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PubMed1995

Malignant transformation by H-ras results in aberrant regulation of ribonucleotide reductase gene expression by transforming growth factor-beta 1.

Ribonucleotide reductase is a key rate-limiting and regulatory step in DNA synthesis and plays a crucial role in the coordination of DNA synthesis, DNA repair, and cell proliferation. The present study demonstrates a link between alterations in TGF-beta 1 regulation during malignant conversion and the expression of ribonucleotide reductase. H-ras-transformed mouse 10T1/2 cell lines exhibiting malignant potential were examined for possible TGF-beta 1-mediated alterations in ribonucleotide reductase expression. Selective induction of ribonucleotide reductase gene expression occurred, since only H-ras-transformed highly metastatic cells exhibited marked elevations in ribonucleotide reductase expression, whereas nontransformed normal 10T1/2 cells were unaffected by TGF-beta 1 treatment. These changes occurred without any detectable modifications in DNA synthesis rates, suggesting that these changes were regulated by a novel mechanism independent of the S-phase of the cell cycle. Furthermore, this TGF-beta 1-mediated regulation of ribonucleotide reductase expression was shown to occur through an autocrine mechanism. TGF-beta 1-modulated regulation of ribonucleotide reductase expression requires de novo protein synthesis and involves, at least in part, transcriptional and post-transcriptional events. Furthermore, evidence is presented to suggest a possible role for protein kinase C-mediated events, protein phosphatases, and G-protein-coupled events in the TGF-beta 1-mediated regulation of ribonucleotide reductase expression in H-ras-transformed malignant cells. TGF-beta 1 regulation of ribonucleotide reductase in highly malignant cells appears to be complex and multifaceted and constitutes an integral part of an altered growth regulatory program.

Animals↗
PubMed1994

Alterations in the cyclic AMP signal transduction pathway regulating ribonucleotide reductase gene expression in malignant H-ras transformed cell lines.

Ribonucleotide reductase is a highly regulated activity responsible for reducing ribonucleotides to deoxyribonucleotides, which are required for DNA synthesis and DNA repair. We have tested the hypothesis that malignant cell populations contain alterations in signal pathways important in controlling the expression of the two genes that code for ribonucleotide reductase, R1 and R2. A series of radiation and H-ras transformed mouse 10T1/2 cell lines with increasing malignant potential were exposed to stimulators of cAMP synthesis (forskolin and cholera toxin), an inhibitor of cAMP degradation (3-isobutyl-1-methylxanthine) and a biologically stable analogue of cAMP (8-bromo-cAMP). Dramatic elevations in the expression of the R1 and R2 genes at the message and protein levels were observed in malignant metastatic populations, which were not detected in the normal parental cell line or in cells capable of benign tumor formation. These changes in ribonucleotide reductase gene expression occurred without any detectable modifications in the rates of DNA synthesis, showing that they were regulated by a novel mechanism independent of the S phase of the cell cycle. Furthermore, studies with forskolin (a stimulator of the protein kinase A signal pathway) and the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (a stimulator of the protein kinase C signal pathway), alone or in combination, indicated that their effects on R1 and R2 gene expression in a highly malignant cell line were greater than when they were tested individually, suggesting that the two pathways modulating R1 and R2 gene expression can cooperate to regulate ribonucleotide reduction, and interestingly this can occur in a synergistic fashion. Also, a direct relationship between H-ras expression and ribonucleotide reductase gene expression was observed; analysis of forskolin mediated elevations in R1 and R2 message levels closely correlated with the levels of H-ras expression in the various cell lines. In total, these studies demonstrate that ribonucleotide reductase expression is controlled by a complex process, and malignant ras transformed cells contain alterations in the regulation of signal transduction pathways that lead to novel modifications in ribonucleotide reductase gene expression. This signal mechanism, which is aberrantly regulated in malignant cells, may be related to regulatory pathways involved in determining ribonucleotide reductase expression in a S phase independent manner during periods of DNA repair.

1-Methyl-3-isobutylxanthine↗
PubMed1972

Purine ribonucleotide biosynthesis, interconversion and catabolism in mouse brain in vitro.

The relative rates of the synthetic, interconversion and catabolic reactions of purine metabolism in chopped mouse cerebrum were studied. The rates of incorporation of [(14)C]adenine and [(14)C]hypoxanthine into purine ribonucleotides were much less than the potential activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, and the rates of incorporation were stimulated by the addition of guanosine to the incubation mixture. The availability of ribose phosphates may be a limiting factor for the formation of ribonucleotides from purine bases. The rate of incorporation of [(14)C]adenosine into purine ribonucleotides was at least seven- to eight-fold higher than that of adenine. The radioactivity in adenine ribonucleotides synthesized from adenine and hypoxanthine was about 100- and ten-fold respectively higher than that in the radioactive guanine ribonucleotides. The conversion of inosinate into guanine ribonucleotides was probably limited by the amount of inosinate available, and the conversion of adenine ribonucleotides into guanine ribonucleotides was probably limited by the activity of adenylate deaminase. The rate of catabolism of [(14)C]adenosine was low in comparison with its rate of utilization for ribonucleotide synthesis. A fraction of the [(14)C]hypoxanthine was catabolized to xanthine and urate. [(14)C]Guanine was completely converted into xanthine, mostly by the guanine deaminase that was released during incubation of chopped mouse cerebrum.

Adenine↗
PubMed1992

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.

3T3 Cells↗
PubMed1980

Adenosine deaminase impairment and ribonucleotide reductase activity and levels in HeLa cells.

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4)-deficient patients recently were found to have abnormally high levels of dATP, a negative allosteric effector of ribonucleotide reductase (ribonucleoside-diphosphate reductase, 2'-deoxyribonucleoside-diphosphate:oxidized thioredoxin 2'-oxidoreductase, EC 1.17.4.1). Therefore it was proposed that the immunodeficiency associated with adenosine deaminase deficiency is mediated through inhibition of ribonucleotide reductase and hence DNA replication. HeLa cells, treated with an adenosine deaminase inhibitor, erythro-9(2-hydroxy-3-nonyl)adenine, and deoxyadenosine to mimic the adenosine deaminase-deficient state, were monitored to determine directly the effects on ribonucleotide reductase activity and levels. A low concentration of erythro-9-(2-hydroxy-3-nonyl)adenine, which did not inhibit cell growth, nevertheless retarded the cells in G2 + M phase of the cell cycle and increased reductase activity. Reductase activity was also elevated in cells treated with a low level of deoxyadenosine which did not affect the cell cycle or cell growth. However, ribonucleotide reductase activity was reduced to one-half of the control value in cells treated with either enough deoxyadenosine to inhibit cell growth or with a combination of erythro-9(2-hydroxy-3-nonyl)adenine and deoxyadenosine, each at concentrations which individually do not inhibit cell growth. Removal of deoxynucleotides, particularly dATP, from these extracts increased ribonucleotide reductase activity to several-fold higher than control values. The reduced activity of ribonucleotide reductase in the simulated adenosine deaminase-deficient HeLa cells provides direct evidence for the thesis that adenosine deaminase deficiency disease is mediated through elevated levels of dATP which inhibit ribonucleotide reductase. In addition, the cell cycle patterns and ribonucleotide reductase levels suggest that the regulatory substance(s) that controls the level of ribonucleotide reductase is not operative until the late S or G2 phase of the cell cycle.

Adenine↗
PubMed1993

Different relationships between cellular adenosine or 3'-deoxyadenosine phosphorylation and cellular adenine ribonucleotide catabolism may be obtained.

Treatment of BALB/c-3T3 mouse fibroblasts with 3'-deoxyadenosine led to a rapid accumulation of 3'-deoxyadenosine phosphates and the kinetics of this process has been determined. Concomitant with accumulation of these compounds, the adenine ribonucleotide pool was reduced. The kinetics of the two processes suggested that they were tightly coupled. The inhibitory effect of relatively high concentrations of coformycin indicated that IMP was an intermediate in the catabolic pathway. Similar experiments with Ehrlich ascites tumor cells were performed in Ringer-Hepes solution at pH 6.5 or 7.5 and with varying concentrations of orthophosphate. The experiments were performed with cells where ATP was [3H]-labeled. This allowed the determination of the catabolism of adenine ribonucleotides to labeled nucleosides under conditions where added adenosine was phosphorylated. The results showed that at low phosphate concentration (5.8 mM) at pH 6.5 adenosine may be phosphorylated at a rate that was completely balanced to the concomitant catabolism of adenine ribonucleotides; that is, there was apparently a tight kinetic coupling between anabolism of adenosine and catabolism of adenine ribonucleotides. With 3'-deoxyadenosine a corresponding effect was obtained although the apparent coupling between phosphorylation of 3'-deoxyadenosine and catabolism of adenine ribonucleotides was not complete. When experiments were performed at the same pH but at high concentration of phosphate (45 mM) there was in contrast no coupling between the two processes; that is, ATP was present in constant amounts while 3'-deoxyadenosine phosphates accumulated at a high rate. In experiments with adenosine under these conditions there was still some although a relatively limited degree of apparent coupling between phosphorylation of adenosine and catabolism of adenine ribonucleotides. In both lines of cells used and with both adenosine and 3'-deoxyadenosine, the main products of the catabolism of adenine ribonucleotides were inosine and hypoxanthine. With 3'-deoxyadenosine there was in addition (about 20%) formation of xanthosine, suggesting that IMP dehydrogenase had also been activated. These results lead to the suggestion that adenosine (or 3'-deoxyadenosine) may be phosphorylated in two ways. 1) Phosphorylation may depend on an adenosine kinase unrelated to catabolism of adenine ribonucleotides. 2) Phosphorylation may be tightly coupled to catabolism of adenine ribonucleotides. A nucleoside phosphotransferase may catalyze the transfer of a phosphoryl group from IMP to adenosine (or 3'-deoxyadenosine) to form AMP (or 3'-dAMP) and inosine, a process that may be tightly coupled to an AMP deaminase reaction. The IMP formed in the latter reaction may not be released but transferred to the phosphotransferase.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗
PubMed1999

Substrate specificity of human glycinamide ribonucleotide transformylase.

The nucleotide substrate specificity of human glycinamide ribonucleotide transformylase, a chemotherapeutic target, has been examined. The enzyme accepts the sarcosyl analog of glycinamide ribonucleotide, carbocyclic glycinamide ribonucleotide, and two phosphonate derivatives of carbocyclic glycinamide ribonucleotide with V/K values, relative to that obtained for beta-glycinamide ribonucleotide, of 1, 27, 1.4, and 2.9%, respectively. Several other analogs of carbocyclic glycinamide ribonucleotide, namely a truncated phosphonate and 2',3'-dideoxy- and 2',3'-dideoxy-2',3'-didehydro-carbocyclic glycinamide ribonucleotide, were inhibitors of the enzyme, competitive against glycinamide ribonucleotide, with Ki values approximately 100 times higher than the Km for -glycinamide ribonucleotide. Although the results of the present study parallel those obtained previously with the avian enzyme (V. D. Antle, D. Liu, B. R. McKellar, C. A. Caperelli, M. Hua, and R. Vince (1996) J. Biol. Chem. 271, 6045-6049), quantitative differences between the two enzyme species have been uncovered.

Animals↗
PubMed1996

Substrate specificity of glycinamide ribonucleotide transformylase from chicken liver.

Several glycinamide ribonucleotide analogs have been prepared and evaluated as substrates and/or inhibitors of glycinamide ribonucleotide transformylase from chicken liver. The side chain modified analogs, in which the glycine side chain, R = CH2NH2, has been replaced by R = CH2NHCH3 and R = CH2CH2NH2, are substrates, with V/K (relative intensity) of 2.4% and 16.3%, respectively. Several carbocyclic analogs of glycinamide ribonucleotide, including the phosphonate derivative of carbocyclic glycinamide ribonucleotide, did not serve as substrates, but were inhibitors of the enzyme, competitive against glycinamide ribonucleotide, with Ki values ranging from 7.4 to 23.6 times the Km for glycinamide ribonucleotide. However, the O-phosphonate analog of carbocyclic glycinamide ribonucleotide did support enzymatic activity, with V/K (relative intensity) of 0.8%. In addition, glycinamide ribonucleoside was neither a substrate for, nor an inhibitor of, glycinamide ribonucleotide transformylase. Furthermore, alpha-glycinamide ribonucleotide had no effect on enzyme activity. These studies have begun to define the structural features of the nucleotide substrate required to support enzymatic activity.

Acyltransferases↗
PubMed1990

Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase, and the significance to DNA synthesis.

Mammalian ribonucleotide reductase, which occupies a key position in the synthesis of DNA, is a highly controlled enzyme activity, because it is solely responsible for the de novo reduction of ribonucleoside diphosphates to their corresponding deoxyribonucleoside diphosphate forms, required for DNA synthesis. Ribonucleotide reductase consists of two dissimilar protein components often called M1 and M2, which are independently regulated during cell proliferation. The M1 component contains multiple effector binding sites and is responsible for the complex allosteric regulation of the enzyme, whereas the M2 protein contains nonheme iron and a unique tyrosyl-free radical required for ribonucleotide reduction. Since the reaction is rate limiting for DNA synthesis, ribonucleotide reductase plays an important role in regulating cell division, and hence, cell proliferation. There are many inhibitors of ribonucleotide reductase and perhaps the most valuable one from a cell biology, biochemistry, and clinical point of view is the hydroxamic acid, hydroxyurea. This drug has also been very useful as a selective agent for isolating a variety of mammalian mutant cell lines altered in ribonucleotide reductase gene expression. Regulatory, structural, and biological characteristics of ribonucleotide reductase are reviewed, including evidence that ribonucleotide reductase, particularly the M2 protein, has an important early role to play in tumor promotion. In addition, modifications in the expressions of genes altered in hydroxyurea-resistant mutants and cultured in the absence or presence of hydroxyurea are discussed, with emphasis on changes in M2 protein, M1 protein, and the iron-storage protein ferritin.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1978

Ribonucleotide reductase from wild type and hydroxyurea-resistant chinese hamster ovary cells.

The kinetic properties of partially purified ribonucleotide reductase from Chinese hamster ovary cells have been investigated. Double reciprocal plots of velocity against substrate concentration were found to be linear for three the substrates tested, and yielded apparent Km values of 0.12 mM for CDP, 0.14 mM for ADP and 0.026 mM for GDP. Hydroxyurea, a potent inhibitor of ribonucleotide reduction, was tested against varying concentrations of ribonucleotide substrates and inhibited the enzyme activity in an uncompetitive fashion. Intercept replots were linear and exhibited Ki values for hydroxyurea of 0.08 mM for CDP reduction, 0.13 mM for ADP reduction and 0.07 mM for GDP reduction. Guanazole, another inhibitor of ribonucleotide reductase, interacted with the enzyme in a similar manner to hydroxyurea showing an uncompetitive pattern of inhibition with CDP reduction and yielding a Ki value of 0.57 mM. Partially purified ribonucleotide reductase from hydroxyurea-resistant cells was compared to enzyme activity from wild type cells. Significant differences were observed in the hydroxyurea Ki values with the three ribonucleotide substrates that were tested. Also, CDP reductase activity from the drug-resistant cells yielded a significantly higher Ki value for guanazole inhibition than the wild type activity. The properties of partially purified ribonucleotide reductase from a somatic cell hybrid constructed from wild type and hydroxyurea-resistant cells was also examined. The Ki value for hydroxyurea inhibition of CDP reductase was intermediate between the Ki values of the parental lines and indicated a codominant expression of hydroxyurea-resistance at the enzyme level. The most logical explanation for these results is that the mutant cells contain a structurally altered ribonucleotide reductase whose activity is less sensitive to inhibition by hydroxyurea or guanazole.

Animals↗
PubMed1992

Transforming growth factor-beta 1 mediated alterations in ribonucleotide reductase gene expression in BALB/c 3T3 fibroblasts.

Transforming growth factor-beta 1 (TGF-beta 1) stimulated DNA synthesis (3-fold) in BALBc/3T3 fibroblasts following 24 hours of growth factor exposure. Since ribonucleotide reductase is important for the coordination of DNA synthesis and cell proliferation, we investigated the hypothesis that cells like BALB/c 3T3, which are TGF-beta 1 responsive, would exhibit modifications in expression of the gene for ribonucleotide reductase following growth factor treatment. We observed 2.6, 4.1, and 4.8-fold increases in ribonucleotide reductase activity following TGF-beta 1 exposure for 6, 12, and 24 hours, respectively. Increased ribonucleotide reductase R2 gene expression (3, 3.7, and 4.5-fold) and R1 gene expression (2,2.5, and 2.6-fold) were observed following 6, 12, and 24 hours of TGF-beta 1 treatment, respectively. Western blots indicated 2.2, 3.1, and 4.1-fold increases in protein R2 levels at 6, 12, and 24 hours exposure to TGF-beta 1, whereas 2.6 and 3.3-fold elevations in R1 protein levels were observed at 12 and 24 hours post-TGF-beta 1 exposure. These TGF-beta 1 mediated modifications in ribonucleotide reductase gene expression occurred, in part, prior to any detectable changes in the rate of DNA synthesis, demonstrating alterations in the normal regulation of ribonucleotide reductase. Furthermore, these alterations could be markedly reduced by prolonged pretreatment with 12-O-tetradecanoylphorbol-13-acetate (R2 gene expression increased by only 1.3, 1.5 and 2.3-fold after 6, 12, and 24 hours of TGF-beta 1 treatment, respectively), suggesting a role for a protein kinase C pathway in the TGF-beta 1 regulated changes in ribonucleotide reductase gene expression. These results indicate for the first time that TGF-beta 1 can regulate the expression of the two genes for ribonucleotide reductase in BALB/c 3T3 fibroblasts, and suggest that regulation of these genes plays an important role in critical events involved in growth factor modulation of normal and transformed cell proliferation.

3T3 Cells↗
PubMedDate not supplied

Ribonucleotide reductase: an important enzyme in the replication of herpes simplex virus type 1 and a target for antiviral chemotherapy.

Herpes simplex virus encodes a ribonucleotide reductase that catalyzes the formation of deoxyribonucleotides from ribonucleotides. The enzyme is not essential for either viral DNA synthesis or replication, yet inhibitors of this enzyme suppress viral replication. To clarify the role of the ribonucleotide reductase in virus infection and to evaluate it as an antiviral target, the metabolism of deoxyribonucleotides in infected cells was examined. Our results show that the cellular ribonucleotide reductase is incapable of generating adequate deoxyribonucleoside triphosphate pools to support efficient virus replication. Additionally, we have shown that the virus is unable to efficiently utilize salvaged deoxyribonucleosides from degraded cellular DNA. A selective inhibitor of the viral ribonucleotide reductase, 2-acetylpyridine thiosemicarbazone, decreased deoxyribonucleotide pools in infected cells, thus inhibiting viral DNA synthesis. This compound also inhibited the cellular ribonucleotide reductase to some extent, thereby enhancing its antiviral activity. The antiviral effects of acyclovir were potentiated by 2-acetylpyridine thiosemicarbazone in the wild-type virus but not in the ribonucleotide reductase mutant, ICP6 delta. Collectively, these data strongly suggest that the viral ribonucleotide reductase is an important enzyme in viral replication and a valid target for antiviral chemotherapy.

Acyclovir↗
PubMed2000

Cellular adaptation to down-regulated iron transport into lymphoid leukaemic cells: effects on the expression of the gene for ribonucleotide reductase.

Ribonucleotide reductase is an iron-containing enzyme that is essential for DNA synthesis. Whereas previous studies have used various iron chelators to examine the relationship between cellular iron metabolism and ribonucleotide reductase activity in cells, they have not elucidated the relationship between iron transport into cells and the expression of the gene for ribonucleotide reductase. To investigate this, we examined ribonucleotide reductase mRNA, protein and enzyme activity in a novel line of CCRF-CEM cells (DFe-T cells) that display an approx. 60% decrease in their uptake of iron compared with the parental wild-type cell line. We found that DFe-T cells displayed an approx. 40% decrease in ribonucleotide reductase specific enzyme activity relative to wild-type cells without a change in their proliferation. Kinetic analysis of CDP reductase activity revealed an approx. 60% decrease in V(max) in DFe-T cells without a change in K(m). Despite the decrease in enzyme activity, the mRNA and protein for the R1 and R2 subunits of ribonucleotide reductase in DFe-T cells were similar to those of wild-type cells. ESR spectroscopy studies revealed that DFe-T cells had a 22% decrease in the tyrosyl free radical of the R2 subunit, suggesting that a larger amount of R2 protein was present as functionally inactive apo-R2 in these cells. Our studies indicate that ribonucleotide reductase activity in CCRF-CEM cells can be down-regulated by more than 50% in response to down-regulated iron transport without an adverse effect on cell proliferation. Furthermore, our studies suggest a regulatory link between ribonucleotide reductase activity and iron transport into these cells.

Adaptation, Physiological↗
PubMed1987

Reversion of hydroxyurea resistance, decline in ribonucleotide reductase activity, and loss of M2 gene amplification.

A key rate-limiting reaction in the synthesis of DNA is catalyzed by ribonucleotide reductase, the enzyme which reduces ribonucleotides to provide the deoxyribonucleotide precursors of DNA. The antitumor agent, hydroxyurea, is a specific inhibitor of this enzyme and has been used in the selection of drug resistant mammalian cell lines altered in ribonucleotide reductase activity. An unstable hydroxyurea resistant population of mammalian cells with elevated ribonucleotide reductase activity has been used to isolate three stable subclones with varying sensitivities to hydroxyurea cytotoxicity and levels of ribonucleotide reductase activities. These subclones have been analyzed at the molecular level with cDNA probes encoding the two nonidentical subunits of ribonucleotide reductase (M1 and M2). Although no significant differences in M1 mRNA levels or gene copy numbers were detected between the three cell lines, a strong correlation between cellular resistance, enzyme activity, M2 mRNA and M2 gene copies was observed. This is the first demonstration that reversion of hydroxyurea resistance is directly linked to a decrease in M2 mRNA levels and M2 gene copy number, and strongly supports the concept that M2 gene amplification is an important mechanism for achieving resistance to this antitumor agent through elevations in ribonucleotide reductase.

Animals↗
PubMed1992

Induction of ribonucleotide reductase activity in cells infected with African swine fever virus.

Infection of Vero cells with African swine fever virus (ASFV) resulted in a marked increase in ribonucleotide reductase activity. The induction of ribonucleotide reductase was detected early after infection and was proportional to the multiplicity of infection. Inhibition of viral DNA replication did not affect the induction of the enzyme. Several characteristics could distinguish the virus-induced from the normal cell enzyme. ASFV-induced ribonucleotide reductase was inhibited by magnesium, was more strongly inhibited by hydroxyurea, and had a fourfold lower Km. The virus-induced enzyme was inhibited by deoxyribonucleoside triphosphates and by ATP. The isolation of hydroxyurea-resistant ASFV mutants provided genetic evidence for the viral origin of the induced ribonucleotide reductase. The resistance to hydroxyurea was due to a threefold overproduction of ribonucleotide reductase, as compared to enzyme induction by wild-type ASFV. Hydroxyurea had similar effect in vitro on ribonucleotide reductases induced by wild-type or mutant virus. The gene for the small subunit of the viral enzyme was mapped within a 2.3-kb fragment by hybridization with an oligonucleotide probe designed from a conserved aminoacid sequence of eukaryotic and viral ribonucleotide reductases.

African Swine Fever Virus↗
PubMed2006

Interaction of cationic vesicle with ribonucleotides (AMP, ADP, and ATP) and physicochemical characterization of DODAB/ribonucleotides complexes.

The interaction between ribonucleotides (AMP, ADP, and ATP) and cationic vesicles prepared from dioctadecyldimethylammonium bromide (DODAB) were investigated in detail. The physicochemical properties of ribonucleotides/cationic lipid complexes were present. Gel exclusion-UV spectroscopic results showed that all the charge ratios of DODAB/ribonucleotides (AMP, ADP, and ATP) are 2:1 when the maximal ribonucleotides were adsorbed onto DODAB, while the molar ratios were different, e.g., 2:1 for DODAB/AMP, 4:1 for DODAB/ADP and 6:1 for DODAB/ATP. These differences may be attributed to the different anion charges of AMP, ADP and ATP. The results demonstrated that ribonucleotides combined with DODAB vesicles with the electrostatic attraction in the complexation of DODAB and ribonucleotides. Transmission electron microscopic results revealed the different extents of aggregation of cationic vesicles in the complexation process of ribonucleotides with cationic lipid. The variation dependence of zeta-potentials or electrophoretic mobilities on vesicle size was also different. The zeta-potentials and electrophoretic mobilities of the DODAB vesicles (0.01 and 0.02 mM) gradually decreased when the ribonucleotide concentration increased. However, the mean diameters of the DODAB vesicles (0.1 and 0.5 mM) gradually increased when the ribonucleotide concentration increased.

Adenosine Diphosphate↗
PubMed1999

Yeast Sml1, a protein inhibitor of ribonucleotide reductase.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides; this step is rate-limiting in DNA precursor synthesis. A number of regulatory mechanisms ensure optimal deoxyribonucleotide pools, which are essential for cell viability. The best studied mechanisms are transcriptional regulation of the RNR genes during the cell cycle and in the response to DNA damage, and the allosteric regulation of ribonucleotide reductase by nucleoside triphosphates. Recently, another mode of RNR regulation has been hypothesized in yeast. A novel protein, Sml1, was shown to bind to the Rnr1 protein of the yeast ribonucleotide reductase; this interaction was proposed to inhibit ribonucleotide reductase activity when DNA synthesis is not required (Zhao, X., Muller, E.G.D., and Rothstein, R. (1998) Mol. Cell 2, 329-340). Here, we use highly purified recombinant proteins to directly demonstrate that the Sml1 protein is a strong inhibitor of yeast RNR. The Sml1p specifically binds to the yeast Rnr1p in a 1:1 ratio with a dissociation constant of 0.4 microM. Interestingly, Sml1p also specifically binds to the mouse ribonucleotide reductase R1 protein. However, the inhibition observed in an in vitro mouse ribonucleotide reductase assay is less pronounced than the inhibition in yeast and probably occurs via a different mechanism.

Animals↗
PubMed1985

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.

Amino Acid Sequence↗