Coupled ribonucleoside diphosphate reduction, channeling, and incorporation into DNA of mammalian cells

There is rapid and specific channeling of ribonucleoside diphosphates into DNA through reactions beginning with ribonucleotide reductase and terminating with DNA polymerase. Lysolecithin-permeabilized Chinese hamster embryo fibroblasts in culture rapidly reduced ribonucleoside diphosphates by ribonucleotide reductase action when dithiothreitol was provided as a reducing agent and incorporated these deoxynucleotides into DNA. The radioactive label provided in ribo-CDP was not diluted by added deoxyribo-CTP during its incorporation into DNA, showing that the ribo-CDP does not pass through a deoxy-CTP pool. Under the conditions that permitted rapid incorporation of ribonucleoside diphosphates, deoxynucleoside triphosphates were very poorly incorporated. Ribonucleotide reductase with the rate-limiting enzyme for the overall process. The Km values for the reductase reaction and the overall process were similar and low enough for saturation by in vivo pools. Natural feedback inhibitors dATP or dTTP inhibited incorporation of labeled ribo-CDP into deoxyribonucleotides and into DNA to the same extent. Ribonucleotide reductase behaved like other enzymes that are associated in a rapidly sedimenting form. It was concentrated in the nucleus during S phase, and most of the enzyme activity in these nuclear extracts was co-sedimented with DNA polymerase on sucrose density gradients. These data support the hypotheses that a physically associated complex of enzymes (replitase) catalyzes the production of deoxynucleotides and their incorporation into DNA in S phase cells.     

Activities of DNA polymerases and RNA polymerases detected in situ in growing and differentiating cells of root cortex.

Activities of DNA polymerases and RNA polymerases were studied by autoradiographic methods in growing and differentiating root cortex cells of Zea mays - a species in which endomitosis occurs - and Tulipa kaufmanniana - in which this process does not occur. In Tulipa kaufmanniana, the highest activity of DNA polymerase appears in the nuclei of meristematic zone during the S phase of the cell cycle. In Zea mays, endomitotic replication of DNA occurs in all growth and differentiation zones and the activity of DNA polymerase in the nuclei is similar to that in the meristematic zone. In both species, nuclear RNA synthesis, measured with 3H uridine incorporation, is highest in the meristematic zone and declines steadily with development. Activity of nuclear RNA polymerase is present in all developmental zones in both species and is similar to that in the meristematic zone. 3H uridine incorporation into nucleoli decreases markedly in both species, whereas the activity of nucleolar RNA polymerase remains at a high level in all root segments in Zea mays and decreases slightly in Tulipa kaufmanniana. It is argued that the differences between the incorporation of 3H uridine and that or 3H UMP may be caused by a reduction of the pool of endogenous ribonucleoside triphosphates. Marked activities of DNA polymerase and RNA polymerase in cytoplasm are possibly related to the growth and division of plastids and mitochondria.     

Organization and expression of leghaemoglobin genes

Leghaemoglobin genes in soybean (Glycine max) are present as a moderately reiterated family of sequences. Since there are identical restriction site patterns of these sequences in DNA isolated from leaf, root, or nodule tissue, the data suggest that no major changes in the organization or methylation of leghaemoglobin genes occur during their induction. Cloned soybean leghaemoglobin-cDNA cross hybridized with RNA from root nodules of kidney bean (Phaseolus vulgaris), and to a lesser extent, of pea (Pisum sativum) indicating sequence homology in the leghaemoglobin genes of these species. Hybridization to the genomic DNA restriction fragments of two other species, Glycine soja and Vicia faba, also indicated interspecies sequence homologies. Several restriction fragments appear to be common to all species examined. The induction of these genes occurs following infection of the plant by Rhizobium and is independent of the appearance of nitrogenase activity in the nodules. The level of expression is, however, influenced by various mutations in Rhizobium that result in the development of ineffective (nonnitrogen fixing) nodules.     

Adaptation of the cells ofEscherichia coli to the presence of hydroxyurea increases the level of inorganic pyrophosphatase activity

Hydroxyurea, an inhibitor of ribonucleoside diphosphate reductase, completely arrested the net synthesis of DNA for 3–4 h, when it was added in 30 mM concentration to growing cultures ofEscherichia coli K12. Thereafter the net synthesis of DNA started again, although slowly, and simultaneously with it the formation of inorganic pyrophosphatase activity was stimulated leading to a 2-fold increase in the specific activity of the enzyme in 2–3 h. Subsequently cell division began again. In this way a new steady state, stable in the presence of hydroxyurea, was reached. This new state was characterized by the high specific activity of inorganic pyrophosphatase, a small but constant amount of DNA/cell mass (1/4 of the normal value), and large elongated cells. All these changes were slowly reversed during 5–6 h, when the cells were transferred into a drug-free medium.The activity of isoleucyl-tRNA synthetase, assayed as a control, did not change significantly in the presence of hydroxyurea.Hydroxyurea had no effect on the activity of inorganic pyrophosphatase in vitro.     

Regulation of ribonucleoside diphosphate reductase synthesis in Escherichia coli: increased enzyme synthesis as a result of inhibition of deoxyribonucleic acid synthesis.

Inhibition of deoxyribonucleic acid (DNA) synthesis in Escherichia coli by chemical inhibitors or by shifting cultures of temperature-sensitive elongation (dnaE and dnaB) or initiation (dnaA) mutants to nonpermissive conditions led to greatly increased synthesis of the enzyme ribonucleoside diphosphate reductase, which catalyzes the first reaction unique to the pathway leading to DNA replication. In contrast to the Gudas and Pardee proposed model for control of the synthesis of DNA repair enzymes, in which both DNA inhibition and DNA degradation are involved, DNA synthesis inhibition in recA, recB, recC, or lex strains results in increased synthesis of ribonucleotide reductase, which suggests that DNA degradation is not required. We propose that inhibition of DNA synthesis causes a cell to accumulate an unknown compound that stimulates the initiation of a new round of DNA replication, and that this same signal is used to induce ribonucleotide reductase synthesis.     

Ribonucleotide reduction and the possible role of cobalamin in evolution

The biological pathways of ribonucleotide reduction are briefly reviewed. The hypothesis is presented that reduction of ribonucleoside triphosphates to their deoxynucleotide analogs through the mediation of vitamin B12 or a similar corrinoid preceded and was necessary for the subsequent development of a DNA-type genome. There are two known biological systems for ribonucleotide reduction: (1) The ribonucleoside diphosphate reduction system which utilizes a nonheme iron ribonucleotide reductase enzyme, thioredoxin and its reductase, and NADPH. This enzyme complex is found in most bacteria, some higher organisms, and in all animals. (2) The ribonucleoside triphosphate reduction system which utilizes adenosyl cobalamin, ribonucleotide reductase and either thioredoxin or a disulfhydryl compound. The cobalamin-dependent reductase is restricted to a few species of bacteria and blue-gree algae. This system is considered more primitive than the iron reductase one based on their differences in distribution, components, and products.     

Does hydroxyurea inhibit DNA replication in mouse cells by more than one mechanism?

Cell-free DNA synthesis was performed in a lysed cell system from mouse cell cultures. The in vitro reaction was totally inhibited by N-ethylmaleimide but unaffected by hydroxyurea or fluorodeoxyuridine when these compounds were added to the incubation mixture. However, in a preparation obtained from cells which had been blocked by hydroxyurea before lysis, the rate of DNA synthesis was markedly reduced. This effect could not have been caused by the depletion of the precursor pools as all necessary triphosphates were added to the in vitro incubation mixture. Analysis by alkaline density gradients showed that the ligation of primary synthesis products is retarded in hydroxyurea-pretreated lysed cells and that small fragments accumulate. These results suggest that hydroxyurea interferes with the processing of early replication products, preventing the formation of longer intermediates. Its mechanism is either independent from the well-known inhibition of ribonucleoside diphosphate reductase or it may be the result of an as-yet-unknown function of this enzyme in a later step of replication. This observation could help to explain why cells appear to be blocked by hydroxyurea in the early part of the S phase (rather than at the G1/S border proper) and also why DNA repair synthesis is relatively insensitive to the drug.     

Nuclear DNA characterization of two species ofVicia:Vicia bithynica L. andVicia narbonensis L.

The speciesVicia bithynica andVicia narbonensis, from the same subgeneric section ofVicia faba, show variations in nuclear DNA content Nuclear DNAs, extracted from root tips of the twoVicia species, were characterized by thermal denaturation, analytical ultracentrifugation and reassociation kinetics. The thermal denaturations of DNA, the number of DNA components reassociating with second order kinetics, the proportion of repeated DNA sequences, the frequency of the repeated DNA classes are reported and compared to previous data onVicia faba DNA. Feulgen absorptions at different thresholds of optical density⁺ of interphase nuclei in cytological preparations of the root meristems ofV. bithynica andV. narbonensis are determined and compared withV. faba analogous determinations. The results, confirming that plant genome is highly flexible, are discussed in relation to other data on the interspecific variations of the nuclear DNA content.     

Selection and characterization of mutant S49 T-lymphoma cell lines resistant to phosphonoformic acid: evidence for inhibition of ribonucleotide reductase

Phosphonoformic acid (PFA) and its congener phosphonoacetic acid (PAA) are inhibitors of viral replication whose mechanism of action appears to be the inhibition of viral DNA polymerase. These drugs inhibit mammalian DNA polymerase to a lesser extent. We sought to characterize the effects of phonoformic acid on mammalian cells by examining mutants of S49 cells (a mouse T-lymphoma line), which were selected by virtue of their resistance to phosphonoformic acid. The 11 mutant lines that were resistant to growth inhibition by 3 mM PFA had a range of growth rates, cell cycle distribution abnormalities, and resistance to the inhibitory effects of thymidine, acycloguanosine (acyclovir), aphidicolin, deoxyadenosine, and novobiocin. Most mutant lines had pools of ribonucleoside triphosphates and deoxyribonucleoside triphosphates similar to those of wild-type S49 cells. However, one line (PFA 3-9) had a greatly elevated dCTP pool. When this mutant line was further characterized, no apparent defect in DNA polymerase alpha activity was seen, but an increased ribonucleotide reductase activity, as assayed by CDP reduction in permeabilized cells, was observed. The CDP reductase activity in the PFA 3-9 cells decreased to wild-type control levels, and the CDP reductase activity of wild-type cells was also greatly reduced when PFA (2-3 mM) was added to permeabilized cells during the enzyme assay. These results demonstrate that PFA can directly inhibit ribonucleotide reductase activity in permeabilized cells. In addition, when PFA was added to exponentially growing cultures of either wild-type or PFA 3-9 mutant cells, the drug caused an arrest in S phase of the cell cycle and a decrease in all four deoxyribonucleotide pools, with the most dramatic decrease in the dCTP pools. The reduction in the dCTP pool level could be reversed by addition of exogenous deoxycytidine, but this reversed PFA toxicity only marginally. These observations suggest that PFA is an inhibitor of mammalian ribonucleotide reductase and that partial resistance to PFA can be effected by mutation to increased CDP reductase activity resulting in a large dCTP pool. This mutation results in less than twofold resistance to PFA, suggesting that other sites of inhibition coexist.     

Effect of 5-fluoro-2′-deoxyuridine and hydroxyurea on the phytohemagglutinin-induced increase of thymidine kinase,replicative DNA polymerase,deoxycytidylate deaminase and CDP reductase activities in human lymphocytes

Summary The inhibitors of DNA synthesis, 5-fluoro-2-deoxyuridine and hydroxyurea, caused an inhibition of thymidine kinase, replicative DNA polymerase and CDP reductase activities in stimulated lymphocytes when they were exposed to the inhibitors during the early transformation period (0–17 hr). However, the enzyme activities were unaffected when the inhibitors were added to cells stimulated for more than 17 hr. As opposed to these enzymes the deoxycytidylate deaminase activity was unaffected by the inhibitors during the entire transformation period (0–28 hr). This indicates a close regulatory mechanism in lymphocytes between DNA synthesis and induction of enzymes involved in DNA replication. The inhibitory mechanism exerted by the inhibitors is for the moment unknown. It might be independent of the well-known inhibition of the target enzymes, thymidylate synthetase and ribonucleoside diphosphate reductase, since there was no immediate apparent correlation in time between depletion of the pool sizes and the inhibition of the enzyme activities.     

3,4-Dichloroaniline is detoxified and exported via different pathways in Arabidopsis and soybean

The metabolic fate of [UL-14C]-3,4-dichloroaniline (DCA) was investigated in Arabidopsis root cultures and soybean plants over a 48 h period following treatment via the root media. DCA was rapidly taken up by both species and metabolised, predominantly to N-malonyl-DCA in soybean and N-glucosyl-DCA in Arabidopsis. Synthesis occurred in the roots and the respective conjugates were largely exported into the culture medium, a smaller proportion being retained within the plant tissue. Once conjugated, the DCA metabolites in the medium were not then readily taken up by roots of either species. The difference in the routes of DCA detoxification in the two plants could be explained partly by the relative activities of the respective conjugating enzymes, soybean containing high DCA-N-malonyltransferase activity, while in Arabidopsis DCA-N-glucosyltransferase activity predominated. A pre-treatment of plants with DCA increased DCA-N-malonyltransferase activity in soybean but not in Arabidopsis, indicating differential regulation of this enzyme in the two plant species. This study demonstrates that DCA can undergo two distinct detoxification mechanisms which both lead to the export of conjugated metabolites from roots into the surrounding medium in contrast to the vacuolar deposition more commonly associated with the metabolism of xenobiotics in plants.     

The mitotic Ca2+-adenosine triphosphatase in proliferating tissue of Vicia faba

The mitotic Ca2+-ATPase was isolated from root tips of Vicia faba. The enzyme shows the same characteristics as the one isolated from a variety of animal cells. The results support the hypothesis that the membrane-bound mitotic Ca2+-ATPase is part of a Ca2+-regulating system universal for both plant and animal cells.     

Ribonucleotide reductase activity in green algae

A ribonucleoside diphosphate reductase is demonstrated in the algae, $\text{Scenedesmus}\text{obliquus}$ and $\text{Chlorella}\text{pyrenoidosa}$. In synchronized cultures an activity maximum at the 12th hour of the cell cycle coincides with maximum DNA production. Induction of reductase activity is prevented by cycloheximide. The enzyme requires dithiols for reduction of CDP $\text{in}\text{vitro}$; it is not significantly stimulated by iron or magnesium ions nor dependent upon deoxyadenosylcobalamin. ATP stimulates the reaction but dATP or dTTP act as inhibitors. The ribonucleotide reductase of green algae differs from the B₁₂-requiring enzyme characterized in $\text{Euglena}\text{gracilis}$.     

The effects of post-treatments with caffeine during S and G2 on the frequencies of chromosomal aberrations induced by thiotepa in root tips of Vicia faba and in human lymphocytes in vitro

The effects of post-treatments with caffeine on the frequencies of chromosomal aberrations induced by the trifunctional alkylating agent thiotepa were studied in human lymphocytes and in root tips of Vicia faba. In lymphocytes the frequency of aberrations induced in G0 or G1 was most strongly increased when the caffeine post-treatments were given during G2. In Vicia faba, on the other hand, the frequency of aberrations induced in early interphase was unaffected by post-treatments with caffeine during G2, but strongly increased when the root tips were exposed to caffeine during the S phase.     

Isolation and properties of a testicular ribonucleic acid polymerase

A procedure is described for isolation in a soluble form of a ribonucleic acid polymerase from rat testes. Evidence is presented that this enzyme catalyzes three distinct reactions: (a) deoxyribonucleic acid-directed synthesis of RNA in the presence of all four major ribonucleoside triphosphates; (b) DNA-primed formation of polyadenylic acid and other ribohomopolymers in the presence of single ribonucleoside triphosphate substrates; (c) synthesis of complementary polyribonucleotides in the presence of various ribohomopolymer primers. The properties of these reactions are discussed with respect to metal ion requirements, affinities for ribonucleoside triphosphates and primer polynucleotides, heat denaturation of DNA primers, and the effects of ionic strength, beta-mercaptoethanol, polyamines, temperature, and inhibitors on the rates and extent of the reactions. The testicular ribonucleic acid polymerase is a very unstable enzyme that can be stabilized by high concentrations of glycerol.     

Activities of DNA polymerases and RNA polymerases detected in situ in growing and differentiating cells of root cortex

Summary Activities of DNA polymerases and RNA polymerases were studied by autoradiographic methods in growing and differentiating root cortex cells ofZea mays — a species in which endomitosis occurs — andTulipa kaufmanniana — in which this process does not occur. InTulipa kaufmanniana, the highest activity of DNA polymerase appears in the nuclei of meristematic zone during the S phase of the cell cycle. InZea mays, endomitotic replication of DNA occurs in all growth and differentiation zones and the activity of DNA polymerase in the nuclei is similar to that in the meristematic zone. In both species, nuclear RNA synthesis, measured with³H uridine incorporation, is highest in the meristematic zone and declines steadily with development. Activity of nuclear RNA polymerase is present in all developmental zones in both species and is similar to that in the meristematic zone.³H uridine incorporation into nucleoli decreases markedly in both species, whereas the activity of nucleolar RNA polymerase remains at a high level in all root segments inZea mays and decreases slightly inTulipa kaufmanniana.It is argued that the differences between the incorporation of³H uridine and that or³H UMP may be caused by a reduction of the pool of endogenous ribonucleoside triphosphates. Marked activities of DNA polymerase and RNA polymerase in cytoplasm are possibly related to the growth and division of plastids and mitochondria.     

Histogenesis and localization of non-specific esterase in root tip

A procedure was developed for satisfactory freeze-sectioning of root tips. The use of Ca formol-fixed material kept and frozen in Holt's syrup is recommended. The existence and different localization of 2 fractions of non—specific esterase was verified in root tips ofVicia faba. The same results were revealed in fixed and unfixed material. The dynamics ofin situ reaction was followed with respect to optimal incubation time. The results with substrates of different chain length support the existence of 2 fraction of the studied enzyme, none of which, concerning substrate specificity, is a lipase. It follows from the present studies inVicia faba and other species (Cucurbita pepo, Lupinus albus, Pisum sativum Zea mays), that non-specific esterase localization is not directly given by histogenesis.     

Bacteriophage-T7-induced DNA-priming protein. A novel enzyme involved in DNA replication.

The T7gene-4 protein has been purified to near homogeneity using a complementation assay in vitro, and it is designated T7 DNA-priming protein (DNA primase). The purified enzyme enables T7 DNA polymerase to initate DNA synthesis on various circular single-stranded DNA templates by a mechanism which involes the synthesis of a very short RNA primer. The oligoribonucleotide, which is linked to the product DNA via a 3':5'-phosphodiester bond, starts with pppA-C and terminates predominantly with AMP. When only ATP and CPT are precursors, the RNA primer is found to be primarily a tetranucleotide of the sequence pppA-C-C-A. Using oligoribonucleotides in place of ribonucleoside triphosphates as chain initators, T7 DNA-priming protein drastically increases the efficiency with which T7 DNA polymerase can utilize particular tetranucleotide primers containing A and C residues. T7 DNA-priming protein also enables T7 DNA polymerase to make use of native or nicked duplex T7 DNA as template-primer. This reaction does not require ribonucleoside triphosphates, although their addition enhances DNA synthesis 2--4 fold. The product formed in their absence is covalently attached to the template DNA and is found to contain a few long branches when examined by electron microscopy. In the presence of ribonucleoside triphosphates most of the newly made product arises from imitation of DNA chains de novo. Incubation of three proteins: T7 DNA-priming protein, T7 DNA polymerase, and T7 DNA-binding protein, with ribonucleoside and deoxyribonucleoside triphosphates, and with phiX174DNA as template leads to the generation of 'rolling circle-like' structures as visualized in the electron microscope. Single-stranded regions at the tail-circle junction indicate that initations can occur de novo on the displaced complementary strand. This is consistent with a discontinuous mode of 'lagging' strand synthesis and suggests that the same proteins may also be responsible for fork propagation in vivo.     

Induced plant responses to pathogen attack. Analysis and heterologous expression of the key enzyme in the biosynthesis of phytoalexins in soybean (Glycine max L. Merr. cv. Harosoy 63).

In soybean (Glycine max L.), pathogen attack induces the formation of glyceollin-type phytoalexins. The biosynthetic key enzyme is a reductase which synthesizes 4,2', 4'-trihydroxychalcone in co-action with chalcone synthase. Screening of a soybean cDNA library from elicitor-induced RNA in lambda gt11 yielded two classes of reductase-specific clones. The deduced proteins match to 100% and 95%, respectively, with 229 amino acids sequenced in the purified plant protein. Four clones of class A were expressed in Escherichia coli, and the proteins were tested for enzyme activity in extracts supplemented with chalcone synthase. All were active in 4,2',4'-trihydroxychalcone formation, and the quantification showed that shorter lengths of the cDNAs at the 5' end correlated with progressively decreasing enzyme activities. Genomic blots with DNA from plants capable of 4,2',4'-trihydroxychalcone synthesis revealed related sequences in bean (Phaseolus vulgaris L.) and peanut (Arachis hypogaea L.), but not in pea (Pisum sativum L.). No hybridization was observed with parsley (Petroselinum crispum) and carrot (Daucus carota) which synthesize other phytoalexins. The reductase protein contains a leucine-zipper motif and reveals a marked similarity with other oxidoreductases most of which are involved in carbohydrate metabolism.