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.     

Nucleotide and thioredoxin specificity of the manganese ribonucleotide reductase from Brevibacterium ammoniagenes

The manganese-containing ribonucleotide reductase previously identified in gram-positive bacteria has been purified and its nucleotide specificity and other requirements were determined. The enzyme isolated from Brevibacterium ammoniagenes is a ribonucleoside-diphosphate reductase which, in the presence of allosteric effectors, reduces all four common substrates at comparable rates; very little activity is observed in the absence of effector nucleotides. Ribonucleoside triphosphates are reduced at 20% the rate of the diphosphates. Cytidine and uridine nucleotide reduction is specifically stimulated by ATP and dATP, adenylate reduction by dGTP, and guanosine nucleotide reduction by dTTP. Unlike the iron-containing ribonucleotide reductase systems, high concentrations of dATP do not inhibit substrate reduction. The new bacterial enzyme tolerates high salt concentrations (up to 250 mM ionic strength) and does not require divalent metal ions for activity in vitro. The presence of thioredoxin has been demonstrated in heat- and acid-treated protein extracts of B. ammoniagenes and the protein was purified to homogeneity. It is very similar to the thioredoxins isolated from other organisms in relative molecular mass (12,000), isoelectric point (4.3) and enzyme-activating properties. In the presence of 0.3 mM dithiothreitol, the bacterial thioredoxin can serve as hydrogen donor for B. ammoniagenes ribonucleotide reductase in vitro, indicating the presence of a functional ribonucleotide reductase-thioredoxin system in these bacteria. The properties described in this and in our preceding paper in this journal [Eur. J. Biochem. 170, 603-611 (1988)] suggest that the B. ammoniagenes ribonucleotide reductase is intermediate in structure and specificity between the deoxyadenosylcobalamin-dependent and the iron-containing enzyme classes and that it is adapted to the specific requirements of deoxyribonucleotide synthesis in this organism.     

Isolation and initial characterization of a series of Chlamydia trachomatis isolates selected for hydroxyurea resistance by a stepwise procedure.

Chlamydiae are obligate intracellular bacteria that are dependent on eukaryotic host cells for ribonucleoside triphosphates but not deoxyribonucleotide triphosphates. Ribonucleotide reductase is the only enzyme known to catalyze the direct conversion of a ribonucleotide to a deoxyribonucleotide. Hydroxyurea inhibits ribonucleotide reductase by inactivating the tyrosine free radical present in the small subunit of the enzyme. In this report, we show that Chlamydia trachomatis growth is inhibited by hydroxyurea in both wild-type mouse L cells and hydroxyurea-resistant mouse L cells. Hydroxyurea was used as a selective agent in culture to isolate, by a stepwise procedure, a series of C. trachomatis isolates with increasing levels of resistance to the cytotoxic effects of the drug. One of the drug-resistant C. trachomatis isolates (L2HR-10.0) was studied in more detail. L2HR-10.0 retained its drug resistance phenotype even after passage in the absence of hydroxyurea for 10 growth cycles. In addition, L2HR-10.0 was cross resistant to guanazole, another inhibitor of ribonucleotide reductase. Results obtained from hydroxyurea inhibition studies using various host cell-parasite combinations indicated that inhibition of host cell and C. trachomatis DNA synthesis by hydroxyurea can occur but need not occur simultaneously. Crude extract prepared from highly purified C. trachomatis reticulate bodies was capable of reducing CDP to dCDP. The CDP reductase activity was not inhibited by monoclonal antibodies to the large and small subunits of mammalian ribonucleotide reductase, suggesting that the activity is chlamydia specific. The CDP reductase activity was inhibited by hydroxyurea. Crude extract prepared from drug-resistant L2HR-10.0 reticulate bodies contained an elevation in ribonucleotide reductase activity. In total, our results indicate that C. trachomatis obtains the precursors for DNA synthesis as ribonucleotides with subsequent conversion to deoxyribonucleotides catalyzed by a chlamydia-specific ribonucleotide reductase.     

Induction of a new ribonucleotide reductase after infection of mouse L cells with pseudorabies virus.

The mammalian ribonucleotide reductase consists of two nonidentical subunits, protein M1 and M2. M1 binds nucleoside triphosphate allosteric effectors, whereas M2 contains a tyrosine free radical essential for activity. The activity of ribonucleotide reductase increased 10-fold in extracts of mouse L cells 6 h after infection with pseudorabies virus. The new activity was not influenced by antibodies against subunit M1 of calf thymus ribonucleotide reductase, whereas the reductase activity in uninfected cells was completely neutralized. Furthermore, packed infected cells (but not mock-infected cells) showed an electron paramagnetic resonance spectrum of the tyrosine free radical of subunit M2 of the cellular ribonucleotide reductase. These data given conclusive evidence that on infection, herpesvirus induces a new or modified ribonucleotide reductase. The virus-induced enzyme showed the same sensitivity to inhibition by hydroxyurea as the cellular reductase. The allosteric regulation of the virus enzyme was completely different from the regulation of the cellular reductase. Thus, CDP reduction catalyzed by the virus enzyme showed no requirement for ATP as a positive effector, and no feedback inhibition was observed by dTTP or dATP. The virus reductase did not even bind to a dATP-Sepharose column which bound the cellular enzyme with high affinity.     

Cell cycle regulation of ribonucleoside diphosphate reductase activity in permeable mouse L cells and in extracts

Ribonucleoside diphosphate reductase (EC1.17.4.1) was previously characterized in exponentially growing mouse L cells selectively permeabilized to small molecules by treatment with dextran sulfate (Kucera and Paulus, 1982b). This characterization has now been extended to cells in specific phases of the cell cycle and in transition between cell cycle phases, with activity studied both in situ (permeabilized cells) and in cell extracts. Cells at various stages in the cell cycle were obtained by unit-gravity sedimentation employing a commercially available reorienting chamber device, by G1 arrest induced by isoleucine limitation, and by metaphase arrest induced by Colcemid. G1 cells from both cycling and noncycling populations had negligible levels of ribonucleotide reductase activity as measured by CDP reduction both in situ and in extracts. When G1 arrested cells were allowed to progress to S phase, ribonucleotide reductase activity increased in parallel with [3H]thymidine incorporation into DNA. Ribonucleotide reductase activity in extracts increased at a somewhat greater rate than in situ activity. S phase ribonucleotide reductase activity measured in situ resembled the previously characterized activity in exponentially growing cells with respect to an absolute dependence on ATP or its analogs as positive allosteric effector, sensitivity to the negative allosteric effector dATP, and low susceptibility to stimulation by NADPH, dithiothreitol, and FeCl3. Disruption of permeabilized cells caused reductase activity to become highly dependent on the presence of both dithiothreitol and FeCl3. As synchronized cultures progressed from S into G2/M phase, no significant change in ribonucleotide reductase activity was seen. On the other hand, when cells that had been arrested in metaphase by Colcemid were allowed to resume cell cycle traversal by removing the drug, in situ ribonucleotide reductase activity decreased by 75% within 2.5 h. This decrease seemed to be a late mitotic event, since it was not correlated with the percentage of cells entering G1 phase. The cause of a subsequent slight increase of in situ ribonucleotide reductase activity is not clear. Parallel measurements of ribonucleotide reductase activity in cell extracts indicated also an initial decline accompanied by increasing dependence on added dithiols and FeCl3, followed by complete activity loss. Our results suggest a cell cycle pattern of ribonucleotide reductase activity that involves negligible levels in G1 phase, a progressive increase of activity upon entry into S phase paralleling overall DNA synthesis, continued retention of significant ribonucleotide reductase activity well into the metaphase period of mitosis, and a very rapid decline in activity during the later phases of mitosis. The periods of increase and decrease of ribonucleotide reductase activity were accompanied by modulation of the properties of the enzyme as indicated by differential changes in enzyme activity measured in situ and in extracts.     

Ribonucleotide reductase of herpes simplex virus type 2 resembles that of herpes simplex virus type 1.

The ribonucleotide reductase (ribonucleoside-diphosphate reductase; EC 1.17.4.1) induced by herpes simplex virus type 2 infection of serum-starved BHK-21 cells was purified to provide a preparation practically free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could significantly deplete the substrates. Certain key properties of the herpes simplex virus type 2 ribonucleotide reductase were examined to define the extent to which it resembled the herpes simplex virus type 1 ribonucleotide reductase. The herpes simplex virus type 2 ribonucleotide reductase was inhibited by ATP and MgCl2 but only weakly inhibited by the ATP X Mg complex. Deoxynucleoside triphosphates were at best only weak inhibitors of this enzyme. ADP was a competitive inhibitor (K'i, 11 microM) of CDP reduction (K'm, 0.5 microM), and CDP was a competitive inhibitor (K'i, 0.4 microM) of ADP reduction (K'm, 8 microM). These key properties closely resemble those observed for similarly purified herpes simplex virus type 1 ribonucleotide reductase and serve to distinguish these virally induced enzymes from other ribonucleotide reductases.     

Deletion of the varicella-zoster virus large subunit of ribonucleotide reductase impairs growth of virus in vitro.

Cells infected with varicella-zoster virus (VZV) express a viral ribonucleotide reductase which is distinct from that present in uninfected cells. VZV open reading frames 18 and 19 (ORF18 and ORF19) are homologous to the herpes simplex virus type 1 genes encoding the small and large subunits of ribonucleotide reductase, respectively. We generated recombinant VZV by transfecting cultured cells with four overlapping cosmid DNAs. To construct a virus lacking ribonucleotide reductase, we deleted 97% of VZV ORF19 from one of the cosmids. Transfection of this cosmid with the other parental cosmids yielded a VZV mutant with a 2.3-kbp deletion confirmed by Southern blot analysis. Virus-specific ribonucleotide reductase activity was not detected in cells infected with VZV lacking ORF19. Infection of melanoma cells with ORF19-deleted VZV resulted in plaques smaller than those produced by infection with the parental VZV. The mutant virus also exhibited a growth rate slightly slower than that of the parental virus. Chemical inhibition of the VZV ribonucleotide reductase has been shown to potentiate the anti-VZV activity of acyclovir. Similarly, the concentration of acyclovir required to inhibit plaque formation by 50% was threefold lower for the VZV ribonucleotide reductase deletion mutants than for parental virus. We conclude that the VZV ribonucleotide reductase large subunit is not essential for virus infection in vitro; however, deletion of the gene impairs the growth of VZV in cell culture and renders the virus more susceptible to inhibition by acyclovir.     

Iron deprivation decreases ribonucleotide reductase activity and DNA synthesis

The effects of the iron-chelator, desferrioxamine, and monoclonal antibodies against transferrin receptors of DNA synthesis and ribonucleotide reductase activity were examined in human leukemia K562 cells. Treatment of the cells with desferrioxamine resulted in decreases of ribonucleotide reductase activity, DNA synthesis, and cell growth. Exposure of the cells to anti-transferrin receptor antibody, 42/6, which blocks iron supplement into cells caused decreases of ribonucleotide reductase activity and DNA synthesis, in a parallel fashion. Decreases of ribonucleotide reductase activity and DNA synthesis by 42/6 were restored by the addition of ferric nitriloacetate. These results indicate that ribonucleotide reductase activity is dependent on the iron-supply and also regulates cell proliferation.     

Subcellular localization of ribonucleotide reductase in Novikoff hepatoma and regenerating rat liver

The subcellular localization of ribonucleotide reductase was ascertained in Novikoff heptoma and normal and regenerating rat tissue. Over 90% of the cellular ribonucleotide reductase is found to be associated with a membrane fraction derived from the postmicrosomal supernatant after centrifugation at 78,000g for 18 hr which bands at 1.3 m sucrose in a discontinuous sucrose gradient. The properties of this particular ribonucleotide reductase are similar to those reported for mammalian ribonucleotide reductase. This membrane fraction, which contains ribonucleotide reductase, had been previously shown to contain a DNA polymerase whose activity is related to cell proliferation. The association of these two enzymes involved in DNA synthesis leads to the suggestion that there may exist a complex of enzymes involved in deoxynucleotide and DNA synthesis in this membrane fraction.     

Reversion of hydroxyurea resistance, decline in ribonucleotide reductase activity, and loss of M2 gene amplification

A key rate-limiting reaction in the synthesis of DNA is catalyzed by ribonucleotide reductase, the enzyme which reduces ribonucleotides to provide the deoxyribonucleotide precursors of DNA. The antitumor agent, hydroxyurea, is a specific inhibitor of this enzyme and has been used in the selection of drug resistant mammalian cell lines altered in ribonucleotide reductase activity. An unstable hydroxyurea resistant population of mammalian cells with elevated ribonucleotide reductase activity has been used to isolate three stable subclones with varying sensitivities to hydroxyurea cytotoxicity and levels of ribonucleotide reductase activities. These subclones have been analyzed at the molecular level with cDNA probes encoding the two nonidentical subunits of ribonucleotide reductase (M1 and M2). Although no significant differences in M1 mRNA levels or gene copy numbers were detected between the three cell lines, a strong correlation between cellular resistance, enzyme activity, M2 mRNA and M2 gene copies was observed. This is the first demonstration that reversion of hydroxyurea resistance is directly linked to a decrease in M2 mRNA levels and M2 gene copy number, and strongly supports the concept that M2 gene amplification is an important mechanism for achieving resistance to this antitumor agent through elevations in ribonucleotide reductase.     

Different cellular distribution of thioredoxin and subunit M1 of ribonucleotide reductase in rat tissues

The cellular distribution of thioredoxin and protein M1 of ribonucleotide reductase in adult rat tissues was investigated with immunohistochemical techniques using specific antisera. Tissues with high or low frequency of either mitotic or meiotic cell divisions were compared. Thioredoxin was demonstrated in many cells types that showed no detectable protein M1 of ribonucleotide reductase. A few cell types with protein M1 immunoreactivity also contained immunoreactive thioredoxin. However, in most cells no such co-localization could be demonstrated. This lack of correlation between cells containing subunit M1 of ribonucleotide reductase and the thioredoxin indicates that thioredoxin is not the physiologist hydrogen donor for ribonucleotide reductase in rat tissues and that the expression of two enzymes is differently regulated.     

Activity of ribonucleotide reductase helps determine how cells repair DNA double strand breaks

Mammalian cells can choose either nonhomologous end joining (NHEJ) or homologous recombination (HR) for repair of chromosome breaks. Of these two pathways, HR alone requires extensive DNA synthesis and thus abundant synthesis precursors (dNTPs). We address here if this differing requirement for dNTPs helps determine how cells choose a repair pathway. Cellular dNTP pools are regulated primarily by changes in ribonucleotide reductase activity. We show that an inhibitor of ribonucleotide reductase (hydroxyurea) hypersensitizes NHEJ-deficient cells, but not wild type or HR-deficient cells, to chromosome breaks introduced by ionizing radiation. Hydroxyurea additionally reduces the frequency of irradiated cells with a marker for an early step in HR, Rad51 foci, consistent with reduced initiation of HR under these conditions. Conversely, promotion of ribonucleotide reductase activity protects NHEJ-deficient cells from ionizing radiation. Importantly, promotion of ribonucleotide reductase activity also increases usage of HR in cells proficient in both NHEJ and HR at a targeted chromosome break. Activity of ribonucleotide reductase is thus an important factor in determining how mammalian cells repair broken chromosomes. This may explain in part why G1/G0 cells, which have reduced ribonucleotide reductase activity, rely more on NHEJ for DSB repair.     

Improvement of a simple method to purify ribonucleotide reductase

The use of an ATP-agarose column to purify ribonucleotide reductase from human D-98 cells was recently reported. The column selectively retains greater than 99.9% of the contaminating nucleoside diphosphate (NDP) kinase from crude preparations of ribonucleotide reductase. It was presently found, however, that extending the length of the column caused the ribonucleotide reductase to dissociate into subunits. One subunit appeared in the low ionic strength buffer wash while the other required 0.5 M KCl for elution. The enzyme could also be recovered intact (non-dissociated) by equilibrating the enzyme preparation and the column with 0.5 M KCl prior to chromatography. Either method greatly improved the overall yield and the specific activity of the ribonucleotide reductase because it prevented the binding and subsequent loss of any of the subunits. In addition, the use of a larger column permitted the gel-filtration properties of the ATP-agarose to separate the bulk of the residual (not bound) NDP kinase from the ribonucleotide reductase.     

Differential effect of hydroxyurea on a ribonucleotide reductase system.

Infection of Escherichia coli with phage T4 induces a large increase in ribonucleotide reductase activity. We show that hydroxyurea inhibits T4-induced CDP, ADP, UDP, and GDP reductase activities in vitro. Moreover, there are significant differences in the degree of inhibition of each ribonucleotide reductase activity. The reductase activities for CDP and ADP are more sensitive to hydroxyurea than those for UDP and GDP, particularly at high hydroxyurea molarities. As little as 5 x 10(-4)M hydroxyurea lowers CDP and ADP reductase activities to 25 to 30% whereas as much as 0.5 M hydroxyurea is needed to lower UDP and GDP reductase activities to 50%.     

The molecular weight of Ehrlich tumor cell ribonucleotide reductase and its subunits: Effector-induced changes

The molecular weights of Ehrlich tumor cell ribonucleotide reductase and its individual components were determined by sedimentation equilibrium in the Beckman Airfuge. The distribution of enzyme after sedimentation equilibrium was determined by measurement of the CDP reductase and ADP reductase activities associated with ribonucleotide reductase. The apparent molecular weight of the intact enzyme was 304,000 when assayed for CDP reductase and 254,000 when assayed for ADP reductase. This difference in apparent molecular weights was statistically significant with a P value of 0.0002. The molecular weights of the individual components of ribonucleotide reductase were determined in a similar fashion by assaying in the presence of an excess of the complementary component. The non-heme iron component had a molecular weight of 81,000 when assayed for either CDP or ADP reductase activity. The effector-binding component had an apparent molecular weight of 127,000 when assayed for CDP reductase and 95,000 when assayed for ADP reductase. This difference in apparent molecular weights was statistically significant with a P value of 0.004. The effectors ATP and dGTP altered the apparent molecular weights of the intact enzyme and individual components. In the presence of ATP the molecular weight of intact CDP reductase was 481,000 while the apparent molecular weight of the effector-binding component of CDP reductase alone was 418,000. In the presence of dGTP, the molecular weight of intact ADP reductase was 293,000 while the apparent molecular weight of the effector-binding component of ADP reductase alone was 154,000. These results indicate that the proportion of the non-heme iron component and the effector-binding component is not equimolar and that the composition of the enzyme is not constant but is altered by the presence of effectors. Our data also suggest that CDP reduction and ADP reduction are catalyzed by different molecular species of the enzyme which apparently have different effector-binding components.     

The small subunit of ribonucleotide reductase is encoded by one of the most abundant translationally regulated maternal RNAs in clam and sea urchin eggs

In both clam oocytes and sea urchin eggs, fertilization triggers the synthesis of a set of proteins specified by stored maternal mRNAs. One of the most abundant of these (p41) has a molecular weight of 41,000. This paper describes the identification of p41 as the small subunit of ribonucleotide reductase, the enzyme that provides the precursors necessary for DNA synthesis. This identification is based mainly on the amino acid sequence deduced from cDNA clones corresponding to p41, which shows homology with a gene in Herpes Simplex virus that is thought to encode the small subunit of viral ribonucleotide reductase. Comparison with the B2 (small) subunit of Escherichia coli ribonucleotide reductase also shows striking homology in certain conserved regions of the molecule. However, our attention was originally drawn to protein p41 because it was specifically retained by an affinity column bearing the monoclonal antibody YL 1/2, which reacts with alpha-tubulin (Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582). The finding that this antibody inhibits the activity of sea urchin embryo ribonucleotide reductase confirmed the identity of p41 as the small subunit. The unexpected binding of the small subunit of ribonucleotide reductase can be accounted for by its carboxy-terminal sequence, which matches the specificity requirements of YL 1/2 as determined by Wehland et al. (Wehland, J., H. C. Schroeder, and K. Weber, 1984, EMBO [Eur. Mol. Biol. Organ.] J., 3:1295-1300). Unlike the small subunit, there is no sign of synthesis of a corresponding large subunit of ribonucleotide reductase after fertilization. Since most enzymes of this type require two subunits for activity, we suspect that the unfertilized oocytes contain a stockpile of large subunits ready for combination with newly made small subunits. Thus, synthesis of the small subunit of ribonucleotide reductase represents a very clear example of the developmental regulation of enzyme activity by control of gene expression at the level of translation.     

Sedimentation analysis of ribonucleotide reductase activity in extracts of Pseudomonas stutzeri

Analysis of ribonucleotide reductase and DNA polymerase activities in extracts of Pseudomonas stutzeri by centrifugation in discontinuous sucrose gradients indicated that these two enzymes are associated with two different high molecular weight cellular components. In addition, 95% of the ribonucleotide reductase activity was pelleted by centrifugation of extracts for 4 hr at 200,000 X G. The reductase activity remained particulate (sedimentable) following sonication whereas some 90% of the DNA polymerase activity was rendered soluble (non-sedimentable) by this technique. This data indicate that the P. stutzeri ribonucleotide reductase is not a cytosolic enzyme, but is associated with a macromolecular component in the cell.     

Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency

From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.     

Ribonucleotide reductase from Escherichia coli: demonstration of a highly active form of the enzyme.

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, proteins B1 and B2. The activity of the enzyme in crude extracts prepared from mechanically disrupted bacteria is very low. Enzyme activity is stimulated 5 to 10-fold by addition of an excess of either subunit. Concentrated extracts from cells lysed gently on Cellophane discs (Schaller et al.) contained 10 to 20-fold higher activity than extracts from mechanically disrupted cells. This activity was not further stimulated by either B1 or B2. The system is suitable for complementation tests for the analysis of temperature-sensitive mutants affecting the ribonucleotide reductase system. Concentrated high-speed supernatants from E. coli treated with lysozyme (Wickner et al.) also contained a high ribonucleotide reductase activity, which was stimulated slightly or not at all by addition of B1 and B2. This active form of the enzyme was unstable and could not be purified. The results suggest that the intracellular form of the enzyme consists of a tight complex of proteins B1 and B2, possibly stabilized by other intracellular structures.