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.The first committed step in DNA biosynthesis occurs by direct reduction of the 2Ј-hydroxyl of ribonucleotides and is catalyzed by the enzyme ribonucleotide reductase. Because this single enzyme is responsible for the production of all four deoxyribonucleotides and because these products are needed only at specific times in cell or viral life cycles, ribonucleotide reductase is highly regulated in both substrate specificity and overall activity.The allosteric properties of the enzyme, as well as the kinetic parameters for all four rNDP 1 substrates, are well characterized for the T4 phage aerobic ribonucleotide reductase (1) and are similar to those of the prototypical enzyme from Escherichia coli. However, to our knowledge, no studies have been done to determine what the simultaneous turnover rates for each of the substrates are under different concentrations and combinations of the enzyme's allosteric effectors and substrates. Of particular interest to our laboratory are the relative rates of formation for each of the four products when the effector and substrate environment of the enzyme mimics the in vivo conditions as measured in T4 phage-infected E. coli (2, 3). In other words, when the known allosteric effectors and substrates are supplied together in a single reaction mixture at their estimated physiological or in vivo concentrations, what are the relative rates of formation of each of the products? This question is of particular interest because, in vivo, T4 ribonucleotide reductase functions as part of an enzyme complex (4) and the possibilitiy that intracellular reaction fluxes are controlled by protein-protein interactions must be considered.Typically, ribonucleotide reductase has been assayed by using only one of the substrates because analysis of the simultaneous turnover of all four substrates by the enzyme...

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.

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.The allosteric regulation of ribonucleotide reductase (RNR) 1 by individual nucleoside triphosphates is well characterized for several forms of the enzyme (1, 2). The three classes of RNR, although quite different in structure (3), have similar allosteric behavior in terms of the effects of modifiers upon substrate specificity. In all classes, ATP and dATP (when it is not inhibitory) activate reduction of the cytidine and uridine nucleotides. Reduction of adenosine and guanosine nucleotides is stimulated by dGTP and dTTP, respectively. These effects are mediated through binding of nucleotides to two different allosteric sites on the dimeric R1 (large) protein: the activity sites, which bind with relatively low affinity ATP (general activator) or dATP (general inhibitor), and the specificity sites, which bind with higher affinity ATP, dATP, dGTP, or dCTP, with the binding of each ligand activating the inhibition of some substrates and inhibiting that of others.However, due primarily to limitations of traditional RNR assay methods, only the isolated effects of individual allosteric effectors have been determined, in kinetic experiments involving single substrates. We have developed an assay procedure (4) that permits simultaneous monitoring of the four RNR activities. With this procedure, the effects of more complex nucleotide enviro...

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.

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. Hydroxyurea (HU)1 inhibits DNA replication in cells that use the class I form of ribonucleotide reductase (RNR) by inactivating the tyrosyl radical required for enzyme activity (1). This inhibition is also observed in viruses such as vaccinia, which encodes a class I RNR that is closely related in structure and regulation to the mammalian cell RNRs. HU inhibits the de novo synthesis of deoxyribonucleotides, thereby starving the host cell and viral replication complexes for precursors. One might expect the inhibition of RNR by a drug like HU to result in an equivalent decrease in the rates of formation of all four products of the enzyme. However, when our laboratory measured the triphosphate forms of these products, the 2Ј-deoxyribonucleoside 5Ј-triphosphates (dNTPs) in vaccinia virus-infected cells, we found that hydroxyurea treatment had different effects upon the four dNTP pools; dATP was the most severely depleted, dCTP and dGTP were depleted to intermediate levels, and dTTP actually accumulated to some 2-fold over control values (2). Similar results had earlier been noted with uninfected mammalian cells (3-5). In vaccinia virus-infected cells, addition of deoxyadenosine to the culture medium, along with an adenosine deaminase inhibitor, reversed the inhibition of virus growth by HU (2). Also, labeling studies that estimated RNR flux rates in vivo confirmed that ADP reduction was the most severely affected of the four RNR activities. These observations suggested that the reduction of ADP is a specific biological target of hydroxyurea in vivo. This finding, in turn, has spurred attempts to ...