Regulation Of Mammalian Ribonucleotide Reductase Research Articles

Comprehensive model for allosteric regulation of mammalian ribonucleotide reductase: refinements and consequences.

Reduction of NDPs by murine ribonucleotide reductase (mRR) requires catalytic (mR1) and free radical-containing (mR2) subunits and is regulated by nucleoside triphosphate allosteric effectors. Here we present the results of several studies that refine the recently presented comprehensive model for the allosteric control of mRR enzymatic activity [Kashlan, O. B., et al. (2002) Biochemistry 41, 462-474], in which nucleotide binding to the specificity site (s-site) drives formation of an active R1(2)R2(2) dimer, ATP or dATP binding to the adenine site (a-site) drives formation of a tetramer, mR1(4a), which isomerizes to an inactive form, mR1(4b), and ATP binding to the hexamerization site (h-site) drives formation of an active R1(6)R2(6) hexamer. Analysis of the a-site D57N variant of mR1, which differs from wild-type mR1 (wt-mR1) in that its RR activity is activated by both ATP and dATP, demonstrates that dATP activation of the D57N variant RR arises from a blockage in the formation of mR1(4b) from mR1(4a), and provides strong evidence that mR1(4a) forms active complexes with mR2(2). We further demonstrate that (a) differences in the effects of ATP versus dATP binding to the a-site of wt-mR1 provide specific mechanisms by which the dATP/ATP ratio in mammalian cells could modulate in vivo RR enzymatic activity, (b) the comprehensive model is valid over a range of Mg(2+) concentrations that include in vivo concentrations, and (c) equilibrium constants derived for the comprehensive model can be used to simulate the distribution of R1 among dimer, tetramer, and hexamer forms in vivo. Such simulations indicate that mR1(6) predominates over mR1(2) in the cytoplasm of normal mammalian cells, where the great majority of RR activity is located, but that mR1(2) may be important for nuclear RR activity and for RR activity in cells in which the level of ATP is depleted.

A Comprehensive Model for the Allosteric Regulation of Mammalian Ribonucleotide Reductase. Functional Consequences of ATP- and dATP-Induced Oligomerization of the Large Subunit

Reduction of NDPs by murine ribonucleotide reductase (mRR) requires catalytic (mR1) and free radical-containing (mR2) subunits and is regulated by nucleoside triphosphate allosteric effectors. Here we present a new, comprehensive, and quantitative model for allosteric control of mRR enzymatic activity based on molecular mass, ligand binding, and enzyme activity studies. In this model, nucleotide binding to the specificity site (s-site) drives formation of an active R1(2)R2(2) dimer, ATP or dATP binding to the adenine-specific site (a-site) results in formation of an inactive tetramer, and ATP binding to the newly described hexamerization site (h-site) drives formation of active R1(6)R2(6) hexamer. In contrast, an earlier phenomenological model [Thelander, L., and Reichard, P. (1979) Annu. Rev. Biochem. 67, 71-98] (the "RT" model) ignores aggregation state changes and mistakenly rationalizes ATP activation versus dATP inhibition as reflecting different functional consequences of ATP versus dATP binding to the a-site. Our results suggest that the R1(6)R2(6) heterohexamer is the major active form of the enzyme in mammalian cells, and that the ATP concentration is the primary modulator of enzyme activity, coupling the rate of DNA biosynthesis with the energetic state of the cell. Using the crystal structure of the Escherichia coliR1 hexamer as a model for the mR1 hexamer, a scheme is presented that rationalizes the slow isomerization of the tetramer form and suggests an explanation for the low enzymatic activity of tetramers complexed with R2. The similar specific activities of R1(2)R2(2) and R1(6)R2(6) are inconsistent with a proposed model for R2(2) docking with R1(2) [Uhlin, U., and Eklund, H. (1994) Nature 370, 533-539], and an alternative is suggested.

Regulation of mammalian ribonucleotide reductase by the tumor promoters and protein phosphatase inhibitors okadaic acid and calyculin A.

A rapid elevation of ribonucleotide reductase activity was observed with BALB c/3T3 fibroblasts treated with 10 nM okadaic acid, a nonphorbol ester tumor promoter and protein phosphatase inhibitor. Northern blot analysis of the two components of ribonucleotide reductase (R1 and R2) showed a marked elevation of R1 and R2 mRNA expression. Western blot analysis with R1 and R2 specific monoclonal antibodies indicated that the increase in ribonucleotide reductase activity was primarily due to the elevation of the R2 rather than the R1 protein during treatment with okadaic acid. The okadaic acid induced elevations in R1 and R2 message levels occurred without a detectable change in the proportion of cells in S phase and were blocked by treatment of cells with actinomycin D, indicating the importance of the reductase transcriptional process in responding to the action of okadaic acid. Furthermore, down-regulation of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate pretreatment abrogated the okadaic acid mediated elevation of ribonucleotide reductase mRNAs, consistent with the involvement of this signal pathway in the regulation of ribonucleotide reductase and the effects of okadaic acid. Treatment of cells with 2.5 nM calyculin A, another non-phorbol ester tumor promoter and protein phosphatase inhibitor, resulted in a rapid elevation of both R1 and R2 mRNA levels within 10 min of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in the activity and regulation of mammalian ribonucleotide reductase by chlorambucil, a DNA damaging agent.

Ribonucleotide reductase provides the four deoxyribonucleotides required for the synthesis of DNA. In this study, we examined the hypothesis that alterations in the regulation of ribonucleotide reductase activity may be necessary to provide the deoxyribonucleotides required for DNA repair, following exposure of mammalian cells to DNA damaging agents such as the antitumor agent chlorambucil. We observed a marked transient increase in ribonucleotide reductase activity within 2 h of exposing BALB/c 3T3 mouse cells to DNA damaging concentrations of chlorambucil. Northern blot analysis showed that elevations in activity were accompanied by transient increases in the mRNA levels of both genes (R1 and R2) that code for ribonucleotide reductase. Western blot analysis indicated that only the protein for the limiting component for enzyme activity, R2, was significantly elevated in chlorambucil treated cultures. The chlorambucil effects upon activity and regulation of ribonucleotide reductase occurred without any detectable changes in the rate of DNA synthesis, as would be expected if the elevation in enzyme activity is required for DNA repair. The chlorambucil-induced elevations in R1 and R2 message levels were blocked by treatment of cells with actinomycin D or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, indicating the importance of the reductase transcriptional process in responding to the action of chlorambucil and providing evidence for the involvement of a protein kinase C pathway in the regulation of mammalian ribonucleotide reductase. In addition to the chlorambucil-induced elevations in enzyme activity, message, and protein levels, the drug was also shown to be an inhibitor of ribonucleotide reductase activity in cell-free preparations. Separation of ribonucleotide components on an affinity column followed by selective exposure of the protein components to chlorambucil showed that both R1 and R2 proteins were targets for chlorambucil, in keeping with the known alkylating abilities of the drug. These observations provide the first direct demonstration of a link between the regulation of mammalian ribonucleotide reductase and the process of DNA repair and contribute to our understanding of the mode of action of a class of drugs represented by chlorambucil, in which chemotherapeutic activity has been attributed to DNA damaging effects.

Oxygen dependent regulation of mammalian ribonucleotide reductase in vivo and possible significance for replicon initiation

The intracellular concentration of the M2 specific free tyrosyl radical of ribonucleotide reductase in cultured Ehrlich ascites cells was estimated by EPR spectroscopy during imposition and after reversal of hypoxia. Under the same conditions, the intracellular intensity of CDP reduction was estimated indirectly by measuring the incorporation of radioactivity into DNA from labeled ribo- and deoxyribo-cytidine, respectively. The radical concentration distinctly decreased under hypoxia and reincreased upon reaeration. At the same time, the CDP reduction was greatly diminished and reactivated, respectively. These observations are interpreted in the sense of an O 2 dependent regulation of the intracellular activity of ribonucleotide reductase. The O 2 dependent deactivation and reactivation of the enzyme was temporally related to a specific suppression or a burst-like release of replicon initiations, respectively. Addition of 100 μM dCtd to hypoxic cells could substitute for reoxygenation with respect to triggering replicon initiations. A possible implication of the intracellular ribonucleotide reductase activity and the size of the dCTP pool in replicon initiation is dicussed.

Cell cycle-dependent regulation of mammalian ribonucleotide reductase. The S phase-correlated increase in subunit M2 is regulated by de novo protein synthesis.

Ribonucleotide reductase in mammalian cells is composed of two nonidentical subunits, proteins M1 and M2. Protein M2 contains a tyrosyl free radical, essential for activity, which can be quantified directly in frozen, packed cells by EPR spectroscopy. A 3-7-fold increase in the concentration of tyrosyl radical-containing M2 subunit was observed when mouse mammary tumor TA 3 cells passed from the G1 to the S phase of the cell cycle. Similar results were obtained with cells synchronized by isoleucine starvation or separated by centrifugal elutriation. Addition of deuterated tyrosine to cells give rise to a different EPR signal in newly synthesized protein M2. Pulse-chase experiments with deuterated tyrosine showed unequivocally that the S phase-correlated increase in radical-containing M2 subunit was due to de novo protein synthesis. Labeled M2 molecules disappeared with a half-life of 3 h, and therefore new molecules must be synthesized at a high rate during the S phase. In contrast, after hydroxyurea inactivation, cells rapidly regenerated the tyrosyl radical in already existing protein M2 molecules. This enzyme activation mechanism is clearly different from the one responsible for regulating protein M2 activity during the cell cycle.

Studies of mammalian ribonucleotide reductase activity in intact permeabilized cells: A genetic approach

We are using a genetic approach to study the complex properties of mammalian ribonucleotide reductase activity. Cytotoxic drugs (hydroxyurea, guanzole and N-carbamoyloxyurea) whose intracellular site of action is ribnonucleotide reductase have been used as selective agents in culture to isolate drug resistant cell lines with specific alterations in reductase activity. In general, cell lines resistant to “hydroxyurea-like” drugs exhibit genetic properties satisfying the majority of the criteria for classification as authentic somatic cell mutants. To study the mammalian enzyme properties, in a situation resembling physiological conditions, we have developed a convenient assay system for ribonucleotide reductase activity in whole cells. The procedure is relatively easy to perform, can accurately determine enzyme activity in as few as 10 6 cells grown conveniently on the surface of a tissue culture plate, and unlike cell-free enzyme preparations, activity is linear at low enzyme concentrations. The procedure can be adapted to investigate activity in a relatively small number of intact cells, it is very useful for studying cultures like human diploid fibroblasts, where large quantities of cells containing high enzyme activity may be difficult to obtain. Using this inact cell assay procedure, we have characterized the reductase activity in CHO cells with respect to pyrimidine (CDP) and purine (ADP) substrates, negative and positive effectors, and drug inhibition with the three antitumor agents employed as selective agents for isolating drug resistant cell lines. Some differences in the mode of action of N-carbamoyloxyurea when compared to hydroxyurea or guanazole were noticed and indicate the potential usefulness of the intact cell assay system for examining the action of specific drugs (e.g., 65) on intracellular enzyme activity. Intracellular ribonucleotide reductase activity was examined in the drug resistant cell lines and three different mutant classes were identified. 1. Cell lines containing an apparent structural alteration to the enzyme which results in reductase activity being less sensitive to drug inhibition. 2. Mutants containing elevated levels of reductase activity with a wild type sensitivity to the drug. 3. Cell lines containing a combination of the first two alterations described above; these mutants apparently contain elevated levels of a ribonucleotide reductase enzyme which has a structural alteration rendering it less sensitive to drug inhibition. Furthermore, some recent preliminary work suggests that additional mutant types with alterations in reductase activity can be isolated. The significance of these mutations in providing a better understanding of the regulation of mammalian ribonucleotide reductase was discussed.

Functional Regulation of Mammalian Ribonucleotide Reductase

Evidence is presented to substantiate the contention that the reduction of ribonucleotides to deoxyribonucleotides is a critical and rate-controlling step in the pathway leading to DNA synthesis and cell replication. One main piece of evidence is the demonstration of a close correlation between ribonucleotide reductase and tumor growth rates in a spectrum of rat hepatomas of varying growth rates. The physiological hydrogen donor for this reaction, thioredoxin-thioredoxin reductase, is found at comparable levels in both fast-growing tumor and normal liver. The modulation of ribonucleotide reductase activity was also examined during the structural and functional development of several rat organs. The various organs have different profiles of activity which are probably related to functional differentiation. For example, during liver development, activity is at a high level in the late fetal stage, but drops sharply at birth and becomes non-detectable in 3–4 weeks, while in the developing spleen and thymus, activity is low at birth and reaches a maximum about one week after birth and then declines. However, activity is still maintained in the adult state. It is suggested that heightened activity may be associated with an intense period of cellular replication related to the process of differentiation. The dramatic fluctuations in ribonucleotide reductase activity observed in tumors and during neonatal development are shown to be due to de novo enzyme synthesis and not to enzyme activation. A protein synthesis inhibitor, cycloheximide, prevents increases in enzyme activity in developing organs and causes a rapid decrease in enzyme activity in Novikoff hepatoma. The inhibition of DNA-directed RNA synthesis by actinomycin D also prevents increases in enzymatic activity in developing organs but has no effect on Novikoff tumor activity. Subcellular fractionation of Novikoff hepatoma and 24-hr regenerating liver reveals that ribonucleotide reductase is associated with a smooth membrane component of the cytoplasm. Thymidine kinase activity is also found with this subcellular membrane fraction. These data, in conjunction with data of Baril et al. (7, 8) that a DNA polymerase is associated with this smooth membrane organelle, suggest that there may exist a complex of several DNA synthetic enzymes in a functional unit.