Non-specific Nuclease Activity Research Articles

An in vitro assay to study regulated mRNA stability.

The examination of posttranscriptional regulation of mRNA in mammalian cells is critical to discovering the role that mRNA plays in the initiation and maintenance of cellular processes. The complexity of the system defies a holistic approach and, therefore, we have devised an in vitro mRNA turnover assay that enables us to elucidate the factors involved in mRNA deadenylation and degradation. Our system, using an S100 HeLa extract and in vitro transcribed RNAs, accurately mimics the end products of mRNA turnover, which have been previously described using in vivo studies and, in addition, allows for the detailed study of factors that may play a role in regulated deadenylation and degradation. Another important aspect of our system is the ease with which it can be manipulated. We can provide any synthetic RNA molecule to the assay to test for specific sequence activity. Furthermore, the results are clear and accurately interpretable. We have demonstrated that our in vitro system accurately deadenylates and decays a capped and polyadenylated RNA molecule in a processive manner without nonspecific nuclease activity. Finally, we have demonstrated regulated instability in vitro using the AU-rich elements (AREs) from tumor necrosis factor-alpha (TNF-alpha) and granulocyte macrophage colony stimulating factor (GM-CSF) embedded within the RNA molecule. The presence of the AREs increased the deadenylation and the decay rates seen in vivo. We feel that this system can be expanded and adapted to examine a variety of mRNA regulatory events in mammalian cells.

Domain III function of Mu transposase analysed by directed placement of subunits within the transpososome.

Assembly of the functional tetrameric form of Mu transposase (MuA protein) at the two att ends of Mu depends on interaction of MuA with multiple att and enhancer sites on supercoiled DNA, and is stimulated by MuB protein. The N-terminal domain I of MuA harbours distinct regions for interaction with the att ends and enhancer; the C-terminal domain III contains separate regions essential for tetramer assembly and interaction with MuB protein (IIIalpha and IIIbeta, respectively). Although the central domain II (the 'DDE' domain) of MuA harbours the known catalytic DDE residues, a 26 amino acid peptide within IIIalpha also has a non-specific DNA binding and nuclease activity which has been implicated in catalysis. One model proposes that active sites for Mu transposition are assembled by sharing structural/catalytic residues between domains II and III present on separate MuA monomers within the MuA tetramer. We have used substrates with altered att sites and mixtures of MuA proteins with either wild-type or altered att DNA binding specificities, to create tetrameric arrangements wherein specific MuA subunits are nonfunctional in II, IIIalpha or IIIbeta domains. From the ability of these oriented tetramers to carry out DNA cleavage and strand transfer we conclude that domain IIIalpha or IIIbeta function is not unique to a specific subunit within the tetramer, indicative of a structural rather than a catalytic function for domain III in Mu transposition.

Nucleotide Excision Repair in Oocyte Nuclear Extracts from Xenopus laevis

We have developed efficient DNA repair extracts derived from the unusually large nuclei of Xenopus oocytes. These extracts use nucleotide excision repair (NER) to completely remove bulky adducts from DNA. There is very little or no synthesis on control, undamaged DNA, indicating the extracts do not have significant nonspecific nuclease activity, and repair of cyclobutane pyrimidine dimers (CPDs) occurs in the dark, indicating that NER, and not photolyase, is responsible for CPD repair. The extracts can be inactivated with antibodies specific to repair proteins and then repair activity can be restored by adding purified recombinant protein. Here we describe detailed protocols for preparing Xenopus nuclear repair extracts.

Immobilization of the restriction endonuclease EcoRI usefulness of a polyclonal antibody support

The restriction enzyme EcoRI has been immobilized by using an immunoaffinity-based procedure on Sepharose 4B. The antibody support, prepared by coupling the γ-globulin fraction of serum of immunized rabbits to CNBr-activated Sepharose-4B, was highly effective in binding EcoRI from solution although only about half of the bound activity appeared to be expressed by the immobilized preparations. Restriction activity of EcoRI immobilized on the antibody support was indistinguishable from that of soluble enzyme on the linear λ-DNA and supercoiled plasmids pBR322 and pBHLUC P. Binding to the antibody support markedly enhanced the resistance of EcoRI to heat inactivation, and the preparation, unlike the native enzyme, retained significant activity after exposure to a temperature of 65°C. It was also possible to immobilize EcoRI directly from the cell lysates of Escherichia coli pMB4, an EcoRI overproducing strain. The immobilized preparation did not possess nonspecific nuclease activity that was prominent in the lysates, suggesting specificity in the binding of EcoRI to the antibody support.

DNA repair activity in protein extracts from rat tissues

Among DNA repair pathways, nucleotide excision repair (NER) is able to recognize and process a wide variety of DNA lesions. The NER mechanism can be summarized in two stages: incision/excision of the lesion and DNA repair synthesis. Here, we have assessed the repair synthesis activity of protein extracts from different rat tissues by an in vitro biochemical assay that reproduces the entire NER reaction. The protein extraction procedure was adapted to rat tissues and the biochemical parameters of the assay (high salt concentration, addition of EGTA) in order to minimize non-specific nuclease activity which allows the measurement of repair activity. Using this repair assay we detected a small increase in the extent of repair synthesis in liver compared to brain and lung tissue protein extracts. Similar results were obtained using a derivative assay that allows the measurement of the incision activity of tissue protein extracts with lower incision activity in lung tissue extract.

DNA and RNA strand scission by copper, zinc and manganese superoxide dismutases.

Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O(2-)-, to O2 and H2O2, play a major role in protecting cells from toxicity of oxidative stress. However, cells overexpressing either form of the enzyme show signs of toxicity, suggesting that too much SOD may be injurious to the cell. To elucidate the possible mechanism of this cytotoxicity, the effect of SOD on DNA and RNA strand scission was studied. High purity preparations of Cu/ZnSOD and MnSOD were tested in an in vitro assay in which DNA cleavage was measured by conversion of phage phi X174 supercoiled double-stranded DNA to open circular and linear forms. Both types of SOD were able to induce DNA strand scission generating single- and double-strand breaks in a process that required oxygen and the presence of fully active enzyme. The DNA strand scission could be prevented by specific anti-SOD antibodies added directly or used for immunodepletion of SOD. Requirement for oxygen and the effect of Fe(II) and Fe(III) ions suggest that cleavage of DNA may be in part mediated by hydroxyl radicals formed in Fenton-type reactions where enzyme-bound transition metals serve as a catalyst by first being reduced by superoxide and then oxidized by H2O2. Another mechanism was probably operative in this system, since in the presence of magnesium DNA cleavage by SOD was oxygen independent and not affected by sodium cyanide. It is postulated that SOD, by having a similar structure to the active center of zinc-containing nucleases, is capable of exhibiting non-specific nuclease activity causing hydrolysis of the phosphodiester bonds of DNA and RNA. Both types of SOD were shown to effectively cleave RNA. These findings may help explain the origin of pathology of certain hereditary diseases genetically linked to Cu/ZnSOD gene.

Efficient magnesium-dependent human immunodeficiency virus type 1 integrase activity

The integrase protein from human immunodeficiency virus type 1 (HIV-1) has generally been reported to require Mn2+ for efficient in vitro activity. We have reexamined the divalent metal ion requirements of HIV-1 integrase and find that the protein is capable of promoting efficient 3' processing and DNA strand transfer with either Mn2+ or Mg2+. The metal ion preference depended upon the reaction conditions. HIV-1 integrase displayed significantly less nonspecific nuclease activity in reaction mixtures containing Mg2+ than it did under the previously described reaction conditions with mixtures containing Mn2+.

A novel DNA binding and nuclease activity in domain III of Mu transposase: evidence for a catalytic region involved in donor cleavage.

The Mu A protein is a 75 kDa transposase organized into three structural domains. By severing the C-terminal region (domain III) from the remainder of the protein, we unmasked a novel non-specific DNA binding and nuclease activity in this region. Deletion analysis localized both activities to a 26 amino acid stretch (aa 575-600) which remarkably remained active in DNA binding and cleavage. The two activities were shown to be tightly linked by site-directed mutagenesis. To study the importance of these activities in the transposition process, an intact mutant transposase lacking the DNA binding and nuclease activity of domain III was constructed and purified. The mutant transposase was indistinguishable from wild-type Mu A in binding affinity for both the Mu ends and the enhancer, and in strand transfer activity when the cleavage step was bypassed. In contrast, the mutant transposase displayed defects in both synapsis and donor cleavage. Our results strongly suggest that the 26 amino acid region in domain III carries catalytic residues required for donor DNA cleavage by Mu A protein. Furthermore, our data suggest that an active site for donor cleavage activity in the Mu tetramer is assembled from domain II (metal ion binding) in one A monomer and domain III (DNA cleavage) in a separate A monomer. This proposal for active site assembly is in agreement with the recently proposed domain sharing model by Yang et al. (Yang, J.Y., Kim, K., Jayaram, M. and Harshey, R.M. [1995] EMBO J., 14, 2374-2384).

Isolation of SagI, a new HaeIII isoschizomer from Streptococcus agalactiae.

A new HaeIII isoschizomer from Streptococcus agalactiae was isolated by a single-step purification method. The highly active restriction endonuclease, SagI, was free of nonspecific nuclease activity and was suitable for use in molecular biology procedures. The rapid isolation procedure may be applicable for the recovery of other restriction endonucleases from bacteria.

From mutation mapping to phenotype cloning.

From mutation mapping to phenotype cloning.

Biochemical characterization of P22 phage-modified Escherichia coli RecBCD enzyme.

The biochemical properties of phage P22 Abc-modified RecBCD enzyme from Escherichia coli have been examined. RecBCD purified from a cell that expresses Abc (anti-RecBCD) contains all three RecBCD subunits and the 11.6-kDa Abc protein in equal stoichiometric amounts. Abc depresses the rate of RecBCD double-stranded DNA exonuclease, helicase, and ATPase activities about 3-4-fold, yet it has no effect on the rate of the single-stranded DNA exonuclease activity. Abc induces a slight increase in the ATP-independent single-stranded DNA endonuclease activity and does not induce dimerization of the RecBCD trimer. Abc-modified RecBCD helicase activity possesses reduced but significant processivity (10 kilobase pairs) relative to the native enzyme (30 kilobase pairs). In the absence of ATP, Abc-modified RecBCD shows a 2-4-fold higher affinity for double-stranded DNA ends. The RecBCD-binding Gam protein from bacteriophage lambda inhibits binding of both native and Abc-modified RecBCD to double-stranded DNA ends. Finally, unlike the native enzyme, the nonspecific nuclease activity of Abc-modified RecBCD is not suppressed by Chi sites in vitro. These findings are discussed in terms of the recombination-deficient phenotype of cells expressing Abc in vivo and the relationship between Abc-modified RecBCD and two mutant RecBCD's previously characterized: the RecBCD-K117Q and RecB2109CD mutant enzymes.

Rapid screening of lactic acid bacteria for restriction endonuclease activity

A rapid microscale heparin Sepharose CL-6B affinity gel procedure was developed for detecting restriction endonuclease (RE-Nase) activity in a variety of lactic acid bacteria. RE-Nase-containing extracts free of DNA, RNA and nonspecific nuclease activity can be produced for forty or more strains daily and only 10–12 ml of each log phase culture was required for screening. RE-Nase activity was detected in several streptococci and lactobacilli. With appropriate modifications, this procedure should allow rapid detection of RE-Nase activity in other bacterial species.

The Mutant recBCD Enzyme, recB 2109CD Enzyme, Has Helicase Activity but Does Not Promote Efficient Joint Molecule Formation in Vitro

The Escherichia coli recB 2109CD enzyme displays a defect in homologous recombination. In vitro, it possesses significant levels of non-specific nuclease activity but is deficient in χ-dependent nicking activity. To determine whether an alteration in helicase activity contributes further to its in vivo defect, the ability of recB 2109CD enzyme to unwind dsDNA was examined. The mutant enzyme is able to unwind DNA but has a k cat which is one-third that of the wild-type enzyme. While the K m for DNA ends of the wild-type and mutant enzymes at low NaCl concentration are essentially equivalent, the K m for ATP of recB 2109CD enzyme is nearly six times greater. The processivity of unwinding (i.e. the average length of DNA unwound before recB 2109CD enzyme dissociates from the DNA substrate) at 1 mM-Mg 2+ ion and 1 mM-ATP is approximately 13 kb/end, whereas that of wild-type recBCD enzyme is 30 kb/end. In an assay which requires the co-ordinate actions of the recBCD, recA, and SSB proteins, joint molecule formation in the presence of recB 2109CD enzyme is up to sixfold slower and proceeds to a lower extent than that mediated by the wild-type enzyme. We conclude that although the reduced helicase activity of the mutant recBCD enzyme may contribute to its recombination deficiency, its defect in the χ-dependent attenuation of non-specific nuclease activity is primarily responsible for the recombination-deficiency of E. coli strains bearing the recB2109 mutation.

Deoxyribonuclease activity in Streptococcus bovis

Deoxyribonuclease activity was tested with lambda bacteriophage DNA as a substrate in three Streptococcus bovis strains isolated from the rumen of sheep and cow. Non-specific nuclease activity was detected in the cell extract of Streptococcus bovis BM 114. Specific endonuclease activity was detected in the cell extract of the strain Streptococcus bovis II/1, isolated from the rumen of a sheep. A rapid technique is proposed for the detection of endonuclease activity of rumen bacteria

‘Star’ activity and complete loss of specificity of CeqI endonuclease

Restriction endonuclease CeqI, an isoschizomer of EcoRV, exhibits 'star' activity, a relaxation of specificity in the presence of Mn2+, dimethyl sulphoxide or glycerol. The enzyme cleaves a set of sequences that differ from the canonical GATATC by only one nucleotide in positions 2, 3, 4 or 5. Two of these sequences are not cleaved if modified by dam methylase. A further loss of specificity can be observed in circumstances less favourable for the enzyme, namely low-ionic-strength buffers of pH values below 6.0 or above 9.4. This activity seems to cleave DNA at any sequence, producing a smear instead of well-defined bands. Partial renaturation of the denatured enzyme gives rise to a similar non-specific nuclease activity.

An enzymatic activity in uninfected cells that cleaves the linkage between poliovirion RNA and the 5′ terminal protein

The 5′ terminal protein (VPg) on poliovirion RNA can be removed by cell-free extracts from a variety of uninfected cells. This soluble enzymatic activity is found in both nuclear and cytoplasmic extracts of HeLa cells and is activated by Mg ++. The enzyme activity cleaves the tyrosine-phosphate bond that links the protein to the RNA. In a partially purified form it has insufficient nonspecific protease or nuclease activity to account for its action. The existence of this enzyme implies that poliovirus RNA is translated in cell-free extracts in a form that lacks the 5′ terminal protein. The role of this enzyme in the uninfected cell is not known.

Physical study of prophage excision and curing of λ prophage from lysogenic Escherichia coli

A sex factor, F′450(λ), which can be isolated as a covalent circle of DNA, has been examined by alkaline sucrose gradient centrifugation of lysates of induced cells in order to study λ prophage excision. Thermal derepression of the prophage results in loss of F′450(λ) covalent circles, which is mediated by systems involved in excision and initiation of replication. When protocols known to result in prophage curing are used, the F′450(λ) is converted to an F′450 and a λ covalent circle; in normal excision leading to phage development, F′450 covalent circles are not found. We have shown that: (1) excision usually occurs later than initiation of DNA replication of the prophage so that the excised prophage is usually already replicated or in the act of replication; (2) the DNA growing points of the prophage leave the prophage and enter the bacterial DNA; (3) the int and xis genes are involved in the earliest detectable stage of the excision process, i.e. breakage of the DNA at the attachment region; (4) the xis gene product is involved in a weak non-specific nuclease activity in addition to its highly specific activity in excision; and (5) the excision system fails to attack a single attachment site.

Mechanism of action of specific soluble inhibitors of protein synthesis in differentiated cells

We have previously isolated from reticulocyte pH 5 fraction a soluble protein which inhibited serumalbumin cell free synthesis and from liver pH 5 fraction a protein which inhibited hemoglobin synthesis. None of these proteins act on the homologous system. These proteins acted neither on aminoacyl tRNA synthesis nor on the release and assembly of polypeptide chains. They are devoid of any non-specific nuclease activity. When reticulocyte RNA was incubated with liver inhibitor and purified, it retained its specific stimulatory activity on hemoglobin synthesis. This excludes the possibility that the inhibitor has acted by a specific nuclease activity on messenger RNA. An evidence was given of a binding of reticulocyte inhibitor with liver RNAs and preferentially with RNAs which sediment in the region of the messenger RNAs. Some binding was also observed with a viral RNA. These inhibitors may play a role in the selection of messenger RNAs in differentiated cells in which certain genes are transcribed but not translated into proteins. Nous avons isolé de la fraction pH 5 de réticulocyte une protéine soluble qui inhibe la synthèse acellulaire de sérumalbumine et de la fraction pH 5 de foie une protéien qui inhibe la synthèse acellulaire d'hémoglobine. Aucune de ces deux protéines n'agit sur le système homologue. Ces inhibiteurs n'agissent ni sur la synthèse des aminoacyl tRNA synthétases, ni sur la libération et l'union des chaînes polypeptidiques. Ils sont dépourvus d'activité ribonucléasiques non spécifique. Si l'on incube du RNA de réticulocyte avec l'inhibiteur de foie et qu'on purifie cet RNA, on constate qu'il a conservé le pouvoir de stimuler spécifiquement la synthèse de l'hémoglobine. Ceci permet d'exclure tout mécanisme impliquant une action nucléasique des inhibiteurs sur les RNA messangers. La démonstration directe d'une liaison entre l'inhibiteur de réticulocyte et le RNA de foie a été donnée. Cette liaison se fait préférentiellement avec les RNA qui sédimentent comme les RNA messagers. Une combinaison avec du RNA viral a également été observée. Ces inhibiteurs peuvent jouer un rôle par la sélection des RNA messagers dans les cellules différenciées chez lesquelles certains gènes sont transcrits mais non traduits en protéines. Wir haben aus der pH 5 — Fraktion der Retikulozyten ein lösliches Protein getrennt, welches die zellenlose Synthese des Serumalbumins hemmt, und aus der pH 5 — Fraktion der Leber ein Protein isoliert, welches die zellenlose Synthese des Hämoglobins inhibiert. Keines dieser beiden Proteine wirkt auf das homologe System. Diese Inhibitoren wirken weder auf die Synthese der Aminoacyl-tRNA-Synthetasen noch auf das Freisetzen und die Kopplung der Polypeptidketten. Sie weisen keine aspezifische Ribonuklease-Aktivität auf Wenn man die Retikulozyt-RNA mit dem Leberinhibitor inkubiert und wenn man diese RNA reinigt, so beobachtet man, dass sie das spezifische Anregungsvermögen in der Hämoglobinsynthese beibehalten hat. Dadurch wird jeder Mechanismus, der auf einer Nukleasewirkung der Inhibitoren auf die Messenger-RNA begründet wäre, ausgeschlossen. Der direkte Nachweis einer Bindung zwischen dem Retikulozytinhibitor und der Leber-RNA ist gegeben worden. Diese Bindung erfolgt vorzugsweise mit den RNA, welche wie die Messenger-RNA sedimentieren. Eine Bindung mit Viren-RNA ist ebenfalls beobachtet worden. Diese Inhibitoren können eine Rolle in der Selektion der Messenger-RNA in differenzierten Zellen, bei welchen gewisse Gene « umgeschriebenaber nicht zu Proteinen « übersetztwerden, spielen.