The c-Myc target gene Rcl (C6orf108) encodes a novel enzyme, deoxynucleoside 5'-monophosphate N-glycosidase

RCL is a c-Myc target with tumorigenic potential. Genome annotation predicted that RCL belonged to the N-deoxyribosyltransferase family. However, its putative relationship to this class of enzymes did not lead to its precise biochemical function. The purified native or N-terminal His-tagged recombinant rat RCL protein expressed in Escherichia coli exhibits the same enzyme activity, deoxynucleoside 5'-monophosphate N-glycosidase, never before described. dGMP appears to be the best substrate. RCL opens a new route in the nucleotide catabolic pathways by cleaving the N-glycosidic bond of deoxynucleoside 5'-monophosphates to yield two reaction products, deoxyribose 5-phosphate and purine or pyrimidine base. Biochemical studies show marked differences in the terms of the structure and catalytic mechanism between RCL and of its closest enzyme family neighbor, N-deoxyribosyltransferase. The reaction products of this novel enzyme activity have been implicated in purine or pyrimidine salvage, glycolysis, and angiogenesis, and hence are all highly relevant for tumorigenesis.     

Isolation and characterization of a bacteriophage T5 mutant deficient in deoxynucleoside 5''-monophosphatase activity.

A bacteriophage T5 mutant has been isolated that is completely deficient in the induction of deoxynucleoside 5'-monophosphatase activity during infection of Escherichia coli F. The mutant bacteriophage has been shown to be deficient in the excretion of the final products of DNA degradation during infection of E. coli F, and about 30% of the host DNA's thymine residues were reinocorporated into phage DNA. During infection with this mutant, host DNA degradation to trichloroacetic acid-soluble products was normal, host DNA synthesis was shut off normally, and second-step transfer was not delayed. However, induction of early phage enzymes and production of DNA and phage were delayed by 5 to 15 min but eventually reached normal levels. The mutant's phenotype strongly suggests that the enzyme's role is to act at the final stage in the T5-induced system of host DNA degradation by hydrolyzing deoxynucleoside 5'-monophosphates to deoxynucleosides and free phosphate; failure to do this may delay expression of the second-step-transfer DNA.     

Properties of herpes simplex virus DNA polymerase and characterization of its associated exonuclease activity.

Herpes simplex virus (HSV) DNA polymerase was isolated on a large-scale from African green monkey kidney cells infected with HSV type 1 (HSV-1) strain Angelotti. After DNA-cellulose chromatography the enzyme showed a specific activity of 48,000 units/mg protein. Three major single polypeptides with molecular weights of 144,000, 74,000 and 29,000 were copurified with the enzyme activity at the DNA-cellulose ste. By its chromatographic behavior and by template studies, the HSV DNA polymerase activity was clearly distinguishable from cellular alpha, beta and gamma DNA polymerase activities. Two exonucleolytic activities were found in the DNA-cellulose enzyme preparation. The main exonucleolytic activity, which degraded both single-stranded and double-stranded DNA to deoxynucleoside 5'-monophosphates, was separated by subsequent velocity sedimentation. The remaining exonucleolytic activity was not separable from the HSV DNA polymerase by several chromatographic steps and by velocity sedimentation at high ionic strength. This novel exonuclease and HSV DNA polymerase were equally sensitive both to phosphonoacetic acid and Zn2+ ions, inhibitors of the viral polymerase. Similar to the 3'-to-5'-exonuclease of procaryotic DNA polymerases and mammalian DNA polymerase delta, the HSV-polymerase-associated exonuclease catalyzed the removal of 3'-terminal nucleotides from the primer/template as well as the template-dependent conversion of deoxynucleoside triphosphates to monophosphates.     

DNase VII of human placenta. Mechanism studies

The mechanism of the human placental DNase VII, described previously (Hollis, G. F., and Grossman, L. (1981) J. Biol. Chem. 256, 8074-8079) has been investigated in further detail. The enzyme initiates exonucleolytic hydrolysis from the 3'-end of DNA in a nonprocessive, or distributive, manner, regardless of whether the carbohydrate moiety associated with the 3'-terminal nucleotide contains H or OH at its 2' and 3' positions. DNase VII does not have associated RNase H activity; however, it is capable of removing 3'-terminal ribonucleotides. The enzyme also can hydrolyze DNA containing a terminal nucleotide lacking a purine or pyrimidine as well as termini containing noncomplementary nucleotides. DNase VII activity is product-inhibited by deoxynucleoside 5'-monophosphates. From kinetic studies, the mononucleotide deoxyadenylic acid is a noncompetitive inhibitor with a Ki = 0.3 mM. The resemblance of DNase VII to the 3'----5' exonuclease activity of Escherichia coli DNA polymerase I and its possible role in excision repair and proofreading are discussed.     

5'-nucleotidase: an ecto-enzyme of frog skeletal muscle.

Using 1-4C-labeled AMP and IMP as substrates, 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity was detected at the external surface of frog skeletal muscle with the active site facing toward the extracellular space. The enzyme was firmly bound to the muscle membrane. Its activity was dependent on Ca2+ or Mg2+ and was inhibited by non-radioactive ribonucleoside 5'-monophosphates, or theophylline, while adenosine 3'-monophosphate and p-nitrophenylphosphate had little or no effect. 5'-Nucleotidase with similar properties was also found in the isolated plasma membrane fraction of the muscle.     

A kinetic study of the soluble 5''-nucleotidase of rat liver.

1. The kinetic properties of the 5'-nucleotidase (EC 3.1.3.5) present in the cytosol of rat liver were investigated in relation to the conversion of adenine nucleotides into uric acid, with particular reference to the stimulation of this process by fructose. The enzyme was assayed by the release of Pi and by a new and more sensitive radiochemical procedure. 2. When IMP was used as substrate, the partially purified enzyme displayed almost hyperbolic kinetics (h = 1.1) with S0.5 = 1.2 mM. Similar kinetics were observed with GMP and other nucleoside 5'-monophosphates, except AMP. 3. Vmax. of the enzyme for AMP was about the same as for IMP, but the kinetics were sigmoidal (h = 1.6) with S 0.5 = 10 mM. 4. The hydrolysis of IMP was inhibited competitively by GMP. IMP, at concentrations up to 0.5 mM, had a paradoxical stimulatory action on the hydrolysis of 2-5 mM-AMP and was inhibitory at higher concentrations. 5. The activity of the enzyme towards AMP and IMP was stimulated by ATP and GTP, and inhibited by Pi. Activators and inhibitor approximately cancelled each others' effects. At pH 7.4, the enzymic activity with 0.2 mM-AMP was undetectable under physiological conditions. 6. It is concluded that, in the liver cell, AMP is not hydrolysed by the soluble 5'-nucleotidase, but that its degradation requires prior deamination to IMP.     

Mechanisms of selective inhibition of 3' to 5' exonuclease activity of Escherichia coli DNA polymerase I by nucleoside 5'-monophosphates.

The 3' to 5' exonuclease activity of Escherichia coli DNA polymerase I can be selectively inhibited by nucleoside 5'-monophosphates, wherease the DNA polymerase activity is not inhibited. The results of kinetic studies show that nucleotides containing a free 3'-hydroxy group and a 5'-phosphoryl group are competitive inhibitors of the 3' to 5' exonuclease. Previous studies by Huberman and Kornberg [Huberman, J., and Kornberg, A. (1970), J. Biol. Chem. 245, 5326] have demonstrated a binding site for nucleoside 5'-monophosphates on DNA polymerase I. The Kdissoc values for nucleoside 5'-monophosphates determined in that study are comparable to the Ki values determined in the present study, suggesting that the specific binding site for nucleoside 5'-monophosphates represents the inhibitor site of the 3' to 5' exonuclease activity. We propose that (1) the binding site for nucleoside 5'-monophosphates on DNA polymerase I may represent the product site of the 3' to 5' exonuclease activity. (2) the primer terminus site for the 3' to 5' exonuclease activity is distinct from the primer terminus site for the polymerase activity, and (3) nucleoside 5'-monophosphates bind at the primer terminus site for the 3' to 5' exonuclease activity.     

Inactivation of avian myeloblastosis virus DNA polymerase by specific binding of pyridoxal 5'-phosphate to deoxynucleoside triphosphate binding site.

Avian myeloblastosis virus (AMV) DNA polymerase is inactivated by preincubation with pyridoxal 5'-phosphate. This inactivation is relatively specific since various pyridoxal-5'-P analogs cause no inactivation. This effect is reversible but can be made irreversible by reduction with sodium borohydride; the reduced pyridoxal-5'-P adduct exhibits a new absorbance maximum at 325 nm and a fluorescence emission at 392 nm when excited at 325 nm. The evidence presented suggests the formation of a Schiff base between pyridoxal-5'-P and a nucleophilic residue of AMV DNA polymerase. The presence of a deoxynucleoside 5'-triphosphate (dTTP) protected the enzyme from inactivation. Reduction of the pyridoxal-5'-P enzyme complex in the presence or absence of a deoxynucleoside 5'-triphosphate showed that the alpha subunit possesses five reactive amino groups, one of which is essential for catalytic activity; the beta subunit has three reactive amino groups which are not involved in the deoxynucleoside binding site.     

Direct determination of uracil in [32P,uracil-3H]poly(dA.dT) and bisulfite-treated phage PM2 DNA

A simple but effective technique for determining the presence of uracil existing as either A:U base pairs or G:U base pairs in DNA was developed. DNA is degraded to deoxynucleoside 3'-monophosphates by a combination of micrococcal nuclease and spleen phosphodiesterase. The monophosphates are converted to 5'-end-labeled 32P-labeled diphosphates in a reaction catalyzed by T4 polynucleotide kinase. The resultant product is then converted to 5'-end-labeled deoxynucleoside monophosphates by P1 nuclease digestion, which specifically removes 3'-phosphates. Successful separation of labeled dUMP from conventional bases in DNA is achieved by two-dimensional polyethyleneimine chromatography, with its detection determined by autoradiography and liquid scintillation counting. The sensitivity of the technique described can detect a minimum 1 X 10(-16) mol of dUMP in DNA. Additionally, the detection of 5-methylcytosine in placental DNA demonstrates the flexibility of the technique for the analysis of modified bases in DNA.     

Exonucleolytic proofreading enhances the fidelity of DNA synthesis by chick embryo DNA polymerase-gamma

The high fidelity of chick embryo DNA polymerase-gamma (pol-gamma) observed during in vitro DNA synthesis (Kunkel, T. A. (1985) J. Biol. Chem. 260, 12866-12874) has led us to examine this DNA polymerase for the presence of an exonuclease activity capable of proofreading errors. Highly purified chick embryo pol-gamma preparations do contain exonuclease activity capable of digesting radiolabeled DNA in a 3'----5' direction, releasing deoxynucleoside 5'-monophosphates. The polymerase and exonuclease activities cosediment during centrifugation in a glycerol gradient containing 0.5 M KCl. In the absence of dNTP substrates, this exonuclease excises both matched and mismatched primer termini, with a preference for mismatched bases. Excision is inhibited by the addition of nucleoside 5'-monophosphates to the digestion reaction. In the presence of dNTP substrates to permit competition between excision and polymerization from the mismatched primer, the exonuclease excises mismatched bases from preformed terminal mispairs with greater than 98% efficiency. The preference for excision over polymerization can be diminished by addition of either high concentrations of dNTP substrates or nucleoside 5'-monophosphates to the exonuclease/polymerase reaction. To determine if this exonuclease is capable of proofreading misinsertions produced during a normal polymerization reaction, a sensitive base substitution fidelity assay was developed based on reversion of an M13mp2 lacZ alpha nonsense codon. In this assay using reaction conditions that permit highly active exonucleolytic proofreading, pol-gamma exhibits a fidelity of less than one error for every 260,000 bases polymerized. As for terminal mismatch excision, fidelity is reduced by the addition to the synthesis reaction of high concentrations of dNTP substrates or nucleoside 5'-monophosphates, both hallmarks of exonucleolytic proofreading by prokaryotic enzymes. Taken together, these observations suggest that the 3'----5' exonuclease present in highly purified chick embryo pol-gamma preparations proofreads base substitution errors during DNA synthesis. It remains to be determined if the polymerase and exonuclease activities reside in the same or different polypeptides.     

Membrane-bound nucleotidase of Bacillus cereus.

A membrane-bound nucleotidase of Bacillus cereus T was solubilized by digestion with trypsin and subsequently purified more than 300-fold. The purified nucleotidase was most active on ribonucleoside 5'-monophosphates and was slightly less active (40 to 60%) on deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates. In addition to hydrolytic activity, the nucleotidase preparation possessed phosphotransferase activity by which phosphate is transferred from a phosphate donor to the 5' position of nucleosides.     

5'-Nucleotidase from rat heart

5'-Nucleotidase has been extracted from rat heart and purified to apparent homogeneity. The enzyme is a glycoprotein. Gel electrophoresis in the presence of sodium dodecyl sulfate indicates that the apparent molecular weight of the subunit is 74 000 at several different gel concentrations. Cross-linking of the native enzyme with dimethylpimelimidate followed by gel electrophoresis shows that the enzyme is a dimer. The enzyme hydrolyzes all nucleoside 5'-monophosphates tested. A comparison of Vmax/Km for 14 different substrates shows that AMP is the best substrate. The enzyme shows lowest Km values for AMPS, AMP, isoAMP, GMP, and IMP. It shows no activity with nucleoside 2'- and 3'-monophosphates, sugar phosphates, and p-nitrophenyl phosphate, even when tested at high enzyme concentrations. The optimum activity of the enzyme occurs at pH 7.5 with AMP as substrate. Above this pH, buffer ions affect the activity in a complex manner, a second optimum being observed under some conditions. Magnesium ions activate the enzyme above pH 7.5 in the presence of some buffer ions but not of others. Magnesium ions show only a slight activation when the reaction is run in diethanolamine buffer, pH 9.5, at 30 degrees C; the activation in this buffer is considerably greater when the reaction is run at 37 degrees C. The enzyme is strongly inhibited by free ADP, maximum inhibition occurring below pH 6. The ADP inhibition is diminished as the pH is raised above 6, becoming negligible above pH9. The enzyme is inhibited by EDTA. The inhibition is partially reversed when the EDTA is removed from the enzyme by gel filtration. This as well as other evidence indicates that the enzyme contains a tightly bound metal ion.     

A guanosine 3':5'-monophosphate-sensitive nuclease from Bacillus brevis.

In toluene-treated cells of Bacillus brevis, newly synthesized RNA is rapidly degraded in a reaction that is inhibited by cyclic guanosine 3':5'-monophosphate (cGMP) and by 1,10-phenanthroline. This appears to be due to a ribonuclease found in cell-free extracts of B. brevis which is inhibited by cGMP and related compounds as well as by 1,10-phenanthroline. The cGMP-sensitive nuclease hydrolyzes synthetic polynucleotides, yielding nucleoside 5'-monophosphates as the sole products, even during the early stages of hydrolysis. Synthetic polynucleotides terminated by a 3'-phosphate are resistant to hydrolysis. While with 3'-hydrolysis of the polymer. The enzyme is therefore an exonuclease that degrades polynucleotides from the 3' end to product 5'-mononucleotides. It also acts on denatured but not on native DNA. Activity is greatest in the presence of Mn2+ and is not affected by the presence of monovalent cations. 1,10-Phenanthroline, but not 1,7-phenanthroline, inhibits the nuclease even when Mn2+ is present in excess. The inhibition of the enzyme by cGMP is noncompetitive, and cGMP itself is not hydrolyzed. The sensitivity of the nuclease to inhibition depends strikingly on the nature of the substrate and is lost when the enzyme is assayed at high pH. These observations suggest that cGMP inhibits the nuclease by combining with an allosteric site on the enzyme. Although cGMP was found to be the most effective inhibitor, other nucleoside 3':5'-monophosphates and derivatives of 5'-GMP can also inhibit the nuclease. Since measurements of cGMP in B. brevis have not revealed detectable amounts (less than 5 times 10-8 M), the substance that modulates the activity of the nuclease under physiological conditions remains to be identified.     

Contribution of 3' leads to 5' exonuclease activity of DNA polymerase III holoenzyme from Escherichia coli to specificity

The effects of deoxynucleoside monophosphates on the 3' leads to 5' exonuclease activity of DNA polymerase III holoenzyme have been correlated with their effects on the fidelity of DNA replication. In particular, dGMP inhibits the proofreading activity of the enzyme and decreases the fidelity in those cases where a "following nucleotide effect" is also noted. This is strong evidence for proofreading. However, the absence of the effects of proofreading inhibitors or following nucleotides need not be evidence against the occurrence of proofreading: a theoretical analysis shows that these effects may not be observed even though there is active proofreading. This is suggested to be the case with the phage T4 enzyme system. The proofreading activity of Pol III appears to be directed primarily towards removing purine x pyrimidine-mediated rather than purine x purine-mediated misincorporations. recA protein inhibits the proofreading activity of Pol III on synthetic templates containing mismatched 3' termini. This is paralleled by a decrease in the fidelity of DNA replication in vitro. The inhibition is increased in the presence of dGMP or dAMP but there is no further increase in the infidelity of replication. The presence of both dNMPs and recA protein does not enable Pol III to copy past pyrimidine photodimers.     

Structural basis for substrate specificity of the human mitochondrial deoxyribonucleotidase

The human mitochondrial deoxyribonucleotidase catalyzes the dephosphorylation of thymidine and deoxyuridine monophosphates and participates in the regulation of the dTTP pool in mitochondria. We present seven structures of the inactive D41N variant of this enzyme in complex with thymidine 3'-monophosphate, thymidine 5'-monophosphate, deoxyuridine 5'-monophosphate, uridine 5'-monophosphate, deoxyguanosine 5'-monophosphate, uridine 2'-monophosphate, and the 5'-monophosphate of the nucleoside analog 3'-deoxy 2'3'-didehydrothymidine, and we draw conclusions about the substrate specificity based on comparisons with enzyme activities. We show that the enzyme's specificity for the deoxyribo form of nucleoside 5'-monophosphates is due to Ile-133, Phe-49, and Phe-102, which surround the 2' position of the sugar and cause an energetically unfavorable environment for the 2'-hydroxyl group of ribonucleoside 5'-monophosphates. The close binding of the 3'-hydroxyl group of nucleoside 5'-monophosphates to the enzyme indicates that nucleoside analog drugs that are substituted with a bulky group at this position will not be good substrates for this enzyme.     

Observations on the pyridoxal 5'-phosphate inhibition of DNA polymerases.

Pyridoxal 5'-phosphate at concentrations greater than 0.5 mM inhibits polymerization of deoxynucleoside triphosphate catalyzed by a variety of DNA polymerases. The requirement for a phosphate as well as aldehyde moiety of pyridoxal phosphate for inhibition to occur is clearly shown by the fact that neither pyridoxal nor pyridoxamine phosphate are effective inhibitors. Since the addition of nonenzyme protein or increasing the amount of template primer exerted no protective effect, there appears to be specific affinity between pyridoxal phosphate and polymerase protein. The deoxynucleoside triphosphates, however, could reverse the inhibition. The binding of pyridoxal 5'-phosphate to enzyme appears to be mediated through classical Schiff base formation between the pyridoxal phosphate and the free amino group(s) present at the active site of the polymerase protein. Kinetic studies indicate that inhibition by pyridoxal phosphate is competitive with respect to substrate deoxynucleoside triphosphate(s).     

5'-Nucleotidase activity of lymphocyte plasma membranes. Effect of concanavalin A]

The 5'-nucleotidase properties of isolated lymphocyte plasma membranes from young pig mesenteric nodes are described; nucleosides-5'-monophosphates are the substrates of this specific enzyme. Concanavalin A inhibits this enzyme; on the same membranes this mitogen does not affect alkaline phosphatase and activates the membrane bound (Ca2+) ATPase. The 5'-nucleotidase inhibition is due to a specific interaction of Con A with carbohydrate groups of the membrane; its high positive cooperativity suggests that the lectin promotes reorganization of the membrane bound 5'-nucleotidase. Solubilization of the 5'-nucleotidase does not prevent the effect of Con A and the solubilized enzyme is firmly bound by Con A-Sepharose 4B; these results suggest that Con A inhibits the enzyme by a direct interaction and that 5'-nucleotidase can be considered as an eventual receptor for the lectin.     

Partial purification and properties of an AMP-specific soluble 5''-nucleotidase from pigeon heart.

A soluble 5'-nucleotidase was purified 200-fold from pigeon heart. The enzyme (1) had an apparent molecular mass close to 150 kDa, (2) had a neutral pH optimum and hydrolysed a wide range of nucleoside 5'-monophosphates with a 15-fold preference for AMP over IMP, (3) at near-physiological concentrations of AMP was activated by ADP but not by ATP, (4) was inhibited by high Mg2+ concentration and high ionic strength, (5) was weakly inhibited by p-nitrophenol phosphate and Pi, and (6) was non-competitively inhibited more potently by 5'-deoxy-5'-isobutylthioinosine than by 5'-deoxy-5'-isobutylthioadenosine, but not by [alpha,beta-methylene]ADP. The data show that the enzyme is distinct from the ecto-5'-nucleotidase and from the previously purified IMP-specific 5'-nucleotidase. They also predict that the enzyme is activated during ATP catabolism and hence will generate a more-than-linear increase in the adenosine-formation rate in response to an increase in cytosolic AMP concentration.     

Purification and properties of 5'-nucleotidase from the membrane of Vibrio costicola, a moderately halophilic bacterium.

Two different Mg2+-dependent adenosine 5'-triphosphate-hydrolyzing activities were detected in membranes of Vibrio costicola, a novel 5'-nucleotidase and an N,N'-dicyclohexylcarbodiimide-sensitive adenosine triphosphatase. The former and the latter had different requirements for Mg2+ and were selectively assayed in the membranes by using, respectively, 20 and 2 mM Mg2+. The two enzymes were extracted with a combination of Triton X-100 and octylglucoside, separated on a diethylaminoethyl cellulose column, and purified on glycerol gradients. The purified 5'-nucleotidase consisted of one major polypeptide of 70,000 daltons when analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate. The purified 5'-nucleotidase was similar in substrate specificities, divalent cation specificities, and pH profiles to the membrane-bound N,N'-dicyclohexylcarbodiimide-insensitive nucleotide-phosphohydrolyzing activity. The enzyme hydrolyzed nucleoside 5'-tri, 5'-di, and 5'-monophosphates at comparable rates. Inorganic pyrophosphate, p-nitrophenyl phosphate, glucose 6-phosphate, beta-glycerophosphate, adenosine 5'-diphosphate glucose, adenosine 3'-monophosphate, and cyclic adenosine 3',5'-monophosphate were not hydrolyzed, either im membranes or by the purified 5'-nucleotides. Actions of NaCl and KCl on the activity of the 5'-nucleotidase were studied. The enzyme was activated by both NaCl and KCl; the activation profiles however, were different for the membrane-bound and purified 5'-nucleotidase. The purified enzyme, unlike the membrane-bound enzyme, was markedly stimulated by high concentrations of NaCl (up to 3 M).