Trypanosoma brucei CTP synthetase: a target for the treatment of African sleeping sickness.

The drugs in clinical use against African sleeping sickness are toxic, costly, or inefficient. We show that Trypanosoma brucei, which causes this disease, has very low levels of CTP, which are due to a limited capacity for de novo synthesis and the lack of salvage pathways. The CTP synthetase inhibitors 6-diazo-5-oxo-l-norleucine (DON) and alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) reduced the parasite CTP levels even further and inhibited trypanosome proliferation in vitro and in T. brucei-infected mice. In mammalian cells, DON mainly inhibits de novo purine biosynthesis, a pathway lacking in trypanosomes. We could rescue DON-treated human and mouse fibroblasts by the addition of the purine base hypoxanthine to the growth medium. For treatment of sleeping sickness, we propose the use of CTP synthetase inhibitors alone or in combination with appropriate nucleosides or bases.

Cross-talk between the allosteric effector-binding sites in mouse ribonucleotide reductase.

We compared the allosteric regulation and effector binding properties of wild type R1 protein and R1 protein with a mutation in the "activity site" (D57N) of mouse ribonucleotide reductase. Wild type R1 had two effector-binding sites per polypeptide chain: one site (activity site) for dATP and ATP, with dATP-inhibiting and ATP-stimulating catalytic activity; and a second site (specificity site) for dATP, ATP, dTTP, and dGTP, directing substrate specificity. Binding of dATP to the specificity site had a 20-fold higher affinity than to the activity site. In all these respects, mouse R1 resembles Escherichia coli R1. Results with D57N were complicated by the instability of the protein, but two major changes were apparent. First, enzyme activity was stimulated by both dATP and ATP, suggesting that D57N no longer distinguished between the two nucleotides. Second, the two binding sites for dATP both had the same low affinity for the nucleotide, similar to that of the activity site of wild type R1. Thus the mutation in the activity site had decreased the affinity for dATP at the specificity site, demonstrating the interaction between the two sites.

Yeast ribonucleotide reductase has a heterodimeric iron-radical-containing subunit.

Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides. Eukaryotes have an alpha(2)beta(2) form of RNR consisting of two homodimeric subunits, proteins R1 (alpha(2)) and R2 (beta(2)). The R1 protein is the business end of the enzyme containing the active site and the binding sites for allosteric effectors. The R2 protein is a radical storage device containing an iron center-generated tyrosyl free radical. Previous work has identified an RNR protein in yeast, Rnr4p, which is homologous to other R2 proteins but lacks a number of conserved amino acid residues involved in iron binding. Using highly purified recombinant yeast RNR proteins, we demonstrate that the crucial role of Rnr4p (beta') is to fold correctly and stabilize the radical-storing Rnr2p by forming a stable 1:1 Rnr2p/Rnr4p complex. This complex sediments at 5.6 S as a betabeta' heterodimer in a sucrose gradient. In the presence of Rnr1p, both polypeptides of the Rnr2p/Rnr4p heterodimer cosediment at 9.7 S as expected for an alpha(2)betabeta' heterotetramer, where Rnr4p plays an important role in the interaction between the alpha(2) and the betabeta ' subunits. The specific activity of the Rnr2p complexed with Rnr4p is 2,250 nmol deoxycytidine 5'-diphosphate formed per min per mg, whereas the homodimer of Rnr2p shows no activity. This difference in activity may be a consequence of the different conformations of the inactive homodimeric Rnr2p and the active Rnr4p-bound form, as shown by CD spectroscopy. Taken together, our results show that the Rnr2p/Rnr4p heterodimer is the active form of the yeast RNR small subunit.

5'-Phosphoramidates and 5'-diphosphates of 2'-O-allyl-beta-D-arabinofuranosyluracil, -cytosine, and -adenine: inhibition of ribonucleotide reductase.

Continuing our studies on ribonucleotide reductase (RNR) mechanism-based inhibitors, we have now prepared the diphosphates (DP) of 2'-O-allyl-1-beta-D-arabinofuranosyl-uracil and -cytosine and 2'-O-allyl-9-beta-D-arabinofuranosyl-adenine and evaluated their inhibitory activity against recombinant murine RNR. 2'-O-Allyl-araUDP proved to be inhibitory to RNR at an IC(50) of 100 microM, whereas 2'-O-allyl-araCDP was only marginally active (IC(50) 1 mM) and 2'-O-allyl-araADP was completely inactive. The susceptibility of the parent nucleosides to phosphorylation by thymidine kinase and 2'-deoxycytidine kinase was also investigated, and all nucleosides proved to be poor substrates for the above-cited kinases. Moreover, prodrugs of 2'-O-allyl-araU and -araC monophosphates, namely 2'-O-allyl-5'-(phenylethoxy-L-alanyl phosphate)-araU and -araC, were prepared and tested against tumor cell proliferation but proved to be inactive. A molecular modeling study has been conducted in order to explain our results. The data confirm that for both the natural and analogue nucleoside diphosphates, the principal determinant interaction with the active site of RNR is with the diphosphate group, which forms strong hydrogen bonds with Glu623, Thr624, Ser625, and Thr209. Our findings indicate that the poor phosphorylation may represent an explanation for the lack of marked in vitro cytostatic activity of the test compounds.

Synthesis, cytostatic activity and inhibition of ribonucleotide reductase by 5'-phosphoramidates and 5'-diphosphates, of 2'-O-allyl-arabinofuranosyl nucleosides.

The diphosphates of a series of 2'-O-allyl-1-beta-D-arabinofuranosyl derivatives, previously obtained by us, have been prepared and tested for their inhibitory activity in an in vitro assay using R1 and R2 subunits of the purified recombinant mouse ribonucleotide reductase (RNR). 2'-O-Allyl-araU diphosphate proved to be inhibitory, with an IC50 of 100 microM. The 5'-phosphoramidate pronucleotide of 2'-O-allyl-araU was also prepared and tested for inhibition of tumor cell proliferation.

The iron-oxygen reconstitution reaction in protein R2-Tyr-177 mutants of mouse ribonucleotide reductase. Epr and electron nuclear double resonance studies on a new transient tryptophan radical.

The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity. We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals. In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 degrees C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in both mutants that do not have successor radicals as Trp. in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E. coli Y122F or native mouse protein R2. All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates.

Two YY-1-binding proximal elements regulate the promoter strength of the TATA-less mouse ribonucleotide reductase R1 gene.

Ribonucleotide reductase is essential for DNA synthesis. In mammalian cells, the enzyme consists of two non-identical subunits, proteins R1 and R2. The expression of the mouse R1 and R2 genes is strictly correlated to S phase. Using promoter-reporter gene constructs, we have defined a region of the TATA-less mouse ribonucleotide reductase R1 gene promoter that correlates reporter gene expression to S phase. This is demonstrated in stably transformed cells both synchronized by serum starvation and separated by centrifugal elutriation, suggesting that the R1 gene expression during the cell cycle is mainly regulated at the transcriptional level. The region contains four protein-binding DNA elements, beta (nucleotides -189 to -167), alpha (-98 to -76), Inr (-4 to +16), and gamma (+34 to +61), together regulating promoter activity. The nearly identical upstream elements, alpha and beta, each form three DNA-protein complexes in gel shift assays. We have identified YY1 as a component in at least one of the complexes using supershift antibodies and a yeast one-hybrid screening of a mouse cDNA library using the alpha element as a target. Transient transfection assays demonstrate that the alpha and beta elements are mainly important for the R1 promoter strength and suggest that YY1 functions as an activator.

The three-dimensional structure of mammalian ribonucleotide reductase protein R2 reveals a more-accessible iron-radical site than Escherichia coli R2.

The three-dimensional structure of mouse ribonucleotide reductase R2 has been determined at 2.3 A resolution using molecular replacement and refined to an R-value of 19.1% (Rfree = 25%) with good stereo-chemistry. The overall tertiary structure architecture of mouse R2 is similar to that from Escherichia coli R2. However, several important structural differences are observed. Unlike the E. coli protein, the mouse dimer is completely devoid of beta-strands. The sequences differ significantly between the mouse and E. coli R2s, but there is high sequence identity among the eukaryotic R2 proteins, and the identities are localized over the whole sequence. Therefore, the three-dimensional structures of other mammalian ribonucleotide reductase R2 proteins are expected to be very similar to that of the mouse enzyme. In mouse R2 a narrow hydrophobic channel leads to the proposed binding site for molecular oxygen near to the iron-radical site in the interior of the protein. In E. coli R2 this channel is blocked by the phenyl ring of a tyrosine residue, which in mouse R2 is a serine. These structural variations may explain the observed differences in sensitivity to radical scavengers. The structure determination is based on diffraction data from crystals grown at pH 4.7. Unexpectedly, the protein is not iron-free, but contains one iron ion bound at one of the dinuclear iron sites. This ferric ion is bound with partial occupancy and is coordinated by three glutamic acids (one bidentate) and one histidine in a bipyramidal coordination that has a free apical coordination position. Soaking of crystals in a solution of ferrous salt at pH 4.7 increased the occupancy on the already occupied site, but without any detectable binding at the second site.

A kinetic study on the influence of nucleoside triphosphate effectors on subunit interaction in mouse ribonucleotide reductase.

For enzymatic activity, mouse ribonucleotide reductase must form a heterodimeric complex composed of homodimeric R1 and R2 proteins. Both substrate specificity and overall activity are regulated by the allosteric effectors ATP, dATP, dTTP, and dGTP, which bind to two different sites found on R1, the activity site and the substrate specificity site. We have used biosensor technique to directly observe the effects of these nucleotides on R1/R2 interactions. In the absence of effectors, positive cooperativity was observed with a Hill coefficient of 1.8 and a KD of 0.5 microM. In the presence of dTTP or dGTP, there was no cooperativity and subunit interaction was observed at a much lower R1 concentration. The highest R1/R2 affinity was in the presence of dATP or ATP with KDs of 0.05-0.1 microM. In all experiments, the molar stoichiometry between the subunits was close to 1:1. Our data support a model whereby binding of any of the effectors to the substrate specificity site promotes formation of the R1 dimer, which we believe is prerequisite for binding to the R2 dimer. Additional binding of either ATP (a positive effector) or dATP (a negative effector) to the activity site further increases R1/R2 association. We propose that binding of ATP or dATP to the activity site controls enzyme activity, not by changing the aggregation state of the R1/R2 proteins as proposed earlier, but rather by locally influencing the long range electron transport between the catalytic site of R1 and the tyrosyl free radical of R2.

Role of a proximal NF-Y binding promoter element in S phase-specific expression of mouse ribonucleotide reductase R2 gene.

Cell cycle-regulated transcription of the R2 gene of mouse ribonucleotide reductase was earlier shown to be controlled at the level of elongation by an S phase-specific release from a transcriptional block. However, the R2 promoter is activated very early when quiescent cells start to proliferate, and this activation is dependent on three upstream sequences located nucleotide -672 to nucleotide -527 from the transcription start. In this study, we use R2-luciferase reporter gene constructs and gel shift assays to demonstrate that, in addition to the upstream sequences, a proximal CCAAT element specifically binding the transcription factor NF-Y is required for continuous activity of the R2 promoter through the S phase. When the CCAAT element is deleted or mutated, promoter activity induced by the upstream elements decays before cells enter S phase, and the transcriptional block is released. This is a clear example of how changing of a proximal sequence element can alter not only the quantitative but also the qualitative response to upstream transcription activation domains.

The critical C-terminus of the small subunit of herpes simplex virus ribonucleotide reductase is mobile and conformationally similar to C-terminal peptides.

The C-terminus of the small subunit of class I ribonucleotide reductases is essential for subunit association and enzymatic activity. 1H NMR analysis of the small subunit (2 x 38 kDa as a homodimer) of herpes simplex virus ribonucleotide reductase shows that this critical binding site is mobile and exposed in relation to the rest of the protein. Assignments of six C-terminal amino acids are made by comparing the TOCSY and NOESY spectra of the small subunit with the spectra of an identical protein truncated by seven amino acids at the C-terminus and the spectra of an analogous 15 amino acid peptide. The mobility of the C-terminus may be important for subunit recognition and could be general for other ribonucleotide reductases. The spectral comparisons also suggest that the six C-terminal amino acids of the small subunit and peptide are conformationally similar. This observation may be important for the design of inhibitors of ribonucleotide reductase subunit association.

Purification, characterization, and localization of subunit interaction area of recombinant mouse ribonucleotide reductase R1 subunit.

Mammalian ribonucleotide reductase is a heterotetramer formed by the two non-identical homodimers proteins R1 and R2. We have succeeded in expressing the 90-kDa mouse R1 protein in Escherichia coli in an active, soluble form using the T7 RNA polymerase pET vector system. To avoid inclusion bodies, the bacteria were grown at 15 degrees C with minimal concentration of the inducer isopropyl-1-thio-beta-D-galactopyranoside. After a rapid purification procedure, approximately 20 mg of pure R1 protein were obtained per liter of bacterial culture. The concentrated R1 protein solution had a pinkish red color. Spectroscopy in combination with iron and labile sulfur analyses demonstrated that the color originated from an iron-sulfur complex. However, all attempts to demonstrate a function of this complex have been inconclusive. A comparison of the recombinant R1 protein with the corresponding protein purified from calf thymus showed no evidence for glycosylation. Circular dichroism spectroscopy indicated an alpha-helical content of 50%. A flexible COOH-terminal tail of 7 residues in the R2 protein was earlier shown to be essential for binding to the R1 protein. Using a peptide protection assay and photoaffinity labeling, we now show that the R2 protein tail interacts with a region close to the carboxyl terminus of the R1 protein.

Role of ribonucleotide reductase in inhibition of mammalian cell growth by potent iron chelators.

Ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, the latter of which contains an iron-tyrosyl free radical center essential for activity. We have studied the in vivo effects on the R2 protein of the potent iron chelators parabactin and desferrioxamine using R2-overproducing mouse cells with a tyrosyl free radical signal easily quantifiable by EPR spectroscopy. Both chelators inhibited cell growth, and the inhibition was reversible by iron. Furthermore, both chelators, which penetrate cells and chelate the intracellular iron pool, caused a disappearance of the R2 tyrosyl free radical. In parallel, there was an accumulation of apo-R2 protein in the inhibited cells. In vitro studies using pure, 59Fe-labeled recombinant mouse R2 protein unexpectedly showed that its iron center is labile at physiological temperatures and that iron is spontaneously lost from the protein even in the absence of chelators in a temperature-dependent process. Our conclusion is that parabactin or desferrioxamine inhibits ribonucleotide reduction and cell growth not by directly attacking the iron-radical center of the R2 protein, but instead by chelating the intracellular iron pool. This prevents the regeneration of the iron-radical center both in newly synthesized apo-R2 protein and in apo-R2 protein continuously formed from active R2 protein by the loss of iron.

Reduction and loss of the iron center in the reaction of the small subunit of mouse ribonucleotide reductase with hydroxyurea.

Ribonucleotide reductase is a key enzyme for DNA synthesis in living cells, and the mechanisms for its reactions with inhibitors are of interest because the inhibitors are potential antiproliferative agents. Protein R2, the small subunit of mouse ribonucleotide reductase, contains a pair of mu-oxo-bridged ferric ions and a tyrosyl free radical in each of its two polypeptide chains. Light absorption spectroscopy was used to probe the reactions of these redox centers with hydroxyurea (HU), a potent inhibitor of iron containing ribonucleotide reductases. In Escherichia coli protein R2, HU reacts with the tyrosyl radical without affecting the iron center. In contrast to the case for the E. coli protein, HU destroys the specific absorbance bands of both the iron center and the radical on a similar time scale in mouse protein R2, and this is accompanied by release of iron from the protein. Anaerobic experiments with the iron chelator bathophenanthroline present during the HU reaction indicate that the iron is released from the mouse R2 protein in the ferrous form after treatment with HU. The reduced iron center, formed by reaction of Fe2+ with mouse apoprotein R2 under anaerobic conditions, was found to be much less stable than the native Fe3+ site in the presence of suitable iron chelators. The observations are of importance for understanding the mode of action of HU on mammalian cells and for the general question of the stability of the iron center of mouse protein R2 in different redox states.

The role of herpes simplex virus ribonucleotide reductase small subunit carboxyl terminus in subunit interaction and formation of iron-tyrosyl center structure.

Herpes simplex virus ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, which are required together for activity. Active R2 protein contains a tyrosyl free radical and a binuclear iron center. A truncated form of the R2 subunit, lacking 7 amino acid residues in the carboxyl terminus, was constructed, overexpressed in Escherichia coli and purified to homogeneity. In the presence of ferrous iron and oxygen, the truncated protein readily generated similar amounts of tyrosyl free radical as the intact protein. However, the radical showed differences in EPR characteristics in the truncated protein compared with the normal one, indicating an altered structural arrangement of the radical relative to the iron center. The truncated R2* protein was completely devoid of binding affinity to the R1 protein, demonstrating that the subunit interaction is totally dependent on the 7 outermost carboxyl-terminal amino acids of protein R2.

Inactivation of ribonucleotide reductase by nitric oxide.

Ribonucleotide reductase has been demonstrated to be inhibited by NO synthase product(s). The experiments reported here show that nitric oxide generated from sodium nitroprusside, S-nitrosoglutathione and the sydnonimine SIN-1 inhibits ribonucleotide reductase activity present in cytosolic extracts of TA3 mammary tumor cells. Stable derivatives of these nitric oxide donors were either inactive or much less inhibitory. EPR experiments show that the tyrosyl radical of the small subunit of E. Coli or mammalian ribonucleotide reductase is efficiently scavenged by these NO donors.

Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit.

Overexpression of recombinant mouse and herpes simplex virus ribonucleotide reductase small subunit (protein R2) has been obtained by using the T7 RNA polymerase expression system. Both proteins, which constitute about 30% of the soluble Escherichia coli proteins, have been purified to homogeneity by a rapid and simple procedure. At this stage, few of the molecules contain the iron-tyrosyl free-radical center necessary for activity; however, addition of ferrous iron and oxygen under controlled conditions resulted in a mouse R2 protein containing 0.8 radical and 2 irons per polypeptide chain. In this reaction, one oxygen molecule was needed to generate each tyrosyl radical. Both proteins had full enzymatic activity. EPR spectroscopy showed that iron-center/radical interactions are considerably stronger in both mouse and viral proteins than in E. coli protein R2. CD spectra showed that the bacterial protein contains 70% alpha-helical structure compared to only about 50% in the mouse and viral proteins. Light absorption spectra between 310 and 600 nm indicate close similarity of the mu-oxo-bridged binuclear iron centers in all three R2 proteins. Furthermore, the paramagnetically shifted iron ligand proton NMR resonances show that the antiferromagnetic coupling and ligand arrangement in the iron center are nearly identical in all three species.

Paracetamol inhibits replicative DNA synthesis and induces sister chromatid exchange and chromosomal aberrations by inhibition of ribonucleotide reductase.

Effects of paracetamol have been studied in a hydroxyurea (HU)-resistant mouse mammary tumour cell line TA3H2, shown to overproduce the small subunit of ribonucleotide reductase. These TA3H2 cells were much more resistant than the TA3H (wild-type) cells towards the inhibitory effect of paracetamol on cell growth, IC50 0.55 mM paracetamol for the wild-type compared to 2.7 mM for the HU-resistant cells. The reduced cell growth was due to an inhibition of replicative DNA synthesis, judged from an increased percentage of cells in S-phase measured by flow cytometry. Furthermore, in the wild-type cells, the increase in the number of cells in S phase was already observed at 0.1 mM while in the HU-resistant cell line this effect was first seen at 3.0 mM paracetamol. HU inhibits ribonucleotide reductase by destroying a tyrosyl free radical located on the small subunit of the enzyme. By electron paramagnetic resonance we demonstrate that paracetamol added to crude cell extracts of HU-resistant cells also immediately destroys this radical. These results show that paracetamol reduces DNA synthesis by a specific inhibition of ribonucleotide reductase. A concentration-dependent induction of sister chromatid exchanges was found both with paracetamol (1.0-10 mM) and HU (0.3-3 mM) in wild-type cells whereas no such increase was observed in HU-resistant cells. Paracetamol (1 mM for 2 h) also increased the number of chromosomal aberrations CAs in wild-type cells (i.e. chromatid breaks and chromatid exchanges). The frequency of CAs was not increased in HU-resistant cells at paracetamol concentrations up to 10 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations of ribonucleotide reductase activity following induction of the nitrite-generating pathway in adenocarcinoma cells.

The murine adenocarcinoma cell line TA 3 synthesized nitrite from L-arginine upon stimulation with gamma-interferon (IFN-gamma) associated with tumor necrosis factor (TNF), and/or bacterial lipopolysaccharide (LPS), but not with IFN-gamma, TNF, or LPS added separately. Induction of the NO2(-)-generating activity caused an inhibition of DNA synthesis in TA 3 cells. This inhibition was prevented by the L-arginine analog N omega-nitro-L-arginine, which inhibited under the same conditions nitrite production by TA 3 cells. The TA 3 M2 subclone, selected for enhanced ribonucleotide reductase activity, was found to be less sensitive than the wild phenotype TA 3 WT to the cytostatic activity mediated by the NO2(-)-generating system. Cytosolic preparations from TA 3 M2 cells treated for 24 or 48 h with IFN-gamma, TNF, and LPS exhibited a reduced ribonucleotide reductase activity, compared to untreated control cells. No reduction in ribonucleotide reductase activity was observed when N omega-nitro-L-arginine was added to treated cells. Addition of L-arginine, NADPH, and tetrahydrobiopterin into cytosolic extracts from 24-h treated TA 3 M2 cells triggered the synthesis of metabolic products from the NO2(-)-generating pathway. This resulted in a dramatic inhibition of the residual ribonucleotide reductase activity present in the extracts. The inhibition was reversed by NG-monomethyl-L-arginine, another specific inhibitor of the NO2(-)-generating activity. No L-arginine-dependent inhibition of ribonucleotide reductase activity was observed using extracts from untreated cells that did not express NO2(-)-generating activity. These results demonstrate that, in an acellular preparation, molecules derived from the NO2(-)-generating pathway exert an inhibitory effect on the ribonucleotide reductase enzyme. This negative action might explain the inhibition of DNA synthesis induced in adenocarcinoma cells by the NO2(-)-generating pathway.

Molecular mechanisms responsible for the drug-induced posttranscriptional modulation of ribonucleotide reductase levels in a hydroxyurea-resistant mouse L cell line.

Ribonucleotide reductase, which catalyzes the formation of deoxyribonucleotides from ribonucleoside diphosphate precursors, is the rate-limiting enzyme in DNA synthesis. The enzyme consists of two nonidentical subunits called M1 and M2, both of which are required for activity. Hydroxyurea, a specific inhibitor of DNA synthesis, acts by destroying the unique tyrosyl free radical of protein M2. Previously, we have described a mouse L cell line which exhibited a stable resistance to high concentrations of hydroxyurea. This mutant cell line contains elevated quantities of both proteins M1 and M2 as a result of corresponding increases in the levels of mRNAs for both subunits. Interestingly, both M1 and M2 protein levels were further elevated when mutant cells were cultured in the presence of hydroxyurea, and this elevation was not accompanied by increases in their corresponding mRNAs. These results indicated that hydroxyurea can modulate ribonucleotide reductase expression posttranscriptionally. In this report, we show that the level of both subunits of ribonucleotide reductase responds to hydroxyurea in a drug concentration dependent manner. Furthermore, results from kinetic studies indicate that protein M2 levels rise much more rapidly than protein M1. Pulse-chase experiments indicated that the half-lives of both the M1 and M2 polypeptides are increased by approximately 2-fold when the mutant cells are cultured in the presence of hydroxyurea. We also present evidence indicating that exposure of these cells to hydroxyurea leads to a relatively slow but specific increase in the rate of biosynthesis of both proteins M1 and M2, as assayed by pulse labeling.(ABSTRACT TRUNCATED AT 250 WORDS)