Mouse ribonucleotide reductase control: influence of substrate binding upon interactions with allosteric effectors.

Using ribonucleotide reductase encoded by vaccinia virus as a model for the mammalian enzyme, our laboratory developed an assay that allows simultaneous monitoring of the reduction of ADP, CDP, GDP, and UDP. That study found ADP reduction to be specifically inhibited by ADP itself. To learn whether this effect is significant for cellular regulation, we have analyzed recombinant mouse ribonucleotide reductase. We report that allosteric control properties originally described in single-substrate assays operate also under our four-substrate assay conditions. Three distinctions from the vaccinia enzyme were seen: 1) higher sensitivity to allosteric modifiers; 2) higher activity with UDP as substrate; and 3) significant inhibition by ADP of GDP reduction as well as that of ADP itself. Studies of the effects of ADP and other substrates upon binding of effectors indicate that binding of ribonucleoside diphosphates at the catalytic site influences dNTP binding at the specificity site. We also examined the activities of hybrid ribonucleotide reductases, composed of a mouse subunit combined with a vaccinia subunit. As previously reported, a vaccinia R1/mouse R2 hybrid has low but significant activity. Surprisingly, a mouse R1/vaccinia R2 hybrid was more active than either mouse R1/R2 or vaccinia R1/R2, possibly explaining why mutations affecting vaccinia ribonucleotide reductase have only small effects upon viral DNA replication.

Ribonucleotide reductase, a possible agent in deoxyribonucleotide pool asymmetries induced by hypoxia.

While investigating the basis for marked natural asymmetries in deoxyribonucleoside triphosphate (dNTP) pools in mammalian cells, we observed that culturing V79 hamster lung cells in a 2% oxygen atmosphere causes 2-3-fold expansions of the dATP, dGTP, and dTTP pools, whereas dCTP declines by a comparable amount. Others have made similar observations and have proposed that, because O(2) is required for formation of the catalytically essential oxygen-bridged iron center in ribonucleotide reductase, dCTP depletion at low oxygen tension results from direct or indirect effects upon ribonucleotide reductase. We have tested the hypothesis that oxygen limitation affects ribonucleotide specificity using recombinant mouse ribonucleotide reductase and an assay that permits simultaneous monitoring of the reduction of all four nucleotide substrates. Preincubation and assay of the enzyme in an anaerobic chamber caused only partial activity loss. Accordingly, we treated the enzyme with hydroxyurea, followed by removal of the hydroxyurea and exposure to atmospheres of varying oxygen content. The activity was totally depleted by hydroxyurea treatment and nearly fully regained by exposure to air. By the criterion of activities regained at different oxygen tensions, we found CDP reduction not to be specifically sensitive to oxygen depletion; however, GDP reduction was specifically sensitive. The basis for the differential response to reactivation by O(2) is not known, but it evidently does not involve varying rates of reactivation of different allosteric forms of the enzyme or altered response to allosteric effectors at reduced oxygen tension.

Metabolic functions of microbial nucleoside diphosphate kinases.

This article summarizes research from our laboratory on two aspects of the biochemistry of nucleoside diphosphate kinase from Escherichia coli--first, its interactions with several T4 bacteriophage-coded enzymes, as part of a multienzyme complex for deoxyribonucleoside triphosphate biosynthesis. We identify some of the specific interactions and discuss whether the complex is linked physically or functionally with the T4 DNA replication machinery, or replisome. Second, we discuss phenotypes of an E. coli mutant strain carrying a targeted deletion of ndk, the structural gene for nucleoside diphosphate kinase. How do bacteria lacking this essential housekeeping enzyme synthesize nucleoside triphosphates? In view of the specific interactions of nucleoside diphosphate kinase with T4 enzymes of DNA metabolism, how does T4 multiply after infection of this host? Finally, the ndk disruption strain has highly biased nucleoside triphosphate pools, including elevations of the CTP and dCTP pools of 7- and 23-fold, respectively. Accompanied by these biased nucleotide pools is a strong mutator phenotype. What is the biochemical basis for the pool abnormalities and what are the mutagenic mechanisms? We conclude with brief references to related work in other laboratories.

Allosteric regulation of vaccinia virus ribonucleotide reductase, analyzed by simultaneous monitoring of its four activities.

As determined by simultaneous monitoring of its four activities, vaccinia virus-coded ribonucleoside diphosphate (rNDP) reductase shows responses to individual nucleoside triphosphate effectors-ATP, dATP, dGTP, and dTTP-similar to those previously reported for rNDP reductase of mouse, which the viral enzyme closely resembles. This investigation uses the vaccinia enzyme as a readily available and convenient model for understanding the cellular enzyme. As previously reported for T4 phage aerobic rNDP reductase, we found the relative activities of ADP, CDP, GDP, and UDP reduction to be reasonably close to the proportions of the four deoxyribonucleotides in the vaccinia virus genome, but only when the four substrates and the four allosteric effectors were all provided at their approximate intracellular concentrations. GDP reductase levels were somewhat higher, proportionately, than the representation of dGMP in vaccinia virus DNA. To understand this behavior and also to evaluate possible relationships between ribonucleotide reductase control and the very low dGTP pools seen in eukaryotic cells, we carried out substrate saturation experiments with a "bioproportional" mixture containing the four rNDP substrates at their relative in vivo concentrations as determined from rNDP pool measurements. Reduction of the two purine substrates was inhibited at high concentrations of this mixture, and data suggest that ADP acts as a specific inhibitor of its own reduction and that of GDP. Use of the four-substrate assay revealed also that a mixture of vaccinia virus R1 protein and mouse R2 protein is catalytically active, making this the first reported chimeric rNDP reductase to show biological activity.

Differential effects of hydroxyurea upon deoxyribonucleoside triphosphate pools, analyzed with vaccinia virus ribonucleotide reductase.

Hydroxyurea inhibits DNA synthesis by destroying the catalytically essential free radical of class I ribonucleoside diphosphate (rNDP) reductase, thereby blocking the de novo synthesis of deoxyribonucleotides. In mammalian cells, including those infected by vaccinia virus, hydroxyurea treatment causes a differential depletion of the four deoxyribonucleoside triphosphate pools, suggesting that the activities of rNDP reductase are differentially sensitive to hydroxyurea. In the presence of different substrates and allosteric modifiers, we measured rates of free radical destruction in the vaccinia virus-coded rNDP reductase, by following absorbance at 417 nm as a function of time after hydroxyurea addition. Also, we followed enzyme activity directly, by using a recently developed assay that allows simultaneous monitoring of the four activities, in the presence of substrates and effectors at concentrations that approximate the intracellular environment. We found the primary determinant of radical loss to be not the ensemble of allosteric ligands bound but the activity of the enzyme. Nucleoside triphosphate effectors accelerated radical decay, compared with rates seen with the free enzyme. Adding substrate to the holoenzyme, under conditions where the enzymatic reaction is proceeding, further accelerated radical decay. Alternative models are discussed, to account for selective depletion of purine nucleotide pools by hydroxyurea.

Studies on some trace and minor elements in blood. A survey of the Kalpakkam (India) population. Part I: Standardization of analytical methods using ICP-MS and AAS.

Blood is one of the widely used specimens for biological trace element research because of its biological significance and ease of sampling. We have conducted a study of the blood of the Kalpakkam township population for trace and minor elements. For this purpose, analytical methods have been developed and standardized in our laboratory for the elemental analysis of blood plasma and red cells. Inductively coupled plasma-mass spectrometry (ICP-MS), a relatively new technique, has been applied for the analysis of trace elements. Details regarding spectral interference and matrix interference encountered in the analysis of blood and the methods of correcting them have been discussed. Flame atomic absorption spectrometry (AAS)/atomic emission spectrometry (AES) has been applied for the determination of minor elements. Precision and accuracy of these methods have also been discussed.

Studies on some trace and minor elements in blood. A survey of the Kalpakkam (India) population. Part III: Studies on dietary intake and its correlation to blood levels.

In our studies on elemental levels in blood of the Kalpakkam population, it was found that the reference values for many elements were normal, but some deficiency with respect to Se was noticed. As a followup study, the dietary ingredients of the local population were analyzed for trace and minor elements to assess the dietary intake of these elements. Details of the analytical methods developed using the technique of inductively coupled plasma-mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) have been described. The dietary intake of many of these trace and minor elements were found to be quite adequate according to the recommended dietary allowance (RDA) levels prescribed, except for Se and Zn. The dietary intake of Se was found to be in the range 20-50 micrograms/d (as opposed to the RDA of 50-200 micrograms/d), whereas the intake of Zn was found to be in the range 8-10 mg/d (as opposed to the RDA of 15 mg/d). Although the deficiency of Se intake was reflected in the blood, that of Zn was not, probably owing to the high level of homeostasis for this element. Fish and egg were found to be rich sources of Se, followed by cereals and pulses, which were found to be the major sources of Zn.

Studies on some trace and minor elements in blood. A survey of the Kalpakkam (India) population. Part II: Reference values for plasma and red cell, and correlation with coronary risk index.

Since data on the trace element levels in Indian population are lacking, we chose to conduct a survey of the Kalpakkam township population. People in the age group 40-55 were included in this study. Reference values for trace and minor elements of the blood of the Kalpakkam population were arrived at by carrying out the analysis of plasma and red cells of healthy subjects of the Kalpakkam population. Although the "reference values" for many elements were found to be normal and comparable to values available in the literature, slight deficiency with respect to Se was noticed. Subjects with high coronary risk index were also included in the study to assess the possible correlation of elemental and lipid profile. A study of box plots showed that the elements Se, Mg, Na, K, and Fe show significant differences between "high risk" coronary risk index (CRI > 5) and "no risk" (CRI < 4.5). In the plasma, the levels of Mg, Na, and K were found to be less in the high-risk group. In red cells, the amount of Se, Fe, and K were found to be significantly less in the "high-risk" group as compared to the "no-risk" group.

Regulation of T4 phage aerobic ribonucleotide reductase. Simultaneous assay of the four activities.

We have devised an assay procedure that permits simultaneous monitoring of the four activities of ribonucleotide reductase. Using this assay, we have compared the reduction of all four substrates by the T4 bacteriophage aerobic ribonucleotide reductase within different allosteric environments. Specifically, we compared the relative turnover rates by the enzyme when activated with "in vivo" concentrations of the known allosteric effectors versus activation by ATP alone. Consistent with the known allosteric properties of this enzyme, our results show that ATP does act as a general activator, although the rate of purine nucleotide reduction was approximately 5% of the rate for the pyrimidine nucleotides. However, addition of the allosteric effectors at their estimated physiological concentrations dramatically changed the relative rates of substrate reduction, creating a more "balanced" pool of products. Addition of the substrates at their respective in vivo concentrations further pushed rates of product formation toward a ratio similar to the base composition of the T4 genome. The similarity of the product profile produced under in vivo conditions to the genomic composition of T4 phage is discussed.

Vaccinia virus G4L gene encodes a second glutaredoxin.

Vaccinia virus (VV) was previously shown to encode a functional glutaredoxin, the product of the o2l gene, which is synthesized late in infection, after the onset of DNA replication. Here we report that an open reading frame in the VV genome designated as g4l encodes a protein that has sequence similarity to glutaredoxins and possesses thioltransferase and dehydroascorbate reductase activities. G4L protein in infected cells can be detected as early as 4 hr after infection and is constitutively expressed up to 24 hr postinfection. A protein homologous to G4L and retaining the predicted glutaredoxin active center is encoded by the recently sequenced Molluscum Contagiosum virus (MCV), whereas O2L protein is not conserved, suggesting that the glutaredoxin activity of G4L may be involved in replication of all poxviruses.

Protein-protein interactions involving T4 phage-coded deoxycytidylate deaminase and thymidylate synthase.

The enzymes deoxycytidylate deaminase (EC) and thymidylate synthase (EC) are functionally associated with one another, since they catalyze sequential reactions. In T4 coliphage infection the two enzymes are found in dNTP synthetase, a multienzyme complex for deoxyribonucleotide biosynthesis. Protein-protein interactions involving the phage-coded forms of these two enzymes have been explored in three experiments that use the respective purified protein as an affinity ligand. First, an extract of radiolabeled T4 proteins was passed through a column of immobilized enzyme (either dTMP synthase or dCMP deaminase), and the specifically bound proteins were identified. Second, two mutant form of dCMP deaminase (H90N and H94N), altered in presumed zinc-binding sites, were analyzed similarly, with the results suggesting that some, but not all, interactions require normal structure near the catalytic site. Third, affinity chromatography using either enzyme as the immobilized ligand, revealed interactions between the two purified enzymes in the absence of other proteins. In these experiments we noted a significant effect of dCTP, an allosteric modifier of dCMP deaminase, upon the interactions.

Effects of T4 phage infection and anaerobiosis upon nucleotide pools and mutagenesis in nucleoside diphosphokinase-defective Escherichia coli strains.

Bacteriophage T4 encodes nearly all of its own enzymes for synthesizing DNA and its precursors. An exception is nucleoside diphosphokinase (ndk gene product), which catalyzes the synthesis of ribonucleoside triphosphates and deoxyribonucleoside triphosphates (dNTPs) from the corresponding diphosphates. Surprisingly, an Escherichia coli ndk deletion strain grows normally and supports T4 infection. As shown elsewhere, these ndk mutant cells display both a mutator phenotype and deoxyribonucleotide pool abnormalities. However, after T4 infection, both dNTP pools and spontaneous mutation frequencies are near normal. An E. coli strain carrying deletions in ndk and pyrA and pyrF, the structural genes for both pyruvate kinases, also grows and supports T4 infection. We examined anaerobic E. coli cultures because of reports that in anaerobiosis, pyruvate kinase represents the major route for nucleoside triphosphate synthesis in the absence of nucleoside diphosphokinase. The dNTP pool imbalances and the mutator phenotype are less pronounced in the anaerobic than in the corresponding aerobic ndk mutant strains. Anaerobic dNTP pool data, which have not been reported before, reveal a disproportionate reduction in dGTP, relative to the other pools, when aerobic and anaerobic conditions are compared. The finding that mutagenesis and pool imbalances are mitigated in both anaerobic and T4-infected cultures provides strong, if circumstantial, evidence that the mutator phenotype of ndk mutant cells is a result of the dNTP imbalance. Also, the viability of these cells indicates the existence of a second enzyme system in addition to nucleoside diphosphokinase for nucleoside triphosphate synthesis.

Identification of a second functional glutaredoxin encoded by the bacteriophage T4 genome.

Thioredoxins and glutaredoxins are small ubiquitous redox proteins that were discovered as hydrogen donors for ribonucleotide reductase, the key enzyme for deoxyribonucleotide biosynthesis. Some organisms encode more than one redox protein. In this study, we demonstrate that an open reading frame in the bacteriophage T4 genome, reported earlier and designated as Y55.7 (Tomaschewski, J., and Rüger, W. (1987) Nucleic Acids Res. 15, 3632-3633), encodes a second functional redox protein. Gene y55.7 was cloned and expressed in Escherichia coli. Purified Y55.7 protein had glutathione-dependent thioltransferase and dehydroascorbate reductase activities indicative of a functional glutaredoxin. The protein is expressed at all stages of the T4 infection cycle and can serve as a hydrogen donor for the phage ribonucleotide reductase in in vitro experiments.

T4 phage gene 32 protein as a candidate organizing factor for the deoxyribonucleoside triphosphate synthetase complex.

After T4 bacteriophage infection of Escherichia coli, the enzymes of deoxyribonucleoside triphosphate biosynthesis form a multienzyme complex that we call T4 deoxyribonucleoside triphosphate (dNTP) synthetase. At least eight phage-coded enzymes and two enzymes of host origin are found in this 1.5-mDa complex. The complex may shuttle dNTPs to DNA replication sites, because replication draws from small pools, which are probably highly localized. Several specific protein-protein contacts within the complex are described in this paper. We have studied protein-protein interactions in the complex by immobilizing individual enzymes and identifying radiolabeled T4 proteins that are retained by columns of these respective affinity ligands. Elsewhere we have described interactions involving three T4 enzymes found in the complex. In this paper we describe similar analysis of five more proteins: dihydrofolate reductase, dCTPase-dUTPase, deoxyribonucleoside monophosphokinase, ribonucleotide reductase, and E. coli nucleoside diphosphokinase,. All eight proteins analyzed to date retain single-strand DNA-binding protein (gp32), the product of T4 gene 32. At least one T4 protein, thymidylate synthase, binds directly to gp32, as shown by affinity chromatographic analysis of the two purified proteins. Among its several roles, gp32 stabilizes single-strand template DNA ahead of a replicating DNA polymerase. Our data suggest a model in which dNTP synthetase complexes, probably more than one per growing DNA chain, are drawn to replication forks via their affinity for gp32 and hence are localized so as to produce dNTPs at their sites of utilization, immediately ahead of growing DNA 3' termini.

The gene for nucleoside diphosphate kinase functions as a mutator gene in Escherichia coli.

Nucleoside diphosphate (NDP) kinase is a key enzyme in the control of cellular concentrations of nucleoside triphosphates, and has been shown to play important roles in various cellular activities such as developmental control, signal transduction and metastasis in eukaryotic systems. In this study, the gene for NDP kinase of Escherichia coli (ndk) was disrupted and surprisingly found to be dispensable without any discernible effects on cell growth or morphology. However, a mutator phenotype was found in ndk-disruption strains; frequencies of spontaneous mutations to rifampicin resistance and nalidixic acid resistant significantly increased. A higher frequency in reversion mutations was observed with use of an amber mutation in the kanamycin-resistance gene in an ndk-disruption strain. Imbalance in dNTP pools, in particular a significant increase of the dCTP content was observed, which is likely to result in the higher spontaneous mutation rates. These results suggest that NDP kinase, although not essential, plays an important role in the appropriate balance of intracellular dNTP pools to maintain a high DNA replication fidelity. Strains with ndk- pykA- pykF- as well as ndk- scs- were constructed without any discernible effect on cell growth, indicating that there is yet another enzyme(s) catalyzing nucleoside triphosphate synthesis, in addition to NDP kinase, pyruvate kinases and succinyl CoA synthetase.

Roles of vaccinia virus ribonucleotide reductase and glutaredoxin in DNA precursor biosynthesis.

To examine the possible role of the vaccinia virus glutaredoxin as a cofactor for viral ribonucleotide reductase, viral growth, DNA synthesis, and dNTP pools were measured in infections of B-SC-40 monkey kidney cells with wild type vaccinia virus and with mutants of vaccinia that lacked a functional reductase or glutaredoxin. In infections of untreated host cells, the lack of viral ribonucleotide reductase or glutaredoxin had only small effects upon virus growth. When host cells were pretreated with alpha-amanitin, which blocks host RNA polymerase II but not viral transcription, viral DNA synthesis was markedly reduced in infections with either of the mutants when compared with wild type infections. Relative to dNTP levels in wild type infections, pools of dCTP, but not of the other dNTPs, were significantly reduced in infections of amanitin-treated cells with either mutant. The parallel depletion of dCTP in the two mutant suggests that the role of glutaredoxin may be to function as a cofactor for viral ribonucleotide reductase. The data suggest that both viral proteins become essential for DNA replication only when levels of the corresponding host cell proteins are depleted.

Natural DNA precursor pool asymmetry and base sequence context as determinants of replication fidelity.

Previous studies showed a complex relationship between nucleotide composition of a gene and the rate of the gene's evolutionary variation. We have investigated mechanisms by constructing M13 phagemids containing part of the Escherichia coli lacZ gene, in which an opal codon is flanked either by nine adenine-thymine base pairs on each side, or by nine guanine-cytosine pairs, or by its wild-type sequence context. Reversions or pseudoreversions within the opal codon yield a lacZ alpha-peptide that can undergo alpha-complementation and yield a blue plaque when plated with a chromogenic substrate. When these constructs were replicated in HeLa cell extracts, in the presence of equimolar deoxyribonucleoside triphosphate (dNTP) mixtures, reversion was near background levels in both the AT-rich and GC-rich contexts. By contrast, when the DNAs were replicated at dNTP concentrations approximating those in HeLa cell nuclei, increases over background were seen in all three contexts. Replication of the phagemids in vivo led to even higher mutation frequencies. Replication in the presence of dGMP, added to inhibit proofreading, caused extraordinarily high reversion frequencies in the GC-flanked opal codon. Apparently, dNTP concentrations approximating intracellular concentrations are mildly but significantly mutagenic, and pool asymmetries and base sequence context both contribute to the natural fidelity of DNA replication.

An immunoblot assay reveals that bacteriophage T4 thymidylate synthase and dihydrofolate reductase are not virion proteins.

Numerous reports describe the phage T4 enzymes thymidylate synthase and dihydrofolate reductase as structural components of the baseplate. However, Y. Wang and C. K. Mathews (J. Virol. 63:4736-4743, 1989) reported that antisera against the respective recombinant enzymes failed to neutralize phage infectivity, in contrast to previous results. Moreover, a deletion mutant lacking the genes for these two enzymes adsorbed normally to host cells. Since these findings tended to undermine the idea of the two enzymes as structural proteins, we developed a quantitative immunoblot assay to resolve the issue directly. Our results show that both enzymes are present only as minor contaminants (< 0.05 copy per phage) and as such cannot be bona fide structural proteins.

Allosteric effectors are required for subunit association in T4 phage ribonucleotide reductase.

Bacteriophage T4 encodes its own aerobic ribonucleotide reductase (RNR), which reduces ribonucleoside diphosphates to the corresponding deoxyribonucleoside diphosphates. T4 RNR is composed of homodimeric large (R1) and small (R2) subunits. Intricate regulation of enzymatic activity is accomplished by the binding of nucleotide effectors to R1. Berglund (Berglund, O. (1972) J. Biol. Chem. 247, 7270-7275) described similarities between T4 RNR and the corresponding enzyme from aerobic Escherichia coli. An important difference, however, is that T4 RNR forms a tight R1.R2 complex, while the E. coli R1 and R2 more readily dissociate. In this study we purified the phage R2 subunit from an overexpression vector constructed by Tseng et al. (Tseng, M., Hilfinger, J., He, P., and Greenberg, R. (1992) J. Bacteriol. 174, 5740-5744) and used this as an immunogen to generate polyclonal antiserum. Using co-immunoprecipitation techniques, we probed in vitro for interactions between the phage-induced R1 and R2 subunits. Our studies indicate that tight binding of the phage RNR subunits is completely dependent upon the known allosteric effectors of the enzyme. Once the R1.R2 holoenzyme has been formed it appears to be remarkably stable when in the presence of dATP. However, if dATP is removed, the R1.R2 complex readily dissociates.

Ribonucleotide reductase: evidence for specific association with HeLa cell mitochondria.

Mammalian mitochondria contain pools of deoxyribonucleoside 5'-triphosphates that behave differently from the much larger whole-cell pools. To investigate the origins of these pools, we analyzed HeLa cell mitochondria for ribonucleotide reductase activity. Three findings suggest specific association of a reductase with mitochondria: (1) enzyme activity in extracts of washed mitochondria, (2) stimulation of that activity by dATP at levels inhibitory to the major cellular activity, and (3) association of immunoreactive material with washed and fractionated mitochondria.