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At least 19 recordsLinked to original sources
PubMed2026

Fluorescent reporter assay reveals ribonucleotides promote mismatch correction in vivo.

Ribonucleotides can serve as a strand discrimination signal in reconstituted in vitro biochemical mismatch repair (MMR) assays, but the influence of ribonucleotides on mismatch correction has not been measured directly in vivo. We have developed a fluorescence-based host cell reactivation assay that reports correction of a mismatch in proximity of a site-specifically incorporated ribonucleotide. A ribonucleotide leads to enhanced mismatch correction. While neither inactivation of a single allele nor knockdown of RNaseH2 is sufficient to suppress ribonucleotide directed MMR, a modest but statistically significant impairment for repair of mismatches in the presence of an embedded ribonucleotide is observed in RNaseH2 knockout cell lines. Reporter plasmids with ribonucleotides located in either the 3' or 5' orientation are robustly repaired in MMR-proficient cells but are weakly repaired in MMR-deficient cells, underscoring their utility as effective MMR reporters. Significant ribonucleotide-enhanced mismatch correction was consistently observed in MMR-deficient cells when the ribonucleotide is in the 3' orientation. The presence of a ribonucleotide led to enhanced MMR even in RNaseH2 knockout cells, suggesting that other enzymes may promote ribonucleotide-directed MMR. Loss of RNaseH2 was not sufficient to confer significant resistance to the alkylating agent, temozolomide, in support of a model in which ribonucleotide-directed repair events make minor contributions to the canonical MMR pathway in mammalian cells. We propose a model in which MMR-independent ribonucleotide enhanced correction of mismatches can proceed by ribonucleotide excision repair when the ribonucleotide is in the 5' direction, and proceeds by an unknown mechanism when the ribonucleotide is in the 3' direction.

DNA Mismatch Repair↗
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↗
PubMed1978

Allosteric regulation of calf thymus ribonucleotide reductase.

Ribonucleotide reductase was purified 3400-fold from calf thymus. The enzyme preparation was essentially free of kinases and phosphatases and therefore allowed a conclusive study of the allosteric regulation of a eukaryotic ribonucleotide reductase to be made for the first time. Comparable maximal activities were obtained for the reduction of all four ribonucleotide substrates in the presence of their optimal stimulatory effectors. These and other results strongly argue for the existence of only one ribonucleotide reductase in mammalian cells. No reduction was observed in the absence of effector. The reduction of CDP and UDP both required ATP, with no stimulatory effect of any other nucleoside triphosphate. The only activator of GDP reduction was dTTP and the only activator of ADP reduction was dGTP. Reduction of the purine ribonucleotides was further stimulated by ATP but only in combination with dTTP or dGTP. The reduction of all four ribonucleotides was strongly inhibited by dATP, the inhibition being partly released by ATP. The data can be integrated into a scheme which links ribonucleotide reduction to DNA synthesis.

Allosteric Regulation↗
PubMed1975

An affinity adsorbent containing deoxyguanosine 5'-triphosphate linked to sepharose and its use for large scale preparation of ribonucleotide reductase of Lactobacillus leichmannii.

P3-(6-(N-Trifluoracetyl)aminohex-1-yl) deoxyguanosine triphosphate has been prepared by the reaction of N-trifluoroacetyl-6-aminohexanol 1-pyrophosphate with the imidazolide of dGMP and has been characterized. This compound and the corresponding free amine, obtained by removal of the protective trigluoroacetyl group, are activators of ribonucleotide reductase of Lactobacillus leichmannii. An affinity adsorbent for the reductase, prepared by reaction of the amine derivative with CNBr-activated Sepharose, contains dGTP covalently attached through the gamma-phosphate via a six-carbon chain to the matrix. The method of synthesis of the dGTP derivative is generally applicable to the synthesis of P3-(omega-aminoalk-1-yl)nucleoside triphosphate esters for the preparation of analogous affinity adsorbents. Ribonucleotide reductase can be rapidly purified to homogeneity, on a large scale, by use of dGTP-Sepharose and conditions for optimum recovery of the enzyme have been determined. The affinity of ribonucleotide reductase and other proteins for dGTP-Sepharose is increased by either raising the ionic strength or lowering the temperature of the eluent. Elution of the enzyme from the adsorbent can be achieved between pH 5.8 and 7.3, whereas at pH 5.3 the reductase is bound extremely tightly and cannot be recovered. Ribonucleotide reductase can be eluted from the adsorbent with dGTP or urea. Elution with urea is carried out at pH 6.3, where the enzyme is stable and maximum recovery is obtained. Affinity chromatography consistently produces ribonucleotide reductase of high specific activity (170-180 units/mg). In the presence of 0.1 to 1.2 M urea or hydroxyurea, the enzyme is inhibited, but allosteric activation is unchanged. No alteration in the structure or function of the reductase was detected when the enzyme was exposed to 2.0 M urea during elution from the affinity adsorbent, but exposure for longer periods causes some inactivation.

Amino Acid Sequence↗
PubMedDate not supplied

[Ribonucleotide reductase in Propionibacterium shermani].

The cell-free extract of Propionibacterium shermanii was found to contain B12-dependent ribonucleotide reductase. The extract of the cells grown under the conditions of the inhibited synthesis of vitamin B12 reduces ribonucleotides with the participation of B12-independent enzyme. The synthesis of B12-dependent apoenzyme of ribonucleotide reductase is partially maintained under these conditions. Both enzymes reduce preferably ribonucleoside diphosphates. The reducing agent of nucleotides in vitro is lipoic acid or dithiothreitol, in the B12-dependent pathway, and NADPH and thioredoxin, in the B12-independent pathway. Only B12-independent ribonucleotide reductase requires Mg2+ ions. Vitamin B12 in the coenzyme form inhibits the activity of B12-independent enzyme.

Cell-Free System↗
PubMed1977

Effect of methotrexate and 5-fluorodeoxyuridine on ribonucleotide reductase activity in mammalian cells.

A number of studies in bacteria have indicated that deoxythymidine 5'-triphosphate may be a repressor or corepressor of ribonucleotide reductase. For determination of whether a similar regulating mechanism exists in mammalian cells, HeLa cells and partially hepatectomized rats were treated with either methotrexate, 5-fluorouracil, or 5-fluorodeoxyuridine in order to block thymidylate synthesis and consequently lower the intracellular pools of deoxythymidine 5'-triphosphate. In HeLa cells there was a significant (360 to 400 percent) increase in reductase activity in both the methotrexate and 5-fluorodeoxyuridine-treated cells. The administration of methotrexate to partially hepatectomized rats resulted in a 2.7-fold enhancement of the hepatectomy-induced increase in reductase activity, and the 5-fluorouracil treatment yielded a 60 percent increment in the increase of ribonucleotide reductase activity after partial hepatectomy. Cycloheximide prevented the increase in reductase activity after the exposure of HeLa cells to methotrexate and 5-fluorodeoxyuridine, indicating that the stimulation of ribonucleotide reductase activity was the result of enhancement of de novo enzyme synthesis rather than of enzyme activation. The data support the thesis that deoxythymidine 5'-triphosphate or a thymidylate metabolite may be involved in the regulation of ribonucleotide reductase levels in mammalian cells.

Animals↗
PubMed1979

Ribonucleotides in DNA newly synthesised in 3T6 cells in vivo.

Within the field of DNA replication, considerable interest has focused in recent years on the mechanism of initiation of synthesis of DNA molecules. In vitro replication systems from Escherichia coli have been instrumental in uncovering a priming function fo9r ribonucleotides on the earliest intermediates of DNA polymerisation in vitro and in identifying the proteins involved. In vitro replication systems from mammalian cells that permit the use of the phosphate-transfer method for detection of RNA-DNA junctions as well as direct labelling of the RNA moiety of the molecules have suggested a similar role for ribonucleotides in DNA synthesis in eukaryotes. However, the existence of this mechanism in mammalian cells in vivo has not been established. Here we report the first evidence that a significant proportion of the earliest intermediates in mammalian DNA polymerisation in vivo do, in fact, possess ribonucleotides, presumably because their synthesis was initiated with one or more ribonucleotides.

Animals↗
PubMed1977

The free radical in ribonucleotide reductase from E. coli.

Protein B2, one of the subunits of ribonucleotide reductase from Escherichia coli, contains a stable free radical. It is characterized by a doublet e.p.r. signal centered around g = 2.0047 and a sharp peak at 410 nm in the optical spectrum. The radical has been assigned to a tyrosyl residue in the protein with its spin density delocalized over the aromatic ring. Protein B2 also contains two antiferromagnetically-coupled high-spin iron(III) atoms, which stabilize the free radical. Protein B1, the other subunit of ribonucleotide reductase, contains two binding sites for substrate molecules, which are the four common ribonucleoside diphosphates. It also contains two classes of allosteric effector-binding sites. ATP and deoxyribonucleoside triphosphates function as effectors. A one-to-one complex of proteins B1 and B2 forms the enzymically-active ribonucleotide reductase. The free radical is, most likely, part of the active site.

Binding Sites↗
PubMed1975

Ribonucleotide reductase from Escherichia coli: demonstration of a highly active form of the enzyme.

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, proteins B1 and B2. The activity of the enzyme in crude extracts prepared from mechanically disrupted bacteria is very low. Enzyme activity is stimulated 5 to 10-fold by addition of an excess of either subunit. Concentrated extracts from cells lysed gently on Cellophane discs (Schaller et al.) contained 10 to 20-fold higher activity than extracts from mechanically disrupted cells. This activity was not further stimulated by either B1 or B2. The system is suitable for complementation tests for the analysis of temperature-sensitive mutants affecting the ribonucleotide reductase system. Concentrated high-speed supernatants from E. coli treated with lysozyme (Wickner et al.) also contained a high ribonucleotide reductase activity, which was stimulated slightly or not at all by addition of B1 and B2. This active form of the enzyme was unstable and could not be purified. The results suggest that the intracellular form of the enzyme consists of a tight complex of proteins B1 and B2, possibly stabilized by other intracellular structures.

Adenosine Triphosphate↗
PubMed1976

Effects of 2-amino-1,3,4-thiadiazole on ribonucleotide pools of leukemia L1210 cells.

The effects of 2-amino-1,3,4-thiadiazole [aminothiadiazole (NSC 4728)] on purine and pyrimidine ribonucleotide pools of L1210 ascites cells in vivo are presented and discussed as they relate to the site of action. Within 1 hr after administration of the drug, the levels of guanosine triphosphate, guanosine diphosphate, adenosine triphosphate, and adenosine diphosphate were reduced, whereas those of inosine monophosphate (IMP) and uridine triphosphate were increased. The most pronounced effects were the lowering of guanine ribonucleotide pools and the elevation of IMP. Aminothiadiazole produced a marked inhibition (approximately 95%) of the incorporation of [8-14C]inosine into guanine nucleotides, whereas only a slight inhibition (approximately 20%) of incorporation into adenine nucleotides was observed. These results suggest that the thiadiazole (or a metabolite thereof) inhibits the conversion of IMP to guanosine monophosphate; this conclusion is reinforced by the observation that mycophenolic acid, a known inhibitor of this conversion, produced effects on ribonucleotide pools similar to those produced by aminothiadiazole. Aminothiadiazole did not inhibit IMP dehydrogenase isolated from L1210 cells. The effects of the thiadiazole on nucleotide pools were prevented by simultaneous administration of nicotinamide. Since nicotinamide is known to prevent or reverse the antileukemic activity of aminothiadiazole, it is probable that the inhibition of synthesis of guanosine monophosphate is related to the antileukemic action of this agent.

Adenosine Diphosphate↗
PubMed1975

Studies on the mechanism of adenosylcobalamin-dependent ribonucleotide reduction by the use of analogs of the coenzyme.

A series of 17 analogs of 5'-deoxy-5'-adenosylcobalamin(adenosylcobalamin) have been synthesized with modifications in the base or ribose moiety of the nucleoside ligand. These analogs have been examined for their effects on reactions catalyzed by the ribonucleotide reductase of Lactobacillus leichmannii. All the analogs are inhibitors of ATP reduction in the presence of adenosylcobalamin as coenzyme, and hence all are bound to the catalytic site. Only the 3-beta-D-ribofuranosyladenine analog (isoadenosylcobalamin) showed substantial activity as a coenzyme in ATP reduction, giving a rate of 59% of that obtained with the adenosylcobalamin. Lesser rates of reduction were obtained with nebularyl-, 2'-deoxyadenosyl-, tubercidyl-, isopropylideneadenosyl-, L-adenosyl-, and ara-adenosylcobalamin, coenzyme activity decreasing in that order. Other analogs showed no significant coenzyme activity. The rate of hydrogen exchange into water from the 5'-methylene group of the nucleoside ligand appeared to parallel the coenzyme activity in those analogs examined, but only the four cobalamins with highest coenzyme activity (adenosyl, isoadenosyl, nebularyl, 2'-deoxyadenosyl) gave detectable amounts of "active coenzyme B12," THe rapidly formed paramagnetic intermediate of ribonucleotide reduction. The enzyme system produced the slowly formed paramagnetic species characterized by a doublet EPR spectrum only with adenosyl- and isoadenosylcobalamin. By contrast the enzymic degradation of analogs to cob(II)alamin and 5'-deoxynucleoside occurred not only with those analogs active as coenzymes and in the exchange reaction but also with a number of coenzymically inactive analogs, and the rate of degradation was unrelated to the rate of ribonucleotide reduction for those analogs with coenzyme activity.

Adenosine Triphosphate↗
PubMed1979

Effect of hydroxyurea on T4 ribonucleotide reductase.

Phage T4-induced ribonucleotide reductase, purified to homogeneity, catalyzes the reduction of the four ribonucleotides CDP, UDP, ADP, and GDP to the corresponding deoxyribonucleotides. The enzyme is an order of magnitude more sensitive to hydroxyurea than the corresponding Escherichia coli enzyme. Fifty per cent inhibition occurs at 10 micrometer hydroxyurea. Inhibition is complete at a high concentration of the drug, and there is no differential effect on the four substrates. Treatment of T4 ribonucleotide reductase or its isolated subunits with hydroxyurea does not lead to their irreversible inactivation.

Coliphages↗
PubMed1979

Specific inhibitors directed at the individual components of ribonucleotide reductase as an approach to combination chemotherapy.

It had been shown previously that the ribonucleotide reductase from mouse tumor consisted of two nonidentical components (Tris and dye fractions, each prepared from the 20 to 40% (NH/)2SO4 protein fraction containing the ribonucleotide reductase activity by blue dextran-Sepharose chromatography). The individual components either separated or present in the intact enzyme can be specifically and independently inhibited by different compounds. The Tris fraction component was inhibited by 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone while the dye fraction component was inactivated by pyridoxal phosphate:BH4- and the dialdehyde derivative of inosine (Inox) and 5'-deoxyinosine prepared by the periodate oxidation of inosine and 5'-deoxyinosine. The intact enzyme could be completely inhibited by any of these compounds. Reductase activity was restored by reconstitution with the exogenous components. The individual components of the reductase in the intact Ehrlich tumor cell could also be specifically inhibited. Activity in the crude cell-free extracts prepared from 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone- or Inox-treated cells was restored by the addition of the appropriate exogenous component. These data suggest that combinations of inhibitors of ribonucleotide reductase which specifically inhibit the components may be useful in the treatment of cancer.

Aldehydes↗
PubMed2026

Bacillus subtilis RNase HII Is Inefficient at Processing Guanosine Monophosphate and Damaged Ribonucleotides.

During one round of DNA replication, nearly 2000 ribonucleoside monophosphates (rNMPs) are incorporated in place of their cognate deoxyribonucleoside monophosphates (dNMPs). Given their high rate of insertion, genomic DNA could contain rNMPs that are damaged or mismatched. Here, we test the activity of Bacillus subtilis and Escherichia coli RNase HII on canonical, mismatched, and damaged rNMPs. We show that E. coli RNase HII is adept at incising most rNMP variants from DNA at similar frequencies, with the exception of an oxidized rNMP, where endoribonuclease activity is sharply reduced. In contrast, B. subtilis RNase HII efficiently incises rAMP, rCMP, and rUMP but is inefficient at processing rGMP in both a canonical and mismatched base pair. We test damaged ribonucleotides and find that B. subtilis RNase HII is refractory to processing abasic and oxidized ribonucleotide lesions. Our work shows that bacterial RNase HII enzymes have different intrinsic endoribonuclease activity toward the repair of canonical, mismatched, and damaged rNMPs, demonstrating that not all rNMP errors provoke efficient resolution. Our finding that B. subtilis RNase HII is recalcitrant to repairing damaged rNMPs resembles what is observed for eukaryotic RNase H2 orthologs, suggesting that other repair processes are necessary to resolve damaged rNMPs.

Bacillus subtilis↗
PubMed1977

Inhibition of mammalian ribonucleotide reductase by a dinucleotide produced in eucaryotic cells.

HS3, a highly phosphorylated dinucleoside originally purified from the fungus Achlya, has been isolated from Chinese hamster ovary cells undergoing glutamine starvation. The HS3 compounds obtained from the fungal and mammalian sources exhibited similar physical and chemical properties. This unusual dinucleotide may be an important regulator of eucaryotic ribonucleoside diphosphate reductase activity; for 50 micrometer HS3, isolated from either mammalian or fungal cells, significantly inhibited CDP reduction in Achlya or hamster cell preparations, but only marginally affected the activity of the enzyme from E. coli. Studies with HS3 isolated from Achlya and partially purified mammalian ribonucleotide reductase indicated that the compound noncompetitively inhibited the reduction of varying concentrations of the substrates CDP, ADP and GDP with Ki values of 23 micrometer, 14 micron and 16 micron respectively. These inhibitor concentrations are well below the estimated intracellular levels of HS3 in glutamine starved cells and suggest that HS3 inhibition of ribonucleotide reduction may be responsible for the rapid inhibition of DNA synthesis seen under these culture conditions.

Adenosine Diphosphate↗
PubMed1977

Ribonucleotide reductase from Escherichia coli. Identification of allosteric effector sites by chromatography on immobilized effectors.

Ribonucleotide reductase is responsible for the production of deoxyribonucleotides by catalyzing the reduction of ribonucleoside diphosphates. The enzyme is allosterically regulated in a complex way by the nucleoside triphosphates, ATP, dTTP, dGTP, dCTP, and dATP. Ribonucleotide reductase consists of two nonidentical subunits, proteins B1 and B2. Both substrates and allosteric effectors bind exclusively to B1. Binding of protein B1 to dTTP or dATP covalently coupled to Sepharose and elution with concentration gradients of the different nucleoside triphosphate effectors gave information about (1) the arrangement of the effector binding sites on protein B1 and (2) the affinity of the effectors for these sites. Protein B1 thus has two classes of effector binding sites. One class binds all effectors, as demonstrated by elution of the protein from dTTP-Sepharose with dATP, dGTP, ATP, or dCTP. The second class binds only dATP or ATP, since dATP and ATP were the only nucleotides which eluted protein B1 from dATP-Sepharose. These results confirm earlier data obtained by dialysis binding experiments. The eluting concentrations obtained for the different nucleoside triphosphates in experiments with dTTP-Sepharose could be used to calculate unknown dissociation constants for protein B1 -effector binary complexes. This was possible, since a plot of the eluting concentrations vs. known dissociation constants was linear.

Allosteric Site↗
PubMed1979

Isolation and characterization of purine-nucleoside phosphorylase-deficient T-lymphoma cells and secondary mutants with altered ribonucleotide reductase: genetic model for immunodeficiency disease.

The inherited deficiency of purine-nucleoside phosphorylase (PNPase; purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) in humans is associated with a severe deficiency of the T lymphocytes of the immune system. Because of the unsatisfactory nature of previously described model systems, we have selected, cloned, and characterized a mutant mouse T cell lymphoma (S49) completely deficient in PNPase. Of the four substrates of PNPase, only deoxyguanosine at low concentrations is toxic to the PNPase-deficient (NSU-1) cells. In order to delineate the biochemical processes necessary for the sensitivity of the NSU-1 cells to deoxyguanosine, we have isolated a series of secondary mutants resistant to deoxyguanosine from the PNPase-deficient line. One of these mutants is defective in its ability to transport deoxyguanosine into the cell. A second type of mutant cannot phosphorylate the deoxyguanosine and is totally deficient in deoxycytidine kinase activity. A third type of mutant (NSU-1-dGuo-L) can both transport and phosphorylate deoxyguanosine and accumulates dGTP. However, unlike its parent, NSU-1-dGuo-L does not become depleted of dCTP and TTP when exposed to exogenous deoxyguanosine. This observation is accounted for by the fact that the reduction of CDP to dCDP by the ribonucleotide reductase (ribonucleoside-diphosphate reductase, 2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) of NSU-1-dGuo-L cells is not normally sensitive to feedback inhibition by dGTP.Thus, in order to exert its toxicity deoxyguanosine must be transported into the cell, be phosphorylated by deoxycytidine kinase, and be accumulated as dGTP. By inhibiting ribonucleotide reductase, dGTP depletes the cell of dCTP and to some extent TTP, thus preventing the synthesis of DNA, a process necessary for any proliferation-dependent function of T cells.

Animals↗