Purification and characterization of an endo-exonuclease from Podospora anserina mitochondria

The senescence phenotype of Podospora anserina wild-type strains depends on mitochondrial (mt) genome stability. Characterization of activities implicated in the maintenance of the mt DNA is therefore essential for a better understanding of these degenerative processes. To address this question we looked for a nuclease activity in this fungal mitochondria. Here we describe the purification of an endo-exonuclease active on single-stranded, double-stranded and flap DNA. The Podospora nuclease also possesses an RNase H activity. Gel filtration chromatography showed a native molecular mass of 90 kDa for the P. anserina enzyme. The highly purified fraction shows a single polypeptide chain of 49 kDa on SDS-PAGE, indicating that the Podospora enzyme is probably active as a dimer. Purification and sequencing of the endolysine digestion peptides of the Podospora mt nuclease suggested that this enzyme could belong to the 5' structure-specific endo-exonuclease family. The possible involvement of this nuclease in mt DNA recombination during the senescence process is evoked.     

RNase P activity in the mitochondria of Saccharomyces cerevisiae depends on both mitochondrion and nucleus-encoded components.

A requisite step in the biosynthesis of tRNA is the removal of 5' leader sequences from tRNA precursors. We have detected an RNase P activity in yeast mitochondrial extracts that can carry out this reaction on a homologous precursor tRNA. This mitochondrial RNase P was sensitive to both micrococcal nuclease and protease, demonstrating that it requires both a nucleic acid and protein for activity. The presence of RNase P activity in vitro directly correlated with the presence of a locus on yeast mitochondrial DNA previously shown by genetic and biochemical studies to be required for tRNA maturation. The product of the locus, the 9S RNA, and this newly described mitochondrial RNase P activity cofractionated, providing further evidence that the 9S RNA is the RNA component of yeast mitochondrial RNase P.     

Mammalian mitochondrial endonuclease G. Digestion of R-loops and localization in intermembrane space.

Mammalian mitochondria contain strong nuclease activity. Endonuclease G (endoG), which predominantly resides in mitochondria, accounts for a large part of this nuclease activity. It has been proposed to act as an RNase H-like nuclease on RNA.DNA hybrids (R-loops) in the D-loop region where the origins of mitochondrial replication are mapped, providing RNA primers for mtDNA replication. However, in contrast with this proposed activity, endoG has recently been shown to translocate to nuclei on apoptotic stimulation and act as a nuclease without sequence specificity. To clarify the role of endoG in mtDNA replication, we examined its submitochondrial localization and its ability to cleave R-loops. At low concentration, it preferentially produces double-stranded breaks in R-loops, but does not act as an RNase H-like nuclease. In addition, it exists in the mitochondrial intermembrane space, but not in the matrix where mtDNA replication occurs. These results do not support the involvement of endoG in mtDNA replication. Based on the fact that guanine tracts, which are preferential targets of endoG, tend to form triplex structures and that endoG produces double-stranded breaks in R-loops, we propose that three-stranded DNA may be the preferred substrate of endoG.     

A gene required for RNase P activity in Candida (Torulopsis) glabrata mitochondria codes for a 227-nucleotide RNA with homology to bacterial RNase P RNA.

We have mapped a gene in the mitochondrial DNA of Candida (Torulopsis) glabrata and shown that it is required for 5' end maturation of mitochondrial tRNAs. It is located between the tRNAfMet and tRNAPro genes, the same tRNA genes that flank the mitochondrial RNase P RNA gene in the yeast Saccharomyces cerevisiae. The gene is extremely AT rich and codes for AU-rich RNAs that display some sequence homology with the mitochondrial RNase P RNA from S. cerevisiae, including two regions of striking sequence homology between the mitochondrial RNAs and the bacterial RNase P RNAs. RNase P activity that is sensitive to micrococcal nuclease has been detected in mitochondrial extracts of C. glabrata. An RNA of 227 nucleotides that is one of the RNAs encoded by the gene that we mapped cofractionated with this mitochondrial RNase P activity on glycerol gradients. The nuclease sensitivity of the activity, the cofractionation of the RNA with activity, and the homology of the RNA with known RNase P RNAs lead us to propose that the 227-nucleotide RNA is the RNA subunit of the C. glabrata mitochondrial RNase P enzyme.     

Purification of a site-specific endonuclease, I-Sce II, encoded by intron 4 alpha of the mitochondrial coxI gene of Saccharomyces cerevisiae

We have purified to near homogeneity a site-specific, double-stranded DNA endonuclease (I-Sce II) encoded by intron 4 alpha (aI4 alpha) of the yeast mitochondrial coxI gene. Our purification starts with a high salt extract of mitochondria isolated from a yeast strain that overproduces the enzyme because of a block in splicing of aI4 alpha. The final step of purification is an affinity column consisting of covalently bound double-stranded DNA multimers of a synthetic sequence, 5'-TTGGTCATCCAGAAGTAT-3', which contains the I-Sce II cleavage/recognition site. Typical yields of enzyme are 3-5% with a specific activity of approximately 500,000 units/mg, where 1 unit of activity cleaves 50 ng of DNA substrate/h at 30 degrees C. I-Sce II has a monomer molecular mass of 31 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Active enzyme purifies as a 55-kDa species, which we presume to be a homodimer. I-Sce II monomer comigrates with an in vivo synthesized mitochondrial translation product made in the strain that overproduces the enzyme. We conclude that I-Sce II is derived by proteolytic processing of a precursor polypeptide, p62, encoded by an in-frame fusion of coxI exons 1-4 with the downstream aI4 alpha reading frame. I-Sce II is most active at pH 7.5 and at 20-30 degrees C. Endonuclease activity is sensitive to salt and is dependent upon Mg2+ or Mn2+, but is unaffected by inclusion of ATP or GTP. I-Sce II is the first intron-encoded protein to be purified and characterized from yeast mitochondria.     

Yeast DNA polymerases: antigenic relationship, use of RNA primer and associated exonuclease activity.

Highly purified preparation of DNA polymerases A and B from yeast were compared with respect to antigenic relationship, ability to use ribonucleotide primers and associated nuclease activity. The following results were obtained. 1. Antiserum directed against DNA polymerase A inhibits this enzyme but does not interfere with activity of DNA polymerase B or of mitochondrial DNA polymerase, nor does it precipitate the latter two enzymes. 2. DNA polymerase A is capable of using oligo(ribouridylic acid) as a primer for the polymerization of dTMP. This reaction is not catalyzed by polymerase B to any significant extent. 3. Whereas DNA polymerase A is devoid of nuclease activity, DNA polymerase B catalyses an exonucleolytic release of mononucleotide units from the 3' end of polynucleotides. The results of several experiments suggest that this nuclease activity is associated with the DNA polymerase B molecule.     

The DNase activity of RNase T and its application to DNA cloning.

RNase T is one of eight distinct 3'-->5' exoribonucleases present in Escherichia coli. The enzyme plays an important role in stable RNA metabolism, including tRNA end turnover and 3' maturation of most stable RNAs because it is the only RNase that can efficiently remove residues near a double-stranded (ds) stem. In the course of study of its specificity and mechanism, we found that RNase T also has single-strand-specific DNase activity. Purified RNase T degrades both single-stranded (ss)RNA and ssDNA in a non-processive manner. However, in contrast to its action on RNA, RNase T binds ssDNA much more tightly and shows less sequence specificity. As with RNA, DNA secondary structure strongly affects its degradation by RNase T. Thus, RNase T action on a dsDNA with a single-stranded 3'-extension efficiently generates blunt-ended DNA. This property of RNase T suggested that it might be a useful enzyme for blunt-ended DNA cloning. We show here that RNase T provides much higher cloning efficiency than the currently used mung bean nuclease.     

Purification and characterization of an endo-exonuclease from adult flies of Drosophila melanogaster.

An endo-exonuclease (designated nuclease III) has been purified to near homogeneity from adult flies of Drosophila melanogaster. The enzyme degrades single- and double-stranded DNA and RNA. It has a sedimentation co-efficient of 3.1S and a strokes radius of 27A The native form of the purified enzyme appears to be a monomer of 33,600 dalton. It has a pH optimum of 7-8.5 and requires Mg2+ or Mn2+ but not Ca2+ or Co2+ for its activity. The enzyme activity on double-stranded DNA was inhibited 50% by 30 mM NaCl, while its activity on single-stranded DNA required 100 mM NaCl for 50% inhibition. Under the latter conditions, its activity on double-stranded DNA was inhibited approximately 98%. The enzyme degrades DNA to complete acid soluble products which are a mixture of mono- and oligonucleotides with 5'-P and 3'-OH termini. Supercoiled DNA was converted by the enzyme to nicked and subsequently to linear forms in a stepwise fashion under the condition in which the enzyme works optimally on single-stranded DNA. The amino acid composition and amino acid sequencing of tryptic peptides from purified nuclease III is also reported.     

The purification from rat liver of a nuclease hydrolysing ribonucleic acid and deoxyribonucleic acid

1. The purification of a nuclease from rat-liver mitochondria is described. The mitochondria are rendered soluble by treatment with Triton X-100 and, after fractionation with ammonium sulphate and acetone, the active fraction is further purified by chromatography on DEAE-cellulose and Sephadex G-75 to give a purification of over 700-fold. 2. The purified enzyme was only very slightly contaminated with deoxyribonuclease II, phosphodiesterase and phosphomonoesterase. The individual activities of these enzymes did not exceed 0.1% of the activity of the liver nuclease. 3. The purified enzyme attacked RNA more rapidly than denatured DNA and hydrolysed native DNA more slowly than denatured DNA. 4. There is some evidence to suggest that the nucleolytic activity of the purified preparation towards native DNA, denatured DNA and RNA is associated with a single protein. 5. The enzyme is relatively labile but is stabilized in the presence of 20% (w/v) glycerol or 10mm-2-mercaptoethanol.     

Characterization of alkaline nuclease from rat liver mitochondria.

The alkaline nuclease (pH optimum 9.0) has been purified about 500-fold in 25% yield from the extract of rat liver mitochondria. The enzyme cleaves yeast RNA, poly(U), poly(U), poly(C) and denatured DNA to yield oligonucleotides with 5'-phosphoryl and 3'-hydroxyl ends. The enzyme has a molecular weight of about 60 000, a sedimentation coefficient of 4 S and an isoelectric point of 9.0. The behaviors of RNAase activity of the nuclease are identical with those of DNAase activity in column chromatography as well as in catalytic nature. The affinities of RNAase activity for substrate, Mg2+, spermidine and polyvinyl sulfate are lower than those of DNAase activity. The alkaline nuclease activity measured in the homogenate of regenerating rat liver is not significantly changed.     

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.     

The Enzyme and the cDNA Sequence of a Thermolabile and Double-Strand Specific DNase from Northern Shrimps (Pandalus borealis)

Background

We have previously isolated a thermolabile nuclease specific for double-stranded DNA from industrial processing water of Northern shrimps (Pandalus borealis) and developed an application of the enzyme in removal of contaminating DNA in PCR-related technologies.

Methodology/Principal Findings

A 43 kDa nuclease with a high specific activity of hydrolysing linear as well as circular forms of DNA was purified from hepatopancreas of Northern shrimp (Pandalus borealis). The enzyme displayed a substrate preference that was shifted from exclusively double-stranded DNA in the presence of magnesium to also encompass significant activity against single-stranded DNA when calcium was added. No activity against RNA was detected. Although originating from a cold-environment animal, the shrimp DNase has only minor low-temperature activity. Still, the enzyme was irreversibly inactivated by moderate heating with a half-life of 1 min at 65°C. The purified protein was partly sequenced and derived oligonucleotides were used to prime amplification of the encoding cDNA. This cDNA sequence revealed an open reading frame encoding a 404 amino acid protein containing a signal peptide. By sequence similarity the enzyme is predicted to belong to a family of DNA/RNA non-specific nucleases even though this shrimp DNase lacks RNase activity and is highly double-strand specific in some respects. These features are in agreement with those previously established for endonucleases classified as similar to the Kamchatka crab duplex-specific nuclease (Par_DSN). Sequence comparisons and phylogenetic analyses confirmed that the Northern shrimp nuclease resembles the Par_DSN-like nucleases and displays a more distant relationship to the Serratia family of nucleases.

Conclusions/Significance

The shrimp nuclease contains enzyme activity that may be controlled by temperature or buffer compositions. The double-stranded DNA specificity, as well as the thermolabile feature, strengthens its potential for in vitro applications.     

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.     

A novel nuclease from mitochondria of rat liver

Alkaline RNase partially purified from rat liver mitochondria hydrolyzes both RNA and denatured DNA. The behaviors of RNase activity of the nuclease are closely similar to those of the DNase activity. The nuclease has a pH optimum between 9.0 and 9.5, and the activity is absolutely dependent on Mg²⁺ and reversibly inhibited by $\text{p}$-hydroxymercuribenzoate.     

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.     

Studies of transcription in isolated wheat mitochondria and organelle extracts

RNA synthesis has been studied in purified wheat embryo mitochondria and in a mitochondrial lysate depleted of endogeneous DNA. Both systems work optimally at pH 8.5 and at 25°C. Magnesium is a better cofactor than manganese; moreover, in purified mitochondria the latter cation seems to abolish the stimulatory effect of magnesium. The RNA polymerase activity present in the mitochondrial lysate is inhibited strongly by heparin and actinomycin D while alpha-amanitin and rifampicin do not affect the enzyme activity. A low molecular weight form (50 kDa) as well as a larger form (150 kDa) of the wheat mitochondrial RNA polymerase have been obtained by gel filtration. Several DNA templates can be used by the mitochondrial lysate; the best template seems to be poly d(AT) but the mitochondrial DNAs from yeast and wheat, as well as single stranded M13 DNA, can be used efficiently as templates. Multiple RNA species, between 1 and 6 kb in size, were synthesized ‘in organello’.