Differential accumulation of ribonucleotide reductase subunits in clam oocytes: the large subunit is stored as a polypeptide, the small subunit as untranslated mRNA

Within minutes of fertilization of clam oocytes, translation of a set of maternal mRNAs is activated. One of the most abundant of these stored mRNAs encodes the small subunit of ribonucleotide reductase (Standart, N. M., S. J. Bray, E. L. George, T. Hunt, and J. V. Ruderman, 1985, J. Cell Biol., 100:1968-1976). Unfertilized oocytes do not contain any ribonucleotide reductase activity; such activity begins to appear shortly after fertilization. In virtually all organisms, this enzyme is composed of two dissimilar subunits with molecular masses of approximately 44 and 88 kD, both of which are required for activity. This paper reports the identification of the large subunit of clam ribonucleotide reductase isolated by dATP-Sepharose chromatography as a relatively abundant 86-kD polypeptide which is already present in oocytes, and whose level remains constant during early development. The enzyme activity of this large subunit was established in reconstitution assays using the small subunit isolated from embryos by virtue of its binding to the anti-tubulin antibody YL 1/2. Thus the two components of clam ribonucleotide reductase are differentially stored in the oocyte: the small subunit in the form of untranslated mRNA and the large subunit as protein. When fertilization triggers the activation of translation of the maternal mRNA, the newly synthesized small subunit combines with the preformed large subunit to generate active ribonucleotide reductase.     

The small subunit of ribonucleotide reductase is encoded by one of the most abundant translationally regulated maternal RNAs in clam and sea urchin eggs

In both clam oocytes and sea urchin eggs, fertilization triggers the synthesis of a set of proteins specified by stored maternal mRNAs. One of the most abundant of these (p41) has a molecular weight of 41,000. This paper describes the identification of p41 as the small subunit of ribonucleotide reductase, the enzyme that provides the precursors necessary for DNA synthesis. This identification is based mainly on the amino acid sequence deduced from cDNA clones corresponding to p41, which shows homology with a gene in Herpes Simplex virus that is thought to encode the small subunit of viral ribonucleotide reductase. Comparison with the B2 (small) subunit of Escherichia coli ribonucleotide reductase also shows striking homology in certain conserved regions of the molecule. However, our attention was originally drawn to protein p41 because it was specifically retained by an affinity column bearing the monoclonal antibody YL 1/2, which reacts with alpha-tubulin (Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582). The finding that this antibody inhibits the activity of sea urchin embryo ribonucleotide reductase confirmed the identity of p41 as the small subunit. The unexpected binding of the small subunit of ribonucleotide reductase can be accounted for by its carboxy-terminal sequence, which matches the specificity requirements of YL 1/2 as determined by Wehland et al. (Wehland, J., H. C. Schroeder, and K. Weber, 1984, EMBO [Eur. Mol. Biol. Organ.] J., 3:1295-1300). Unlike the small subunit, there is no sign of synthesis of a corresponding large subunit of ribonucleotide reductase after fertilization. Since most enzymes of this type require two subunits for activity, we suspect that the unfertilized oocytes contain a stockpile of large subunits ready for combination with newly made small subunits. Thus, synthesis of the small subunit of ribonucleotide reductase represents a very clear example of the developmental regulation of enzyme activity by control of gene expression at the level of translation.     

Specific inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of their second subunit

Previous studies have shown that herpes virus ribonucleotide reductase can be inhibited by a synthetic nonapeptide whose sequence is identical to the C-terminal of the small subunit of the enzyme. This peptide is able to interfere with normal subunit association that takes place through the C-terminal of the small subunit. In this report, we illustrate that inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of subunit R2 is also observed for the enzyme isolated from Escherichia coli, hamster, and human cells. The nonapeptide corresponding to the bacterial C-terminal sequence was found to inhibit E. coli enzyme with an IC50 of 400 microM, while this peptide had no effect on mammalian ribonucleotide reductase. A corresponding synthetic peptide derived from the C-terminal of the small subunit of the human enzyme inhibited both human and hamster ribonucleotide reductases with IC50 values of 160 and 120 microM, respectively. However, this peptide had no inhibitory activity against the bacterial enzyme. Equivalent peptides derived from herpes virus ribonucleotide reductase had no effect on either the bacterial or mammalian enzymes. Thus, subunit association at the C-terminal of the small subunit appears to be a common feature of ribonucleotide reductases. In addition, the inhibitory phenomenon observed with peptides corresponding to the C-terminal appears not only to be universal, but also specific to the primary sequence of the enzyme.     

Induction of a new ribonucleotide reductase after infection of mouse L cells with pseudorabies virus.

The mammalian ribonucleotide reductase consists of two nonidentical subunits, protein M1 and M2. M1 binds nucleoside triphosphate allosteric effectors, whereas M2 contains a tyrosine free radical essential for activity. The activity of ribonucleotide reductase increased 10-fold in extracts of mouse L cells 6 h after infection with pseudorabies virus. The new activity was not influenced by antibodies against subunit M1 of calf thymus ribonucleotide reductase, whereas the reductase activity in uninfected cells was completely neutralized. Furthermore, packed infected cells (but not mock-infected cells) showed an electron paramagnetic resonance spectrum of the tyrosine free radical of subunit M2 of the cellular ribonucleotide reductase. These data given conclusive evidence that on infection, herpesvirus induces a new or modified ribonucleotide reductase. The virus-induced enzyme showed the same sensitivity to inhibition by hydroxyurea as the cellular reductase. The allosteric regulation of the virus enzyme was completely different from the regulation of the cellular reductase. Thus, CDP reduction catalyzed by the virus enzyme showed no requirement for ATP as a positive effector, and no feedback inhibition was observed by dTTP or dATP. The virus reductase did not even bind to a dATP-Sepharose column which bound the cellular enzyme with high affinity.     

Molecular cloning of the cDNA for a mutant mouse ribonucleotide reductase M1 that produces a dominant mutator phenotype in mammalian cells.

Mammalian ribonucleotide reductase is regulated by the binding of dATP and other nucleotide effectors to allosteric sites on subunit M1. Using mRNA from a mutant mouse T-lymphoma (S49) cell line, we have isolated a cDNA which encodes an altered, dATP feedback-resistant subunit M1. The mutant cDNA contains a single point mutation (a G-to-A transition) at codon 57, converting aspartic acid to asparagine. Proof that this mutation is responsible for the phenotype of dATP feedback resistance is provided by the following evidence. (i) The mutation was detected only in mutant S49 cells containing dATP feedback-resistant ribonucleotide reductase and not in wild-type or other mutant S49 cells. (ii) Transfection of Chinese hamster ovary cells with an expression plasmid containing the mutant M1 cDNA resulted in the production of dATP feedback-resistant ribonucleotide reductase. Transfected CHO cells expressing the mutant M1 cDNA exhibited a 15- to 25-fold increase in the frequency of spontaneous mutation to 6-thioguanine resistance, confirming that dATP feedback-resistant ribonucleotide reductase produces a mutator phenotype in mammalian cells. The availability of a cDNA which encodes dATP feedback-resistant subunit M1 thus provides a means of manipulating by transfection the frequency of spontaneous mutation in mammalian cells.     

Sequence analysis of the large and small subunits of human ribonucleotide reductase.

We have isolated and sequenced overlapping cDNA clones from a breast carcinoma cDNA library containing the entire coding region of both the R1 and R2 subunits of the human ribonucleotide reductase gene. The coding region of the human R1 subunit comprises 2376 nucleotides and predicts a polypeptide of 792 amino acids (calculated molecular mass 90,081). The sequence of this subunit is almost identical to the equivalent mouse ribonucleotide reductase subunit with 97.7% homology between the mouse and human R1 subunit amino acid sequences. The coding region of the human R2 subunit of ribonucleotide reductase comprises 1170 nucleotides and predicts a polypeptide of 389 amino acids (calculated molecular mass 44,883), which is one amino acid shorter than the equivalent mouse subunit. The human and mouse R2 subunits display considerable homology in their carboxy-terminal amino acid sequences, with 96.3% homology downstream of amino acid 68 of the human and mouse R2 proteins. However, the amino-terminal portions of these two proteins are more divergent in sequence, with only 69.2% homology in the first 68 amino acids.     

Herpes simplex virus ribonucleotide reductase: expression in Escherichia coli and purification to homogeneity of a tyrosyl free radical-containing, enzymatically active form of the 38-kilodalton subunit.

Infection of mammalian cells with herpes simplex virus (HSV) induces a virus-encoded ribonucleotide reductase which is different from the cellular enzyme. This essential viral enzyme consists of two nonidentical subunits of 140 and 38 kilodaltons (kDa) which have not previously been purified to homogeneity. The small subunit of ribonucleotide reductases from other species contains a tyrosyl free radical essential for activity. We have cloned the gene for the small subunit of HSV-1 ribonucleotide reductase into a tac expression plasmid vector. After transfection of Escherichia coli, expression of the 38-kDa protein was detected in immunoblots with a specific monoclonal antibody. About 30 micrograms of protein was produced per liter of bacterial culture. The 38-kDa protein was purified to homogeneity in an almost quantitative yield by immunoaffinity chromatography. It contained a tyrosyl free radical which gave a specific electron paramagnetic resonance spectrum identical to that we have observed in HSV-infected mammalian cells and clearly different from that produced by the E. coli and mammalian ribonucleotide reductases. The recombinant 38-kDa subunit had full activity when assayed in the presence of HSV-infected cell extracts deficient in the native 38-kDa subunit.     

Platelet interaction with active and TLCK-inactivated alpha-thrombin.

Purification of ribonucleotide reductase from regenerating rat liver using dATP Sepharose chromatography isolated a subunit of the enzyme which was specific for the reduction of CDP. Activities for the other ribonucleotide diphosphates showed differing distribution in the various fractions suggesting different forms of the enzyme for each ribonucleotide diphosphate.     

Deletion of the varicella-zoster virus large subunit of ribonucleotide reductase impairs growth of virus in vitro.

Cells infected with varicella-zoster virus (VZV) express a viral ribonucleotide reductase which is distinct from that present in uninfected cells. VZV open reading frames 18 and 19 (ORF18 and ORF19) are homologous to the herpes simplex virus type 1 genes encoding the small and large subunits of ribonucleotide reductase, respectively. We generated recombinant VZV by transfecting cultured cells with four overlapping cosmid DNAs. To construct a virus lacking ribonucleotide reductase, we deleted 97% of VZV ORF19 from one of the cosmids. Transfection of this cosmid with the other parental cosmids yielded a VZV mutant with a 2.3-kbp deletion confirmed by Southern blot analysis. Virus-specific ribonucleotide reductase activity was not detected in cells infected with VZV lacking ORF19. Infection of melanoma cells with ORF19-deleted VZV resulted in plaques smaller than those produced by infection with the parental VZV. The mutant virus also exhibited a growth rate slightly slower than that of the parental virus. Chemical inhibition of the VZV ribonucleotide reductase has been shown to potentiate the anti-VZV activity of acyclovir. Similarly, the concentration of acyclovir required to inhibit plaque formation by 50% was threefold lower for the VZV ribonucleotide reductase deletion mutants than for parental virus. We conclude that the VZV ribonucleotide reductase large subunit is not essential for virus infection in vitro; however, deletion of the gene impairs the growth of VZV in cell culture and renders the virus more susceptible to inhibition by acyclovir.     

Different cellular distribution of thioredoxin and subunit M1 of ribonucleotide reductase in rat tissues

The cellular distribution of thioredoxin and protein M1 of ribonucleotide reductase in adult rat tissues was investigated with immunohistochemical techniques using specific antisera. Tissues with high or low frequency of either mitotic or meiotic cell divisions were compared. Thioredoxin was demonstrated in many cells types that showed no detectable protein M1 of ribonucleotide reductase. A few cell types with protein M1 immunoreactivity also contained immunoreactive thioredoxin. However, in most cells no such co-localization could be demonstrated. This lack of correlation between cells containing subunit M1 of ribonucleotide reductase and the thioredoxin indicates that thioredoxin is not the physiologist hydrogen donor for ribonucleotide reductase in rat tissues and that the expression of two enzymes is differently regulated.     

Identification of the stable free radical tyrosine residue in ribonucleotide reductase.

The small subunit of iron-dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue. Tyrosine-122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine. The mutation was introduced with oligonucleotide-directed mutagenesis in an M13 recombinant and verified by DNA sequencing. Purified native and mutant B2 protein were found to have the same size, iron content and iron-related absorption spectrum. The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity. These results identify Tyr122 of E. coli protein B2 as the tyrosyl radical residue. An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits. It contains the entire nrd operon with its own promoter in a 2.3-kb fragment from pBR322. Both the B1 and the B2 subunits were expressed at a 25-35 times higher level as compared to the host strain.     

Relationships between sensitivity to hydroxyurea and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIO) and ribonucleotide reductase RNR2 mRNA levels in strains of Saccharomyces cerevisiae

Ribonucleotide reductase is responsible for providing the deoxyribonucleotide precursors for DNA synthesis. In most species the enzyme consists of a large and a small subunit, both of which are required for activity. In mammalian cells, the small subunit is the site of action of several antitumor agents, including hydroxyurea and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ). The mRNA levels for the small subunit of ribonucleotide reductase (RNR2) and sensitivity to hydroxyurea and MAIQ were determined in four strains of the yeast, Saccharomyces cerevisiae. Two strains exhibited significantly different sensitivities to both hydroxyurea and MAIQ, which closely correlated with differences in the levels of RNR2 mRNA. These results are consistent with recent observations with mammalian cells in culture, and indicate that a common mechanism of resistance to hydroxyurea and related drugs occurs through the elevation in ribonucleotide reductase message levels. A transplason mutagenized strain with marked structural modifications in RNR2 DNA and mRNA showed an extreme hypersensitivity to hydroxyurea but not to MAIQ, providing evidence that the two drugs do not inhibit the RNR2 subunit by the same mechanism. In addition, a yeast strain isolated for low but reproducible resistance to MAIQ exhibited a sensitivity to hydroxyurea similar to the parental wild-type strain, supporting the idea that the two drugs inhibit the activity of RNR2 by unique mechanisms. These yeast strains provide a useful approach for further studies into the regulation of eucaryotic ribonucleotide reduction and drug resistance mechanism involving a key rate-limiting step in DNA synthesis.     

Improvement of a simple method to purify ribonucleotide reductase

The use of an ATP-agarose column to purify ribonucleotide reductase from human D-98 cells was recently reported. The column selectively retains greater than 99.9% of the contaminating nucleoside diphosphate (NDP) kinase from crude preparations of ribonucleotide reductase. It was presently found, however, that extending the length of the column caused the ribonucleotide reductase to dissociate into subunits. One subunit appeared in the low ionic strength buffer wash while the other required 0.5 M KCl for elution. The enzyme could also be recovered intact (non-dissociated) by equilibrating the enzyme preparation and the column with 0.5 M KCl prior to chromatography. Either method greatly improved the overall yield and the specific activity of the ribonucleotide reductase because it prevented the binding and subsequent loss of any of the subunits. In addition, the use of a larger column permitted the gel-filtration properties of the ATP-agarose to separate the bulk of the residual (not bound) NDP kinase from the ribonucleotide reductase.     

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.     

Ribonucleotide reductase activity and deoxyribonucleoside triphosphate metabolism during the cell cycle of S49 wild-type and mutant mouse T-lymphoma cells

We investigated deoxyribonucleoside triphosphate metabolism in S49 mouse T-lymphoma cells synchronized in different phases of the cell cycle. S49 wild-type cultures enriched for G1 phase cells by exposure to dibutyryl cyclic AMP (Bt2cAMP) for 24 h had lower dCTP and dTTP pools but equivalent or increased pools of dATP and dGTP when compared with exponentially growing wild-type cells. Release from Bt2cAMP arrest resulted in a maximum enrichment of S phase occurring 24 h after removal of the Bt2cAMP, and was accompanied by an increase in dCTP and dTTP levels that persisted in colcemid-treated (G2/M phase enriched) cultures. Ribonucleotide reductase activity in permeabilized cells was low in G1 arrested cells, increased in S phase enriched cultures and further increased in G2/M enriched cultures. In cell lines heterozygous for mutations in the allosteric binding sites on the M1 subunit of ribonucleotide reductase, the deoxyribonucleotide pools in S phase enriched cultures were larger than in wild-type S49 cells, suggesting that feedback inhibition of ribonucleotide reductase is an important mechanism limiting the size of deoxyribonucleoside triphosphate pools. The M1 and M2 subunits of ribonucleotide reductase from wild-type S49 cells were identified on two-dimensional polyacrylamide gels, but showed no significant change in intensity during the cell cycle. These data are consistent with allosteric inhibition of ribonucleotide reductase during the G1 phase of the cycle and release of this inhibition during S phase. They suggest that the increase in ribonucleotide reductase activity observed in permeabilized S phase-enriched cultures may not be the result of increased synthesis of either the M1 or M2 subunit of the enzyme.     

Half-site reactivity of the tyrosyl radical of ribonucleotide reductase from Escherichia coli

A C-terminally truncated form of protein B2, the homodimeric small subunit of ribonucleotide reductase from Escherichia coli, was found as the result of an apparently specific proteolysis. Truncated homodimers contain an intact binuclear iron center and a normal tyrosyl radical but have no binding capacity for the other ribonucleotide reductase subunit, protein B1, and are consequently enzymatically inactive. Heterodimers, consisting of one full-length and one truncated polypeptide, formed spontaneously during a chelation-reconstitution cycle and were easily separated from the two homodimeric variants. The heterodimeric form of B2 shows a weak interaction with the B1 subunit resulting in low enzyme activity. Using heterodimers containing deuterated tyrosine on the full-length side and protonated tyrosine on the truncated side, we could demonstrate that the tyrosyl radical was randomly generated in one or the other of the two polypeptide chains of the heterodimeric B2 subunit. The small subunit of ribonucleotide reductase thus conforms to a half-site reactivity.     

Subunit M2 of mammalian ribonucleotide reductase. Characterization of a homogeneous protein isolated from M2-overproducing mouse cells

The M2 subunit of mammalian ribonucleotide reductase was purified to homogeneity from hydroxyurea-resistant, M2-overproducing mouse cells. The purification procedure involved affinity chromatography on an anti-tubulin antibody-Sepharose column and high performance gel permeation chromatography. The pure protein is a dimer of Mr = 88,000, containing stoichiometric amounts of a non-heme iron center and a tyrosyl free radical. The radical is destroyed by hydroxyurea but can readily be regenerated on incubation of the radical-free protein alone with iron-dithiothreitol in the presence of air. The ability to spontaneously regenerate the tyrosyl radical distinguishes protein M2 from the corresponding subunit of Escherichia coli ribonucleotide reductase, protein B2, but apart from that the two proteins are very similar.     

Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.     

Enhancement of ribonucleotide reductase activity at low enzyme concentrations by polyethylene glycol

The estimation of ribonucleotide reductase in cell extracts has been problematical on account of abnormally low activities at low enzyme concentrations, presumably due to subunit dissociation. This problem can be alleviated by assaying the enzyme in the presence of polyethylene glycol. The presence of 15% polyethylene glycol during the assay greatly stimulated ribonucleotide reductase activity at low enzyme concentrations and allowed measurement of enzyme activity in as little as 10(5) mouse L929 cells, a 30-fold enhancement of assay sensitivity. Enzyme activity measured in the presence of 15% polyethylene glycol was proportional to enzyme concentration, thus making possible the accurate measurement of very low levels of ribonucleotide reductase.