Purification and properties of an endonuclease from the mitochondrion of Saccharomyces cerevisiae

An endonuclease, which is found only in the mitochondrion of the yeast Saccharomyces cerevisiae, has been purified. The protein has a sedimentation coefficient of 6.3 S, equivalent to a molecular weight of 105,000. The enzyme is active at pH 7.6, when it degrades single-stranded DNA about 10-times faster than double-stranded DNA, but at pH 5.4 only double-stranded DNA is degraded. In both cases the enzyme acts endonucleolytically, breaking a single phosphodiester bond at a random location within the DNA substrate. Mn2+ or Mg2+ are required for activity; Ca2+ and Zn2+ are ineffective cofactors. Enzyme activity at pH 7.6 is severely inhibited by low concentrations of NaCl or KCl, while activity at pH 5.4 is unaffected by salt. Ethidium bromide inhibits both the DNase activity at pH 5.4 and the activity with single-stranded DNA at pH 7.6, but has no effect on the DNase activity with double-stranded DNA at pH 7.6.     

Purification and characterization of Mycoplasma penetrans Ca2+/Mg2+-dependent endonuclease.

The major nuclease from Mycoplasma penetrans has been purified to homogeneity. The enzyme seems to be present as a membrane-associated precursor of 50 kDa and as a peripheral membrane monomeric polypeptide of 40 kDa that is easily removed by washing of cells with isotonic buffers and in the aqueous phase upon Triton partitioning of Triton X-114-solubilized protein. The 40-kDa nuclease was extracted from M. penetrans cells by Triton X-114 and phase fractionation and was further purified by chromatography on Superdex 75 and chelating Sepharose (Zn2+ form) columns. By gel filtration, the apparent molecular mass was 40 kDa. The purified enzyme exhibits both a nicking activity on superhelical and linear double-stranded DNA and a nuclease activity on RNA and single-stranded DNA. No exonuclease activity was found for this enzyme. This nuclease required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity and exhibited a pH optimum between pH 7 and 8 for DNase activity. It was inhibited by Zn2+, Mn2+, heparin, sodium dodecyl sulfate, and chelator agents such EDTA and EGTA, but no effect was observed with ATP, 2-mercaptoethanol, N-ethylmaleimide, dithiothreitol, nonionic detergents, phenylmethylsulfonyl fluoride, and iodoacetamide. Nuclease activity was inhibited by diethylpyrocarbonate at both pH 6 and 8 and by pepstatin, suggesting the involvement of a histidine and an aspartate in the active site. When added to human lymphoblast nuclei, the purified M. penetrans endonuclease induced internucleosomal fragmentation of the chomatin into oligonucleosomal fragments. On the basis of this result, and taking into account the fact that M. penetrans has the capacity to invade eucaryotic cells, one can suggest, but not assert, that produced Ca2+/Mg2+-dependent endonuclease may alter the nucleic acid metabolism of host cells by DNA and/or RNA degradation and may act as a potential pathogenic determinant.     

A second form of the single-strand specific endonuclease of Neurospora crassa which is associated with a double-strand exonuclease.

A second form of single-strand specific endonuclease, which is stable to heating up to 74 degrees C and does not bind strongly to phosphocellulose, has been partially purified from extracts of mycelia of wild-type Neurospora crassa. The endonuclease is associated with an equally heat-stable exonuclease which degrades linear but not circular double-stranded DNA and does not attack double-stranded RNA. The exonuclease probably also degrades single-stranded DNA. Both endonuclease and exonuclease activities are inhibited by 0.1-0.5 mM ATP. The exonuclease is preferentially inhibited by a variety of agents and preferentially inactivated by trypsin. A DNA-unwinding activity has also been detected in the nuclease preparation. Protease(s) present in the nuclease preparation destroy the DNA-unwinding and exonuclease activities on incubation at 37 degrees C, but do not affect the endonuclease activity. However, the heat-stability and chromatographic properties of the endonuclease are affected by this treatment. The altered properties of the endonuclease are very similar to those of the single-strand specific endonuclease which has been previously described. The combined nuclease activities of the unaltered preparational make up a putative recombination nuclease of N. crassa.     

Alteration by site-directed mutagenesis of the conserved lysine residue in the consensus ATP-binding sequence of the RecB protein of Escherichia coli.

The RecB and RecD subunits of the RecBCD enzyme of Escherichia coli contain amino acid sequences similar to a consensus mononucleotide binding motif found in a large number of other enzymes. We have constructed by site-directed mutagenesis a lysine-to-glutamine mutation in this sequence in the RecB protein. The mutant enzyme (RecB-K29Q-CD) has essentially no nuclease or ATP hydrolysis activity on double-stranded DNA, showing the importance of RecB for unwinding double-stranded DNA. However, ATP hydrolysis stimulated by single-stranded DNA is reduced by only about 5-8-fold compared to the wild-type, nuclease activity on single-stranded DNA is reduced by less than 2-fold, and the nuclease activity of the RecB-K29Q-CD enzyme requires ATP. The effects of the RecB mutation suggest that the RecD protein hydrolyzes ATP and can stimulate the RecBCD enzyme nuclease activity on single-stranded DNA.     

The old exonuclease of bacteriophage P2.

The Old protein of bacteriophage P2 is responsible for interference with the growth of phage lambda and for killing of recBC mutant Escherichia coli. We have purified Old fused to the maltose-binding protein to 95% purity and characterized its enzymatic properties. The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA. The direction of digestion of double-stranded DNA is from 5' to 3', and digestion initiates at either the 5'-phosphoryl or 5'-hydroxyl terminus. The nuclease is active on nicked circular DNA, degrades DNA in a processive manner, and releases 5'-phosphoryl mononucleotides.     

Extracellular nucleases of Lysobacter enzymogenes: production of the enzymes and purification and characterization of an endonuclease

Lysobacter enzymogenes produced a nonspecific extracellular nuclease and an extracellular RNAase when grown in tryptone broth. Both enzyme activities appeared after the exponential growth phase of the organism. The addition of RNA to the medium specifically inhibited the production of the nuclease and the addition of phosphate prevented the synthesis of the RNAase. DNA had no effect on the enzyme production. The Lysobacter nuclease was purified 274-fold and its molecular weight was estimated to be between 22 000 and 28 000. Freshly purified nuclease showed one major protein band and one major activity band on polyacrylamide gels, whereas two major bands were seen after prolonged storage of the enzyme. The nuclease was most active at pH 8.0 and required Mg2+ or Mn2+. Little activity was obtained in the presence of Ca2+. The enzyme degraded double-stranded DNA more rapidly than single-stranded DNA or RNA and was essentially inactive with poly(A) or poly(C) as the substrate. Extensive hydrolysis of double-stranded DNA by the enzyme yielded oligodeoxyribonucleotides with terminal 5'-phosphate groups. The Lysobacter RNAase appeared to have a molecular weight approximately twice that of the nuclease and was specific for ribonucleotide polymers.     

Purification and characterization of an endoribonuclease from nucleoplasm and nucleoli of HeLa cells.

An endoribonuclease has been isolated from HeLa cell nuclei. Approximately 70% of the enzyme appears to be nucleolar bound; 30% is in the nucleoplasm. Studies of the purified enzyme reveal that the enzyme is an endonuclease of estimated molecular weight 16,000. It produces oligonucleotides bearing 5'-phosphate end groups. The enzyme degrades poly(C) and poly(U), as well as rRNA and heterogeneous nuclear RNA, Poly(A), double-stranded RNA, and DNA are not cleaved. The enzyme is heat-labile and is inhibited by 10mM Mg2+ and 50 mM NaCl. The enzyme is probably distinct from previously described nuclear endonucleases.     

An ATP-inhibited endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence: purification and characterization

In our studies on the role of enzymes in plant DNA replication, recombination, and repair, we isolated from cauliflower (Brassica oleracea L. var. botrytis) inflorescences a single-stranded DNA-specific endonuclease that was inhibited by ATP. The endonuclease, designated cauliflower nuclease II, was purified to near homogeneity through six successive column chromatographies. The enzyme is a single polypeptide with a molecular mass of 70 kDa as judged by the results of sodium dodecyl sulfate-polyacry amide gel electrophoresis, activity gel, and gel-filtration column chromatography. The enzyme can cleave a linear or a circular single-stranded DNA but cannot cut or nick a double-stranded DNA. The mode of activity of the nuclease is endonucleolytic and non-processive. Interestingly, the endonuclease activity is strongly inhibited by less than 0.1 mM ATP, although the role of this inhibition is thus far unclear. While ATPγS and GTP can also inhibit the activity, other ribonucleoside triphosphates are much less effective. The optimum pH of the enzyme is 5.6. The enzyme requires an exceptionally high ionic strength, 0.2 M KCI for optimum activity, and without these ions no activity can be detected. The endonuclease activity is stimulated by Ca²⁺, which cannot be replaced by Mg²⁺ or Mn²⁺. The features of the enzyme and its relation to plant DNA metabolism are discussed. Received: 26 March 1998 / Accepted: 4 June 1998     

Purification and properties of two deoxyribonucleases of Pseudomonas aeruginosa.

A survey of the major deoxyribonucleases in Pseudomonas aeruginosa strain PAO was undertaken. Two activities predominated in Brij-58 lysates of this organism. These have been purified from contaminating nuclease activities, and some of their properties have been elucidated. The first was a nuclease that degraded heat-denatured deoxyribonucleic acid (DNA) to mono- and dinucleotides. The activity of this enzyme was confined to single-stranded DNA, and 100% of the substrate was hydrolyzed to acid-soluble material. The Mg2+ optimum is low (1 to 3mM), and the molecular weight is 6 X 10(4). The second predominant activity was an adenosine 5'-triphosphate (ATP)-dependent deoxyribonuclease. This enzyme had an absolute dependence on the presence of ATP Mg2+ concentrations of approximately 10 mM. Five moles of ATP was consumed for each mole of phosphodiester bonds cleaved. The acid-soluble products of the reaction consisted of short oligonucleotides from one to six bases in length. Only 50% of the double-stranded DNA was rendered acid soluble in a limit digest. The molecular weight of this enzyme is 3 X 10(5). The observation of these enzymes in P. aeruginosa is consistent with the possibility that recombinational pathways similar to those of Escherichia coli are operating in this organism.     

Purification and properties of a novel nucleolar exoribonuclease capable of degrading both single-stranded and double-stranded RNA

A ribonuclease that hydrolyzes either linear duplex or single-stranded RNA in an exonucleolytic manner has been partially purified from Ehrlich ascites tumor cell nucleoli and is free from other ribonucleases. The enzyme will also degrade the RNA complement of an RNA X DNA duplex; however, no nuclease activity is observed on linear duplex or single-stranded DNA. The exonuclease acts on RNA nonprocessively from the 3' end releasing 5'-mononucleotides. The enzyme has a broad pH optimum around pH 8.0, requires Mg2+ or Mn2+ (0.06 mM) for optimum activity, and is sensitive to ethylenediaminetetraacetic acid and N-ethylmaleimide inhibition. Monovalent cations including K+, Na+, and NH4+ are inhibitory. Gel filtration studies of this enzyme gave a Stokes radius of 40 A. Sedimentation velocity measurements in glycerol gradients yield a S20,W of 6.0 S. From these values a native molecular weight of 100 000 was calculated. Copurification of the single- and double-stranded activities, identical reaction requirements, and identical heat-inactivation curves strongly suggest that both activities reside with the same enzyme.     

A 3' to 5' exonuclease activity is associated with phage 029 DNA polymerase

Bacteriophage 029 produces its own DNA polymerase which is encoded by gene 2 [Watabe, K. and Ito, J. (1983) Nucleic Acid Res. 11, 8333]. This 029 DNA polymerase has been purified by phospho-cellulose, DEAE-cellulose, double-stranded DNA cellulose chromatography and glycerol gradient centrifugation. An exonuclease activity associated with the DNA polymerase was found through all the steps of the purification. This nuclease preferably degrades single-stranded DNA from the 3' to the 5' terminus direction, suggesting that the enzyme plays a role for proofreading during DNA replication. While DNA polymerase activity isolated from cells infected with temperature sensitive mutant of gene 2 is thermolabile, the nuclease activity is not significantly reduced at the restrictive temperature.     

Purification and properties of a single strand-specific endonuclease from mouse cell mitochondria.

A nuclease was purified from mitochondria of the mouse plasmacytoma cell line, MCP-11 which acts on single-stranded DNA endonucleolytically and appears to have no activity upon native DNA. It degrades unordered RNA somewhat more effectively than it does DNA. The enzyme activity and the major detectable polypeptide migrate to a position corresponding to an Mr of 37,400 on denaturing polyacrylamide gels; in its native form the activity has an S value of 4.7, which corresponds to a molecular weight of roughly 73,000. The single-strand DNase activity has a pH optimum near 7.5, requires a divalent cation and is inhibited by EDTA, phosphate, KCl and NaCl. The enzyme is remarkably similar to fungal mitochondrial enzymes whose absence in various mutants correlates with defective DNA repair and recombination. It reacts weakly with antibody to a form of such an enzyme from Neurospora crassa.     

Purification and characterisation of a DNA helicase, dheI I, from Drosophila melanogaster embryos.

We have purified a DNA helicase (dhel l) from early Drosophila embryos. dhel l co-purifies with the single-stranded DNA binding protein dRP-A over two purification steps, however, the proteins can be separated by their different native molecular weight, with dhel l activity co-sedimenting with a polypeptide of approximately 200 kDa and a sedimentation coefficient of 8.6 S. The enzyme needs ATP hydrolysis and divalent cations for displacement activity. It is very salt sensitive, having a Mg2+ optimum of 0.5 mM and being inhibited by NaCl concentration > 10 mM. Dhel l moves 5'-->3' on the DNA strand to which it is bound. Unwinding activity decreases with increasing length of the double-stranded region suggesting a distributive mode of action. However, addition of dRP-A to the displacement reaction stimulates the activity on substrates with >300 nucleotides double-stranded region suggesting a specific interaction between these two proteins.     

DNA helicase IV from HeLa cells.

Human DNA helicase IV, a novel enzyme, was purified to homogeneity from HeLa cells and characterized. The activity was measured by assaying the unwinding of 32P labeled 17-mer annealed to M13 ss DNA. From 440g of HeLa cells we obtained 0.31 mg of pure protein. Helicase IV was free of DNA topoisomerases, DNA ligase and nuclease activities. The apparent molecular weight is 100 kDa. It requires a divalent cation for activity (Mg2+ = Mn2+ = Zn2+) and the hydrolysis of only ATP or dATP. The activity is destroyed by trypsin and is inhibited by 200 mM KCl or NaCl, 100 mM potassium phosphate, 45 mM ammonium sulfate, 5 mM EDTA, 20 microM ss M13 DNA or 20 microM poly [G] (as phosphate). The enzyme unwinds DNA by moving in the 5' to 3' direction along the bound strand, a polarity opposite to that of the previously described human DNA helicase I (Tuteja et al Nucleic Acids Res. 18, 6785-6792, 1990). It requires more than 84 bases of single-stranded DNA in order to exert its unwinding activity and does not require a replication fork-like structure. Like human DNA helicase I the enzyme can also unwind RNA-DNA hybrid.     

Partial purification and characterization of a double-stranded RNA-specific nuclease from human placenta

A double-stranded RNA-specific nuclease (ds RNase) has been isolated and partially purified from human placenta by DEAE-cellulose and DNA-cellulose column chromatography. Denatured DNA-cellulose retained most of the single-stranded RNA-specific nuclease (ss RNase) activity, whereas the ds RNase came out in the void volume. N-ethylmaleimide at a concentration of 5 mM, selectively inhibited ds RNase activity by 60% under the conditions in which the ss RNase activity was inhibited to an extent of 7%. The ds RNase was specifically inhibited by Penicillium chrysogenum viral ds RNA and by ethidium bromide. The partially purified ds RNase showed requirements for Mg+ whereas Mn2+ and NH4+ ions were inhibitory. The DEAE-enzyme cleaved 32P-labelled 45S ribosomal precursor RNAs from Yoshida ascites sarcoma cells into species that had similar electrophoretic mobilities as the mature rRNAs.     

S1 nuclease of Aspergillus oryzae: a glycoprotein with an associated nucleotidase activity

S₁ nuclease (EC 3.1.30.1) of Aspergillus oryzae has been purified 1600-fold by a procedure designed to remove traces of contaminating phosphatases. The nearly homogeneous enzyme was found to be a glycoprotein with a carbohydrate content of 18%. At pH 4.5 the enzyme preparation hydrolyzed single-stranded DNA, RNA, 3′-AMP, and 2′-AMP at relative rates of 100, 52, 13, and 0.05, respectively. The 3′-nucleotidase activity of this single-strand specific nuclease is inhibited by single-stranded DNA but not by double-stranded DNA. Three forms of the enzyme, with isoelectric points of 3.35, 3.53, and 3.67, were observed on electrofocusing, and each form exhibited the same relative activity on single-stranded DNA and 3′-AMP. Enzymatic hydrolysis of nucleotides occurred over a broad range of pH, with maximal activity at pH 6–7. Ribonucleotides were hydrolyzed approximately 100-fold more rapidly than deoxyribonucleotides. A high degree of base specificity was not observed. The 3′-nucleotidase activity was stimulated by Zn²⁺, but not by other divalent cations tested.     

A mouse DNA repair enzyme (APEX nuclease) having exonuclease and apurinic/apyrimidinic endonuclease activities: purification and characterization.

A mouse repair enzyme having priming activity on bleomycin-damaged DNA for DNA polymerase was purified to apparent homogeneity and characterized. The enzyme extracted from permeabilized mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M potassium phosphate buffer (pH 7.5) was purified by successive chromatographies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time), Sephadex G-100, single-stranded DNA cellulose and hydroxyapatite. The purified enzyme has an Mr of 34,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzymatical studies indicated that it is a multifunctional enzyme having exonuclease, apurinic/apyrimidinic endonuclease and phosphatase activities, similar to Escherichia coli exonuclease III. This enzyme is tentatively designated as APEX nuclease for apurinic/apyrimidinic endonuclease and exonuclease activities. The amino acid composition, amino-terminal amino acid sequence and an internal amino acid sequence of APEX nuclease are determined.     

Purification and properties of the major nuclease from mitochondria of Saccharomyces cerevisiae

The vast majority of nuclease activity in yeast mitochondria is due to a single polypeptide with an apparent molecular weight of 38,000. The enzyme is located in the mitochondrial inner membrane and requires non-ionic detergents for solubilization and activity. A combination of heparin-agarose and Cibacron blue-agarose chromatography was employed to purify the nuclease to approximately 90% homogeneity. The purified enzyme shows multiple activities: 1) RNase activity on single-stranded, but not double-stranded RNA, 2) endonuclease activity on single- and double-stranded DNA, and 3) a 5'-exonuclease activity on double-stranded DNA. Digestion products with DNA contain 5'-phosphorylated termini. Antibody raised against an analogous enzyme purified from Neurospora crassa (Chow, T. Y. K., and Fraser, M. (1983) J. Biol. Chem. 258, 12010-12018) inhibits and immunoprecipitates the yeast enzyme. This antibody inhibits 90-95% of all nuclease activity present in solubilized mitochondria, indicating that the purified nuclease accounts for the bulk of mitochondrial nucleolytic activity. Analysis of a mutant strain in which the gene for the nuclease has been disrupted supports this conclusion and shows that all detectable DNase activity and most nonspecific RNase activity in the mitochondria is due to this single enzyme.     

A single nuclease active site of the Escherichia coli RecBCD enzyme catalyzes single-stranded DNA degradation in both directions

The RecBCD enzyme of Escherichia coli is an ATP-dependent DNA exonuclease and a helicase. Its exonuclease activity is subject to regulation by an octameric nucleotide sequence called chi. In this study, site-directed mutations were made in the carboxyl-terminal nuclease domain of the RecB subunit, and their effects on RecBCD's enzymatic activities were investigated. Mutation of two amino acid residues, Asp(1067) and Lys(1082), abolished nuclease activity on both single- and double-stranded DNA. Together with Asp(1080), these residues compose a motif that is similar to one shown to form the active site of several restriction endonucleases. The nuclease reactions catalyzed by the RecBCD enzyme should therefore follow the same mechanism as these restriction endonucleases. Furthermore, the mutant enzymes were unable to produce chi-specific fragments that are thought to result from the 3'-5' and 5'-3' single-stranded exonuclease activities of the enzyme during its reaction with chi-containing double-stranded DNA. The results show that the nuclease active site in the RecB C-terminal 30-kDa domain is the universal nuclease active site of RecBCD that is responsible for DNA degradation in both directions during the reaction with double-stranded DNA. A novel explanation for the observed nuclease polarity switch and RecBCD-DNA interaction is offered.