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Protein glutathionylation in response to oxidative stress can affect both the stability and activity of target proteins. Mitochondrial thymidine kinase 2 (TK2) is a key enzyme in mitochondrial DNA precursor synthesis. Using an antibody specific for glutathione (GSH), S-glutathionylated TK2 was detected after the addition of glutathione disulfide (GSSG) but not GSH. This was reversed by the addition of dithiothreitol, suggesting that S-glutathionylation of TK2 is reversible. Site-directed mutagenesis of the cysteine residues and subsequent analysis of mutant enzymes demonstrated that Cys-189 and Cys-264 were specifically glutathionylated by GSSG. These cysteine residues do not appear to be part of the active site, as demonstrated by kinetic studies of the mutant enzymes. Treatment of isolated rat mitochondria with hydrogen peroxide resulted in S-glutathionylation of added recombinant TK2. Treatment of intact cells with hydrogen peroxide led to reduction of mitochondrial TK2 activity and protein levels, as well as S-glutathionylation of TK2. Furthermore, the addition of S-glutathionylated recombinant TK2 to mitochondria isolated from hydrogen peroxide-treated cells led to degradation of the S-glutathionylated TK2, which was not observed with unmodified TK2. S-Glutathionylation on Cys-189 was responsible for the observed selective degradation of TK2 in mitochondria. These results strongly suggest that oxidative damage-induced S-glutathionylation and degradation of TK2 have significant impact on mitochondrial DNA precursor synthesis.

Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G(2) phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G(1)/S progression, resulting in a smaller G(2) fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53(-/-) cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G(2) arrest.

RNA polymerases effectively discriminate against deoxyribonucleotides and specifically recognize ribonucleotide substrates most likely through direct hydrogen bonding interaction with the 2'-alpha-hydroxy moieties of ribonucleosides. Therefore, ribonucleoside analogs as inhibitors of viral RNA polymerases have mostly been designed to retain hydrogen bonding potential at this site for optimal inhibitory potency. Here, two novel nucleoside triphosphate analogs are described, which are efficiently incorporated into nascent RNA by the RNA-dependent RNA polymerase NS5B of hepatitis C virus (HCV), causing chain termination, despite the lack of alpha-hydroxy moieties. 2'-deoxy-2'-beta-fluoro-4'-azidocytidine (RO-0622) and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187) were excellent substrates for deoxycytidine kinase and were phosphorylated with efficiencies up to 3-fold higher than deoxycytidine. As compared with previous reports on ribonucleosides, higher levels of triphosphate were formed from RO-9187 in primary human hepatocytes, and both compounds were potent inhibitors of HCV virus replication in the replicon system (IC(50) = 171 +/- 12 nM and 24 +/- 3 nM for RO-9187 and RO-0622, respectively; CC(50) >1 mM for both). Both compounds inhibited RNA synthesis by HCV polymerases from either HCV genotypes 1a and 1b or containing S96T or S282T point mutations with similar potencies, suggesting no cross-resistance with either R1479 (4'-azidocytidine) or 2'-C-methyl nucleosides. Pharmacokinetic studies with RO-9187 in rats and dogs showed that plasma concentrations exceeding HCV replicon IC(50) values 8-150-fold could be achieved by low dose (10 mg/kg) oral administration. Therefore, 2'-alpha-deoxy-4'-azido nucleosides are a new class of antiviral nucleosides with promising preclinical properties as potential medicines for the treatment of HCV infection.

Thymidine kinase 2 (TK2) is a mitochondrial (mt) pyrimidine deoxynucleoside salvage enzyme involved in mtDNA precursor synthesis. The full-length human TK2 cDNA was cloned and sequenced. A discrepancy at amino acid 37 within the mt leader sequence in the DNA compared with the determined peptide sequence was found. Two mutations in the human TK2 gene, His-121 to Asn and Ile-212 to Asn, were recently described in patients with severe mtDNA depletion myopathy (Saada, A., Shaag, A., Mandel, H., Nevo, Y., Eriksson, S., and Elpeleg, O. (2001) Nat. Genet. 29, 342-344). The same mutations in TK2 were introduced, and the mutant enzymes, prepared in recombinant form, were shown to have similar subunit structure to wild type TK2. The I212N mutant showed less than 1% activity as compared with wild type TK2 with all deoxynucleosides. The H121N mutant enzyme had normal K(m) values for thymidine (dThd) and deoxycytidine (dCyd), 6 and 11 microm, respectively, but 2- and 3-fold lower V(max) values as compared with wild type TK2 and markedly increased K(m) values for ATP, leading to decreased enzyme efficiency. Competition experiments revealed that dCyd and dThd interacted differently with the H121N mutant as compared with the wild type enzyme. The consequences of the two point mutations of TK2 and the role of TK2 in mt disorders are discussed.

Globular inclusions of abnormal alpha1-antitrypsin (AAT) in the endoplasmic reticulum of hepatocytes are a characteristic feature of AAT deficiency of the PiZZ phenotype. Monoclonal antibodies, which contain constant specificity and affinity, are often used for the identification of Z-mutation carriers. A mouse monoclonal antibody (ATZ11) raised against PiZZ hepatocytic AAT was successfully used in enzyme-linked immunosorbent assays (ELISA) and in identification of Z-related AAT globular inclusions by immunohistochemical techniques. Using electrophoresis, Western blotting, and ELISA procedures, we have shown in the present study that this monoclonal antibody specifically detects a conformation-dependent neoepitope on both polymerized and elastase-complexed molecular forms of AAT. The antibody has no apparent affinity for native, latent, or cleaved forms of AAT. The antibody ATZ11 illustrates the structural resemblance between the polymerized form of AAT and its complex with elastase and provides evidence that Z-homozygotes beyond the native form may have at least one more circulating molecular form of AAT, i.e. its polymerized form. In addition, staining of endothelial cells with ATZ11 antibody in both M- and Z-AAT individuals shows that AAT attached to endothelial cells is in a polymerized form. The antibody can be a powerful tool for the study of the molecular profile of AAT, not only in Z-deficiency cases but also in other (patho)physiological conditions.

Cytosolic 5'-nucleotidase/phosphotransferase (cN-II), specific for purine monophosphates and their deoxyderivatives, acts through the formation of a phosphoenzyme intermediate. Phosphate may either be released leading to 5'-mononucleotide hydrolysis or be transferred to an appropriate nucleoside acceptor, giving rise to a mononucleotide interconversion. Chemical reagents specifically modifying aspartate and glutamate residues inhibit the enzyme, and this inhibition is partially prevented by cN-II substrates and physiological inhibitors. Peptide mapping experiments with the phosphoenzyme previously treated with tritiated borohydride allowed isolation of a radiolabeled peptide. Sequence analysis demonstrated that radioactivity was associated with a hydroxymethyl derivative that resulted from reduction of the Asp-52-phosphate intermediate. Site-directed mutagenesis experiments confirmed the essential role of Asp-52 in the catalytic machinery of the enzyme and suggested also that Asp-54 assists in the formation of the acyl phosphate species. From sequence alignments we conclude that cytosolic 5'-nucleotidase, along with other nucleotidases, belong to a large superfamily of hydrolases with different substrate specificities and functional roles.

Deoxyguanosine kinase (dGK) initiates the salvage of purine deoxynucleosides in mitochondria and is a key enzyme in mitochondrial DNA precursor synthesis. The active form of the enzyme is a 60-kDa protein normally located in the mitochondrial matrix. Here we describe the subcellular distribution of dGK during apoptosis in human epithelial kidney 293 cells and human lymphoblast Molt-4 cells. Immunological methods were used to monitor dGK as well as other mitochondrial proteins. Surprisingly, dGK was found to relocate to the cytosolic compartment at a similar rate as cytochrome c, a mitochondrial intermembraneous enzyme known to enter the cytosol early in apoptosis. The redistribution of dGK from the mitochondria to the cytosol may be of importance for the activation of apoptotic purine nucleoside cofactors such as dATP and demonstrates that mitochondrial matrix proteins may selectively leak out during apoptosis.

Deoxyguanosine kinase (dGK) is a nuclear gene product that catalyzes the phosphorylation of purine deoxyribonucleosides and their analogues. The human enzyme is located predominantly in the mitochondria, as shown by biochemical fractionation studies and in situ localization of the overexpressed recombinant protein. Here we describe the cloning of mouse dGK cDNA and the identification of a novel amino-terminally truncated isoform that corresponds to about 14% of the total dGK mRNA population in mouse spleen. In situ fluorescence assays suggest that the new isoform cannot translocate into the mitochondria and thus may represent a cytoplasmic enzyme. Expression of mouse dGK mRNA was highly tissue-specific and differed from the tissue distribution observed in humans. Recombinant mouse dGK showed similar specific activity and substrate specificity as compared with the human enzyme. The broad specificity, restricted tissue distribution, and location of mouse dGK in multiple cellular compartments raise new considerations with respect to the role of the individual deoxynucleoside kinases in nucleotide metabolism.

Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxynucleoside salvage pathway in mammalian cells and plays a key role in the activation of several pharmacologically important nucleoside analogs. Using a highly specific polyclonal antibody raised against a C-terminal peptide of the human dCK, we analyzed its subcellular localization by Western blots of biochemically fractionated nuclear and cytoplasmic fractions as well as by in situ immunochemistry. Native dCK was found to be located mainly in the cytoplasm in several cell types, and the enzyme was more concentrated in the perinuclear and cellular membrane area. In contrast, when dCK was overexpressed in the cells, it was mainly located in the nucleus. The results demonstrate that native dCK is a cytoplasmic enzyme. However, it has the ability to enter the nucleus under certain conditions, suggesting the existence of a cytoplasmic retention mechanism that may have an important function in the regulation of the deoxynucleoside salvage pathway.

A 39-43-amino acid residue-long fragment (beta-peptide) from the amyloid precursor protein is the predominant component of amyloid deposits in the brain of individuals with Alzheimer's disease. Serum amyloid P component (SAP) is present in all types of amyloid, including that of Alzheimer's disease. We have used an in vitro model to study the effects of purified SAP on the fibril formation of synthetic Alzheimer beta-peptide 1-42. SAP was found to inhibit fibril formation and to increase the solubility of the peptide in a dose-dependent manner. At a 5:1 molar ratio of A beta 1-42 peptide to SAP, fibril formation was completely inhibited, and approximately 80% of the peptide remained in solution even after 4 days of incubation. At lower SAP concentrations, e.g. at peptide to SAP ratio of 1000:1, short fibrillar like structures, lacking amyloid characteristics, were formed. These structures frequently contained associated SAP molecules, suggesting that SAP binds to the polymerizing peptide in a reaction which prevented further fibril formation.

Saccharomyces cerevisiae mtDNA polymerase, isolated as a single 135-kDa recombinant polypeptide, showed high processivity and a capacity of use poly(dA).oligo(dT), poly(rA).oligo(dT), or primed bacteriophage M13 DNA as a template. In a primer extension assay, the enzyme exhibited an intrinsic 3'-5'-exonuclease activity. By optimizing the polymerization reaction conditions, apparent Km and Vmax values could be determined for the incorporation of dTTP, 2'-3'-dideoxy-TTP (ddTTP), 3'-azido-TTP (AZTTP), 3'-fluoro-TTP, dCTP, 2'-3'-dideoxy-CTP, and didehydro(d4)CTP. The yeast mtDNA polymerase used ddTTP, 3'-fluoro-TTP, and ddCTP almost as efficiently as natural deoxynucleoside trisphosphates. Both 3'AZTTP and d4CTP were each significantly less efficient as substrates. Overall, the kinetic data with mtDNA polymerase were very similar to those of the recombinant human immunodeficiency virus reverse transcriptase control. Terminally incorporated AZTTP or ddTTP was not removed by the 3'-5' exonuclease activity of mtDNA polymerase. This may explain the inhibition of mtDNA replication observed in anti-human immunodeficiency virus treatment with dideoxynucleoside analogs for their effects of mtDNA polymerase could be of value in future rational drug design.

A cDNA encoding mouse deoxycytidine kinase (dCK) (EC 2.7.1.74) was cloned from a mouse T-cell lambda ZAP cDNA library. An insert of 2.8 kilobases (kb) contained the entire coding sequence of 780 base pairs. The protein coding sequence was 88% homologous at the nucleotide level with human dCK cDNA (Chottiner, E. G., Shewach, D. S., Datta, N. S., Ashcraft, E., Gribbin, D., Ginsburg, D., Fox, I. H., and Mitchell, B. S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1531-1535). At the amino acid level the homology was greater with only 16 of the 260 amino acids being different. Northern blot analyses revealed a size of 3.4 kb for mouse dCK mRNA as compared with 2.8 kb for human dCK. Part of the 3'-untranslated region was conserved between human and mouse dCK cDNA in contrast to the remainder of the 3'-sequence which was unrelated and about 500 nucleotides longer in mouse dCK cDNA. Mouse dCK cDNA showed cross-hybridization with several bands in EcoRI-digested genomic DNA from seven different mammalian species and chicken but not with yeast DNA. Both mouse and human dCK were cloned into the T5 promotor pQE30 vector system, expressed in Escherichia coli and purified to homogeneity. The kinetic constants for dCyd phosphorylation were similar for the human and mouse enzymes and also similar to what previously has been observed for dCK purified from human tissues. Mouse dCK was less efficient with regard to dAdo, dGuo, and ddCyd phosphorylation as compared with human dCK when using ATP as phosphate donor in a phosphoryl transfer assay.

Mitochondrial deoxyguanosine kinase (dGK) (EC 2.7.1.113) was purified to apparent homogeneity from bovine brain. The molecular mass of the native protein was 56 kDa, as judged by gel filtration, and one single band of 28 kDa was seen in sodium dodecyl sulfate-gel electrophoresis. 2'-Deoxyguanosine (dGuo) (Km, 7.6 microM), 2'-deoxyinosine, and 2'-deoxyadenosine (Km, 60 microM) were substrates for the enzyme as well as several dGuo analogs containing a lipophilic substituent at C-2'. Carbocyclic dGuo, 9-beta-D-arabinofuranosylguanine, 9-beta-D-arabinofuranosylhypoxanthine, and 9-beta-D-arabinofuranosyladenine were substrates for the enzyme, whereas no 3'-modified dGuo analogs were effective. Interestingly, 2-chloro-2'-deoxyadenosine (CdA) was found to be an efficient substrate for dGK (Km, 85 microM). Subcellular fractionation of human CEM lymphoblasts showed that extracts of mitochondria contain significant CdA phosphorylating activity (71.5 pmol/mg/min) that is not inhibited by excess of 2'-deoxycytidine (dCyd). This contrasts with the CdA phosphorylating activity found in cytosolic extracts, which is carried out by dCyd kinase and strongly inhibited by excess of dCyd. The efficient CdA phosphorylation by mitochondrial dGK is a novel finding that may have far reaching implications for the clinical use of this potent cytostatic drug.

The binding of single- as well as double-stranded DNA to RecA, in the presence of the cofactor analog ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)), leads to about 20% quenching of the tyrosine fluorescence of the protein but to no essential change of the tryptophan fluorescence. The excitation spectrum of the fluorescent DNA analog poly(d epsilon A), complexed with RecA, shows no sign of energy transfer from the tyrosine residues of RecA to the etheno-modified adenine bases of the polynucleotide. From this observation we reject stacking interaction between tyrosine residues and DNA bases. The RecA filament may bind up to three molecules of single-stranded DNA; however, the observed fluorescence change occurs only upon the binding of the first DNA strand, indicating that the binding mode of this first strand is different from those of the others. The fluorescence change is interpreted in terms of a conformational change of the RecA protein promoted by cooperative binding to DNA. A larger quenching (40%) upon the binding of single-stranded DNA is observed in the absence of cofactor. At high salt condition, which induces ATPase activity in RecA just as DNA binding does, the tyrosine fluorescence is more pronounced than at low salt conditions, indicating that the effect induced by high salt is different from the conformational change induced by DNA binding.

The tyrosine fluorescence of the RecA protein is quenched by about 15% upon binding of the cofactor analog adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). This quenching is not observed with a modified RecA in which the tyrosine residue at position 264 (Tyr-264) is replaced for alanine by site-directed mutagenesis, a modification which also results in a decrease of binding affinity of cofactor. This indicates that Tyr-264 is responsible for the fluorescence change and that the residue is close to or within the cofactor binding site. Upon DNA binding, a change of tyrosine fluorescence is observed both with the modified protein and with wild type RecA, indicating that DNA binding affects the environment of other tyrosine residues than Tyr-264. However, the change is significantly smaller in the modified protein, suggesting that both Tyr-264 as well as other residue(s) may be affected by the DNA binding. Changed fluorescence properties of the remaining tyrosine residues as a result of a slightly different DNA binding mode of the modified protein are also possible. Tyr-264 may be an important residue for the allosteric effect induced by the cofactor for the binding of DNA to RecA. In the recent crystal structure of RecA-ADP published by Story and Steitz (Story, R.M., and Steitz, T. A. (1992) Nature 355, 374-376), ADP is stacked with Tyr-103 and does not interact with Tyr-264. The fact that we observe no interaction of ATP gamma S with Tyr-103 (as evidenced from absence of fluorescence change) but instead with Tyr-264 may suggest an important conformational difference between the RecA complexes with, respectively, ADP and ATP.

3'-Azido-3'-deoxythymidine (AZT) is HIV-inhibitory in human macrophages, which is surprising in view of the low AZT phosphorylation reported in macrophage extracts. To elucidate the mechanism of AZT activation, we studied AZT anabolism as well as catabolism in human lymphocytes and macrophages, and compared it to that of thymidine. Thymidine kinase (TK)-specific activity in mitogen-stimulated lymphocytes was 15 times higher than in macrophages. However, the TK activity per cell was only 1.3 times higher, because of the large macrophage cell volume. Total cellular TK activity, but not specific activity, matched the level of intracellular AZT anabolism. The substrate specificity of TK in macrophages strongly suggests that mitochondrial TK2 was the enzyme phosphorylating thymidine and AZT in these cells, whereas it was cytosolic TK1 in stimulated lymphocytes. In vivo thymidine catabolism was extensive, forming thymine and dihydrothymine. In macrophages more than 95% of the added thymidine (0.5 microM) was degraded within 60 min. AZT, in contrast, was not catabolized, which explains the high AZT nucleotide accumulation, a process opposed only by AZTMP excretion. The lack of catabolism together with phosphorylation by TK2 clarifies how AZT can inhibit human immunodeficiency virus in macrophages. The fact that TK2 and not TK1 phosphorylates AZT in macrophages should have important implications for combination chemotherapy.

The two thymidine (dThd) kinases in human cells, the cytosolic, S-phase-specific TK1 and the mitochondrial, constitutively expressed TK2 were purified to homogeneity as judged from sodium dodecyl sulfate-gel electrophoresis. The substrate specificity of TK1 and TK2 toward natural substrates and important nucleoside analogues was compared. With TK1, the Km values for 5-fluorodeoxyuridine (FdUrd), 3'-azido-2',3'-dideoxythymidine (AZT), and 3'-fluoro-2',3'-dideoxythymidine (FLT) were 2.2, 0.6, and 2.1 microM as compared to 0.5 microM for dThd and 9 microM for deoxyuridine (dUrd). With TK2, dUrd, deoxycytidine (dCyd), and 5-fluorodeoxyuridine (FdUrd) were efficiently phosphorylated, but with distinctly different kinetics: Michaelis-Menten kinetics with dCyd, dUrd, and FdUrd; negative cooperativity with dThd. Negative cooperativity was also observed with AZT, although this drug was a very poor substrate for TK2 with a Vmax of 5-6% of that with dThd. FLT, 2',3'-dideoxycytidine (ddCyd), and arabinofuranosylcytosine (araC) were not substrates for TK2, and 2',3'-didehydrodideoxy-thymidine (D4T) was not a substrate for TK1 or TK2. On the other hand, AZT, FLT, and D4T were competitive inhibitors with Ki values of 0.6, 6, and 2073 microM for TK1, and 2, 10, and 78 microM for TK2, respectively. The much lower tolerance for modifications of the deoxyribose moiety of TK2 as compared to TK1 is important for the design of new antiviral nucleoside analogues intended for use in cells with different expression of TK1 and TK2.

Ribonucleotide reductase reduces all four ribonucleoside diphosphates to the deoxyribonucleotides required for DNA synthesis. The enzyme is composed of two nonidentical subunits, M1 and M2. The 89-kilodalton M1 subunit contains at least two allosteric sites which, by binding nucleotide effectors, regulate the catalytic activity and substrate specificity of the enzyme. We now show that in addition, protein M1 contains a substrate-binding (catalytic) site which is specifically photolabeled after UV irradiation in the presence of the natural substrate, [32P]CDP. The photolabeling of protein M1 by [32P]CDP required the presence of the second subunit, protein M2, and ATP, the positive allosteric effector for CDP reduction. The negative effectors, dATP, dGTP, and dTTP, inhibited the photolabeling of wild type protein M1. Deoxy-ATP did not inhibit the labeling of a mutant protein M1 that is resistant to feedback inhibition by dATP. In addition, hydroxyurea and 4-methyl-5-aminoisoquinoline thiosemicarbazone, two inhibitors of ribonucleotide reductase which affect protein M2, also inhibited the [32P]CDP labeling of protein M1. These data provide new insights into the role and interaction of the two ribonucleotide reductase subunits, proteins M1 and M2, and the mechanism of action of the allosteric effectors.

The B1 subunit of Escherichia coli ribonucleotide reductase is coded for by the nrdA gene, of determined structure. Protein B1 contains two types of allosteric binding sites. One type (h-sites) determines the substrate specificity while the other type (l sites) governs the overall activity. The effectors dGTP and dTTP bind only to the h-sites while dATP and ATP bind to both the h- and the l-sites. Protein B1 has been photoaffinity-labeled with radioactive dTTP and dATP using direct UV irradiation. Following tryptic digestion of labeled protein B1 only one peptide labeled with dTTP was found, while several peptides were labeled with dATP. One of the dATP-labeled peptides had chromatographic properties very similar to that labeled with dTTP and this peptide most likely forms part of the h-site of protein B1. Labeling of the l-site could not be conclusively shown since substantial non-specific labeling occurred with dATP. CNBr fragments of dTTP-labeled protein B1 were used to localize the region of nucleotide binding in the deduced primary structure of the nrdA gene. The dTTP label was further localized to a tryptic octapeptide with the sequence Ser-X-Ser-Gln-Gly-Gly-Val-Arg. The labeled amino acid was found at position 2, but the residue itself could not be directly identified. Unexpectedly, this sequence was not found in the earlier reported primary structure of the nrdA gene. However, a recent revised structure of the gene identifies the labeled residue as Cys-289 and fully confirms the rest of the peptide sequence. Thus the present result clearly defines one of the allosteric binding sites in ribonucleotide reductase.

From a mutagenized population of mouse T-lymphoma cells (S49) in continuous culture a cell line has been isolated (Ullman, B., Gudas, L. J., Clift, S. M., Martin, D. W., Jr. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1074-1978) with ribonucleotide reductase activity that is inhibited only 50% by concentrations of dGTP which abolish wild type enzyme activity. Ribonucleotide reductase activity from this dGuo-L cell line retains its normal sensitivity to dATP. The partial sensitivity/partial resistance of the ribonucleotide reductase suggests that the dGuo-L cell line is heterozygous for ribonucleotide reductase, possessing one normal allele and one allele which codes for a dGTP-resistant enzyme. Both homologous and heterologous mixing experiments between the separated nonidentical subunits of ribonucleotide reductase, protein M1 and protein M2, from wild type and dGuo-L cells showed that the dGTP- feedback sensitivity was governed by the source of the protein M1. A partial resolution of two dGuo-L protein M1 components was achieved by chromatography on dextran blue-Sepharose. In order to resolve the two dGuo-L protein M1 components more completely, we introduced into dGuo-L cells a second mutation which conferred resistance of the ribonucleotide reductase to dATP, while the original dGTP resistance was maintained. The chromatography of protein M1 from this latter clone, dGuo-L-Aphid-G5, on dATP-Sepharose resolved two kinetically distinct protein M1 components. The first component was sensitive to dGTP inhibition but stimulated by dATP; the second was absolutely refractory to dGTP but sensitive to dATP inhibition. This confirms the hypothesis that the dGuo-L parent is heterozygous for protein M1, containing one wild type and one mutant allele.

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.

Ribonucleotide reductase is responsible for the production of the deoxyribonucleotides required for DNA synthesis. The enzyme is composed of two dissociable subunits, proteins M1 and M2, which are inactive alone, but are fully active when combined. From mouse S49 T lymphoma cells we have isolated and separated the two subunits and used each for determining the activity of the complementary subunit in extracts from cells of different phases in the cell cycle. Treatment of S49 cells with cAMP analogs (e.g. Bt2cAMP) results in the protein kinase-dependent arrest of the cells in the G1 phase of the cell cycle. Ribonucleotide reductase (holoenzyme) activity fell in S49 cells treated for more than 16 h with Bt2cAMP but was unchanged during short term treatments. The activity of protein M2 was decreased in parallel to the overall activity of ribonucleotide reductase, while protein M1 activity changed less. Removal of bt2cAMP after 24 h exposure resulted in increased holoenzyme and protein M2 activities. Centrifugal elutriation of exponentially growing S49 cells separated cells into a 90% pure G1 cell population a mixture of G1 and early S phase cells and a 95% pure S phase/G2 cell population. The specific catalytic activity of protein M1 was the same in all these fractions while that of protein M2 was decreased 60% in the G1 cell population. These results demonstrate that the ribonucleotide reduction necessary for DNA synthesis is regulated in a cell cycle-dependent fashion by the activity of the protein M2 subunit of ribonucleotide reductase.

Ribonucleotide reductase is responsible for the reduction of the 2'-hydroxy moiety of all four ribonucleoside diphosphates to the corresponding deoxyribonucleotides. The overall activity of the enzyme is regulated by the allosteric effectors ATP (activator) and dATP (inhibitor), and the enzyme's substrate specificity is also controlled by nucleotide effectors. For instance, wild type ribonucleotide reductase from mouse T-lymphoma (S49) cells requires dGTP as a positive effector for ADP reduction. This effect of dGTP causes a reciprocal inhibition of CDP reduction. The dGuo-L mutant cell line, resistant to growth inhibition by exogenous deoxyguanosine, contains a nucleotide-binding subunit, protein M1, that conveys to its CDP reductase an insensitivity to dGTP (and dTTP) inhibition. The dGuo-L protein M1 also shows a decreased capacity to use ADP as a substrate, and therefore, the regulation of the substrate specificity is altered in the mutant protein M1. Another mutant cell line, dGuo-200-1, is resistant to deoxyadenosine and its ribonucleotide reductase is abnormally resistant to inhibition by dATP. The isolated mutant protein M1 from dGuo-200-1 cells has a CDP reductase activity which is stimulated by dATP, unlike the wild type enzyme which is inhibited by dATP. It appears that this mutant enzyme has lost the capacity to distinguish between dATP and ATP, but is still sensitive to regulation by dGTP and dTTP. Thus, the site of protein M1 regulating overall activity is altered in the dGuo-200-1 mutant, while the site regulating substrate specificity is normal. These characteristics of the mutants provide genetic evidence for two independent allosteric domains of protein M1, each responsible for a different aspect of nucleotide sensitivity of ribonucleotide reductase.

Consistent with its specialized role in DNA synthesis, the activity of ribonucleotide reductase is cell cycle-dependent, reaching its maximum during S-phase. This paper demonstrates, however, the levels of the two protein subunits, M1 and M2, of this enzyme vary independently of one another. The level of protein M1 was determined by use of a two-site monoclonal antibody-enzyme immunoassay and found to be constant throughout the cell cycle in bovine kidney MDBK cells. Pulse-chase experiments showed that the half-life of protein M1 was 15 h. This contrasts with our previous results demonstrating an S-phase-correlated increase in the concentration of protein M2 and a half-life of this subunit of 3 h. Therefore, ribonucleotide reductase is controlled during the cell cycle by the level of protein M2.