Effect of Hydroxyurea on Replication of Bacteriophage T4 in Escherichia coli

Hydroxyurea inhibited the replication of bacteriophage T4 in Escherichia coli B. The concentration of hydroxyurea required to inhibit net deoxyribonucleic acid (DNA) synthesis 50% was about 50-fold less than that required in uninfected cells. Even in the presence of high hydroxyurea concentrations, phage DNA was readily synthesized from the products of breakdown of the E. coli DNA, and viable phage were made. Deoxyribonucleotide, but not ribonucleotide, synthesis was strongly inhibited in the presence of hydroxyurea. The data indicate that hydroxyurea specifically inhibits de novo DNA synthesis in E. coli infected with bacteriophage T4 by inhibiting the ribonucleoside diphosphate reductase system, but does not affect DNA synthesis at subsequent steps.     

Isotope-dilution analysis of the effects of deoxyguanosine and deoxyadenosine on the incorpo?ation of thymidine and deoxycytidine by hydroxyurea-treated thymus cells

It is presumed that the dGTP and dATP needed for replicative DNA synthesis can be formed by way of either `salvage' pathways or biosynthesis de novo. This was examined by adding hydroxyurea to cultures of rat thymus cells to inhibit ribonucleoside diphosphate reductase, a key enzyme of the `de novo' pathway. Most of the inhibition of the incorporation of [Me-³H]thymidine and deoxy[5-³H]cytidine by low concentrations of hydroxyurea (100–500μm) was prevented by substrates of the salvage pathway (400μm-deoxyguanosine and, to a lesser extent, 200μm-deoxyadenosine). However, isotope-dilution studies indicated that the purine deoxyribonucleosides prevented inhibition by decreasing pyrimidine deoxyribonucleotide competitor pools. Evidence was obtained that a hydroxyurea-induced increase in the thymidine-competitor pool (probably dTTP) was prevented to an equal extent by deoxyguanosine and by the inhibitor of thymidylate synthase, deoxy-5-fluorouridine. These compounds had almost identical effects on hydroxyurea dose–response curves and on thymidine isotope-dilution plots. The evidence suggests that exogenous purine deoxyribonucleosides cannot prevent the inhibition by hydroxyurea of thymus-cell DNA synthesis. This could mean that, with respect to the metabolism of purine deoxyribonucleotides, ribonucleoside diphosphate reductase is tightly coupled to DNA polymerase in a multienzyme complex. The complex would not permit entry of exogenous metabolic intermediates into the `de novo' pathway, but would still be subject to the regulatory effects of these intermediates. Thus dGTP and dATP formed from exogenous purine deoxyribonucleosides by salvage pathways might deplete pyrimidine deoxyribonucleotide competitor pools by inhibiting relatively hydroxyurea-insensitive activities of ribonucleoside diphosphate reductase.     

Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region.

Recombinant plasmids containing all or part of the genetic region of Escherichia coli coding for the two subunits of ribonucleoside diphosphate reductase (proteins B1 and B2) were constructed with the aid of the multicopy plasmid pBR322. Two of these plasmids (pPS1 and pPS2) appeared to carry both a regulator and the complete structural information for the enzyme and, after transformation of E. coli, directed a 10- to 20-fold overproduction of both proteins B1 and B2. The other plasmids (pPS101 and pPS201) carried structural information for only protein B2. Cells carrying pPS1 and pPS2 showed a 5- to 500-fold increased resistance against the drug hydroxyurea. This establishes that in E. coli the inhibition of deoxyribonucleic acid synthesis by hydroxyurea is fully explained by its action on ribonucleotide reductase.     

Amplification of the genes for both components of ribonucleotide reductase in hydroxyurea resistant mammalian cells

Ribonucleotide reductase catalyzes the formation of deoxyribonucleotides from ribonucleoside diphosphate precursors, and is a rate-limiting step in the synthesis of DNA. The enzyme consists of two dissimilar subunits usually called M1 and M2. The antitumor agent, hydroxyurea, is a specific inhibitor of DNA synthesis and acts by destroying the tyrosyl free radical of the M2 subunit of ribonucleotide reductase. Two highly drug resistant cell lines designated HR-15 and HR-30 were isolated by exposing a population of mouse L cells to increasing concentrations of hydroxyurea. HR-15 and HR-30 cells contained elevated levels of ribonucleotide reductase activity, and were 68 and 103 times, respectively, more resistant than wild type to the cytotoxic effects of hydroxyurea. Northern and Southern blot analysis indicated that the two drug resistant lines contained elevated levels of M2 mRNA and M2 gene copy numbers. Similar studies with M1 specific cDNA demonstrated that HR-15 and HR-30 cell lines also contained increased M1 message levels, and showed M1 gene amplification. Mutant cell lines altered in expression and copy numbers for both the M1 and M2 genes are useful for obtaining information relevant to the regulation of ribonucleotide reductase, and its role in DNA synthesis and cell proliferation.     

Reduction of ribonucleotides by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

Rickettsia prowazekii, an obligate intracellular parasitic bacterium, was shown to have a ribonucleotide reductase that would allow the rickettsiae to obtain the deoxyribonucleotides needed for DNA synthesis from rickettsial ribonucleotides rather than from transport. In the presence of hydroxyurea, R. prowazekii failed to grow in mouse L929 cells or SC2 cells (a hydroxyurea-resistant cell line), which suggested that R. prowazekii contains a functional ribonucleotide reductase. This enzymatic activity was demonstrated by the conversion of ADP to dADP and CDP to dCDP, using (i) a crude extract of Renografin-purified R. prowazekii that had been harvested from infected yolk sacs and (ii) high-performance liquid chromatographic analysis. The rickettsial ribonucleotide reductase utilized ribonucleoside diphosphates as substrates, required magnesium and a reducing agent, and was inhibited by hydroxyurea. ADP reduction was stimulated by dGTP and inhibited by dATP. CDP reduction was stimulated by ATP and adenylylimido-diphosphate and inhibited by dATP and dGTP. These characteristics provided strong evidence that the rickettsial enzyme is a nonheme iron-containing enzyme similar to those found in mammalian cells and aerobic Escherichia coli.     

Induction of the SOS response by hydroxyurea in Escherichia coli K12

Hydroxyurea at concentrations higher than 10(-2) M induced the recA and sfiA genes of E. coli as well as the lambda prophage by a pathway independent of the recBC genes. In addition, the hydroxyurea-mediated induction of the SOS response is accompanied by a recA-dependent decrease on the cellular ATP pool. The presence of the multicopy plasmid pPS2, harboring the nrdAB genes (encoding the ribonucleoside reductase enzyme), abolished the hydroxyurea-induced expression of the recA gene. These data lead us to suggest that induction of the SOS response by hydroxyurea is due to the blocking of DNA replication by the inhibition of the ribonucleoside reductase complex activity.     

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.     

Inhibition of mammalian ribonucleotide reductase by a dinucleotide produced in eucaryotic cells.

HS3, a highly phosphorylated dinucleoside originally purified from the fungus Achlya, has been isolated from Chinese hamster ovary cells undergoing glutamine starvation. The HS3 compounds obtained from the fungal and mammalian sources exhibited similar physical and chemical properties. This unusual dinucleotide may be an important regulator of eucaryotic ribonucleoside diphosphate reductase activity; for 50 micrometer HS3, isolated from either mammalian or fungal cells, significantly inhibited CDP reduction in Achlya or hamster cell preparations, but only marginally affected the activity of the enzyme from E. coli. Studies with HS3 isolated from Achlya and partially purified mammalian ribonucleotide reductase indicated that the compound noncompetitively inhibited the reduction of varying concentrations of the substrates CDP, ADP and GDP with Ki values of 23 micrometer, 14 micron and 16 micron respectively. These inhibitor concentrations are well below the estimated intracellular levels of HS3 in glutamine starved cells and suggest that HS3 inhibition of ribonucleotide reduction may be responsible for the rapid inhibition of DNA synthesis seen under these culture conditions.     

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.     

Mutations in the R2 subunit of ribonucleotide reductase that confer resistance to hydroxyurea

Ribonucleotide reductase is an essential enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides for use in DNA synthesis. Ribonucleotide reductase from Escherichia coli consists of two subunits, R1 and R2. The R2 subunit contains an unusually stable radical at tyrosine 122 that participates in catalysis. Buried deep within a hydrophobic pocket, the radical is inaccessible to solvent although subject to inactivation by radical scavengers. One such scavenger, hydroxyurea, is a highly specific inhibitor of ribonucleotide reductase and therefore of DNA synthesis; thus it is an important anticancer and antiviral agent. The mechanism of radical access remains to be established; however, small molecules may be able to access Tyr-122 directly via channels from the surface of the protein. We used random oligonucleotide mutagenesis to create a library of 200,000 R2 mutants containing random substitutions at five contiguous residues (Ile-74, Ser-75, Asn-76, Leu-77, Lys-78) that partially comprise one side of a channel where Tyr-122 is visible from the protein surface. We subjected this library to increasing concentrations of hydroxyurea and identified mutants that enhance survival more than 1000-fold over wild-type R2 at high drug concentrations. Repetitive selections yielded S75T as the predominant R2 mutant in our library. Purified S75TR2 exhibits a radical half-life that is 50% greater than wild-type R2 in the presence of hydroxyurea. These data represent the first demonstration of R2 protein mutants in E. coli that are highly resistant to hydroxyurea; elucidation of their mechanism of resistance may provide valuable insight into the development of more effective inhibitors.     

Ribonucleoside diphosphate reductase is a component of the replication hyperstructure in Escherichia coli

Although the nrdA101 allele codes for a ribonucleoside diphosphate (rNDP) reductase that is essentially destroyed in less than 2 min at 42 degrees C, and chemical inhibition of the enzyme by hydroxyurea stops DNA synthesis at once, we found that incubation at 42 degrees C of an Escherichia coli strain containing this allele allows DNA replication for about 40min. This suggests that mutant rNDP reductase is protected from thermal inactivation by some hyperstructure. If, together with the temperature upshift, RNA or protein synthesis is inhibited, the thermostability time of the mutant rNDP reductase becomes at least as long as the replication time and residual DNA synthesis becomes a run-out replication producing fully replicated chromosomes. This suggests that cessation of replication in the nrdA101 mutant strain is not the result of inactivation of its gene product but of the activity of a protein reflecting the presence of a partially altered enzyme. The absence of Tus protein, which specifically stops the replication complex by inhibiting replicative helicase activity, allows forks to replicate for a longer time at the restrictive temperature in the nrdA101 mutant strain. We therefore propose that rNDP reductase is a component of the replication complex, and that this association with other proteins protects the protein coded by allele nrdA101 from thermal inactivation.     

Chloroplast DNA synthesis during the cell cycle in cultured cells of Nicotiana tabacum: inhibition by nalidixic acid and hydroxyurea

The effects of nalidixic acid and hydroxyurea on nuclear and chloroplast DNA formation in cultured cells of Nicotiana tabacum were investigated. At low concentrations (5 and 20 micrograms/ml) nalidixic acid, an inhibitor of DNA gyrase, exhibited a greater inhibitory effect on plastid DNA synthesis than on nuclear DNA formation. Since the plastid genome is a circular double-stranded DNA, this is consistent with the proven involvement of a DNA gyrase in the replication of closed circular duplex DNA genomes in procaryotic cells. At a high concentration of nalidixic acid (50 micrograms/ml), DNA synthesis in both the plastid and nuclear compartment was rapidly inhibited. Removal of the drug from the culture medium led to the resumption of DNA synthesis in 8 h. Hydroxyurea, an inhibitor of ribonucleoside diphosphate reductase, also depresses nuclear as well as plastid DNA formation. Removal of hydroxyurea from the blocked cells leads to a burst of nuclear DNA synthesis, suggesting that the cells had been synchronized at the G1/S boundary. The recovery of plastid DNA synthesis occurs within the same time frame as that of nuclear DNA. However, whereas plastid DNA formation is then maintained at a constant rate, nuclear DNA synthesis reaches a peak and subsequently declines. These results indicate that the synthesis of plastid DNA is independent of the cell cycle events governing nuclear DNA formation in cultured plant cells.     

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.     

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.     

Replicative bacteriophage DNA synthesis in plasmolyzed T4-infected cells: evidence for two independent pathways to DNA.

Bacteriophage T4-infected Escherichia coli rendered permeable to nucleotides by sucrose plasmolysis exhibited two apparently separate pathways or channels to T4 DNA with respect to the utilization of exogenously supplied substrates. By one pathway, individual labeled ribonucleotides, thymidine (tdR), and 5-hydroxymethyl-dCMP could be incorporated into phage DNA. Incorporation of each of these labeled compounds was not dependent upon the addition of the other deoxyribonucleotide precursors, suggesting that a functioning de novo pathway to deoxyribonucleotides was being monitored. The second pathway or reaction required all four deoxyribonucleoside triphosphates or the deoxyribonucleoside monophosphates together with ATP. However, in this reaction, dTTP was not replaced by TdR. The two pathways were also distinguished on the basis of their apparent Mg2+ requirements and responses to N-ethylmaleimide, micrococcal nuclease, and to hydroxyurea, which is a specific inhibitor of ribonucleoside diphosphate reductase. Separate products were synthesized by the two channels, as shown by density-gradient experiments and velocity sedimentation analysis. Each of the pathways required the products of the T4 DNA synthesis genes. Furthermore, DNA synthesis by each pathway appeared to be coupled to the functioning of several of the phage-induced enzymes involved in deoxyribonucleotide biosynthesis. Both systems represent replicative phage DNA synthesis as determined by CsCl density-gradient analysis. Autoradiographic and other studies provided evidence that both pathways occur in the same cell. Further studies were carried out on the direct role of dCMP hydroxymethylase in T4 DNA replication. Temperature-shift experiments in plasmolyzed cells using a temperature-sensitive mutant furnished strong evidence that this gene product is necessary in DNA replication and is not functioning by allowing preinitiation of DNA before plasmolysis.     

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.     

Characteristics of a bacteriophage T4-induced complex synthesizing deoxyribonucleotides

A preparation of bacteriophage T4-induced deoxyribonucleotide synthetase complex is described. This very large complex of enzymes can be separated by centrifugation at 100,000 X g, by sucrose step gradient centrifugation, or with molecular exclusion columns. By direct assay and by unidimensional and two-dimensional acrylamide electrophoretic separations the following T4-coded enzymes were shown to be associated with the complex: ribonucleoside diphosphate reductase, dCMP deaminase, dCTP/dUTPase, dCMP hydroxymethylase, dTMP synthetase, and DNA polymerase. Other phage-coded prereplicative proteins related to DNA replication and other phage functions such as the proteins coded by genes 32, 46, rIIA, and rIIB as well as many unidentified proteins were also consistently associated with the isolated fractions. T4 DNA topoisomerase, a membrane-bound enzyme, was found in quantity in all purified fractions of the complex, even in preparations apparently free of membrane and of T4 DNA. The functional integrity of a segment of the complex was followed by measuring the conversion of [5-3H]CDP to the level of 5-hydroxymethyl dCMP. This series of reactions requires the actions of T4-coded ribonucleoside diphosphate reductase and its associated reducing system, dCTP/dUTPase and dCMP hydroxymethylase, 3H being lost to water at the last step. In this reaction sequence an intermediate, [5-3H]dCMP, is maintained at low steady state concentrations, and argument is presented that the synthesis of deoxyribonucleotides is channeled and normally tightly coupled to DNA replication. One of the primary characteristics of this complex is its ready dissociation of dilution into smaller complexes of proteins and to the free forms of the proteins. That the complex is held together by weak electrostatic forces was supported by its sensitivity to dissociation at moderate salt concentrations. Not only the enzymes required in deoxyribonucleotide synthesis but T4 DNA polymerase, T4 DNA topoisomerase, and a number of other proteins dissociate to varying degrees from the larger complexes under these conditions.     

Effect of hydroxyurea on T4 ribonucleotide reductase.

Phage T4-induced ribonucleotide reductase, purified to homogeneity, catalyzes the reduction of the four ribonucleotides CDP, UDP, ADP, and GDP to the corresponding deoxyribonucleotides. The enzyme is an order of magnitude more sensitive to hydroxyurea than the corresponding Escherichia coli enzyme. Fifty per cent inhibition occurs at 10 micrometer hydroxyurea. Inhibition is complete at a high concentration of the drug, and there is no differential effect on the four substrates. Treatment of T4 ribonucleotide reductase or its isolated subunits with hydroxyurea does not lead to their irreversible inactivation.     

Pathways of pyrimidine deoxyribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides.

By measuring the specific activity of deoxyribonucleotides isolated from DNA after the incorporation of 14C-labeled precursors with and without competition from other nucleotide precursors, we defined the major pathways of pyrimidine deoxyribonucleotide synthesis in Mycoplasma mycoides subsp. mycoides. Uracil, guanine, and thymine are required for the synthesis of nucleotides. Cytidine competed effectively with uracil to provide all of the deoxycytidine nucleotide, as well as most of the deoxyribose-1-phosphate, for the synthesis of thymidylate from thymine via thymidine phosphorylase. Each of dUMP, dCMP, and dTMP competed with cytidine for incorporation into DNA thymidylate. Appreciable incorporation of exogenous deoxyribonucleoside 5'-monophosphates into DNA without prior dephosphorylation was observed. Dephosphorylation also occurred since the added deoxyribonucleotide provided phosphate for the synthesis of the other nucleotides in DNA in competition with the 32Pi in the growth medium. Hydroxyurea inhibited cell growth and decreased the intracellular level of dATP, consistent with the action of a ribonucleoside diphosphate reductase with regulatory properties similar to those of the Escherichia coli enzyme.