K-12 chromosome associated with P2 mediated education extend through the structural gene for uridine kinase, udk, and the dcd gene encoding 2'-deoxycytidine 5'-triphosphate deaminase. The lack of uridine kinase makes a positive selection possible for these strains. Due to the dcd mutation, P2 eductants show large alterations in their deoxyribonucleoside triphosphate pools.

Deoxyribonucleotides↗