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.     

Partial characterization of ribonuclease (RNase) activity from the isolated and solubilized brush border of Hymenolepis diminuta

The isolated brush border membrane of Hymenolepis diminuta contained ribonuclease (RNase) activity which was demonstrable using yeast RNA or synthetic homopolymers of adenylic, cytidylic, inosinic, or uridylic acids as substrates. Polyguanylic acid was not hydrolyzed by worm RNase. RNase activity was inhibited by EDTA and divalent cations as well as sulfhydryl blocking and reducing agents. Polyguanylic acid and DNA were also inhibitors of RNase activity; these compounds were not hydrolyzed, but inhibited the hydrolysis of other substrates, possibly by nonproductive substrate binding. Data suggested that RNase (endonuclease) was probably the major enzyme activity in the degradation of long chain polyribonucleotides at the work's surface, while phosphodiesterase (exonuclease) activity did not contribute significantly to the hydrolysis of these compounds.     

A developmentally regulated Streptomyces endoribonuclease resembles ribonuclease E of Escherichia coli

We report that the Streptomyces species S. lividans and S. coelicolor , morphologically complex Gram-positive soil bacteria, contain a developmentally regulated endoribonuclease activity (here named RNase ES) that functionally and immunologically resembles Escherichia coli RNase E. In Streptomyces cells, RNA I — the antisense repressor of replication of ColE1-type plasmids — is cleaved at sites attacked by RNase E. A Mg²⁺-dependent endonuclease that produces RNase E-like cleavages in RNA I and 9S ribosomal RNA was identified in S. lividans cell extracts. A Streptomyces peptide migrating at 70 kDa in SDS/polyacrylamide gels binds to RNase E substrates and reacts with three separate anti-RNase E monoclonal antibodies; the endonucleolytic cleavage activity co-purified with the immunoreactive 70 kDa peptide. We show that RNase ES activity is regulated during the Streptomyces life cycle: activity increased as cells progressed from exponential growth to stationary phase in liquid culture, or from mycelial growth to sporulation on solid media. While mutations that interfere with S. coelicolor development late in its life cycle did not prevent this developmentally associated increase in RNase ES activity, the increase was blocked by a mutation ( bldA ) that interferes early with both morphological and physiological differentiation.     

Escherichia coli rna gene encoding RNase I: cloning, overexpression, subcellular distribution of the enzyme, and use of an rna deletion to identify additional RNases.

The cloning and overexpression of the Escherichia coli rna gene encoding RNase I are described. Only a single copy of the rna gene is present on the E. coli chromosome. Although cells with as much as a 100-fold increase in RNase I activity were constructed, little effect on cell growth was observed. Overexpressed RNase I was found in the periplasmic space to the same degree (approximately 85%) as wild-type enzyme, suggesting no limitation in RNase I transport. The rna clone was used to identify a deletion strain totally lacking the rna gene. The normal growth of this strain showed that RNase I is not essential for cell viability. Extracts from the RNase I deletion strain still retained a low level of RNase activity in the presence of EDTA, conclusively demonstrating the existence of additional EDTA-active RNases in E. coli. The possibility of a RNase I inhibitor is also discussed.     

E. coli RNase I exhibits a strong Ca2+-dependent inherent double-stranded RNase activity

Since its initial characterization, Escherichia coli RNase I has been described as a single-strand specific RNA endonuclease that cleaves its substrate in a largely sequence independent manner. Here, we describe a strong calcium (Ca²⁺)-dependent activity of RNase I on double-stranded RNA (dsRNA), and a Ca²⁺-dependent novel hybridase activity, digesting the RNA strand in a DNA:RNA hybrid. Surprisingly, Ca²⁺ does not affect the activity of RNase I on single stranded RNA (ssRNA), suggesting a specific role for Ca²⁺ in the modulation of RNase I activity. Mutation of a previously overlooked Ca²⁺ binding site on RNase I resulted in a gain-of-function enzyme that is highly active on dsRNA and could no longer be stimulated by the metal. In summary, our data imply that native RNase I contains a bound Ca²⁺, allowing it to target both single- and double-stranded RNAs, thus having a broader substrate specificity than originally proposed for this traditional enzyme. In addition, the finding that the dsRNase activity, and not the ssRNase activity, is associated with the Ca²⁺-dependency of RNase I may be useful as a tool in applied molecular biology.     

Studies on an Endonuclease Activity Associated with the Bacteriophage T7 DNA-Membrane Complex

Infection of Escherichia coli with bacteriophage T7 results in the formation of an endonuclease which is selectively associated with the T7 DNA-membrane complex. A specificity of association with the complex is indicated by the finding that the enzyme is completely resolved from a previously described T7 endonuclease I. When membrane complexes containing (3)H-labeled in vivo synthesized DNA are incubated in the standard reaction mixture a specific cleavage product is formed which is about one-fourth the size of T7 DNA. The endonuclease associated with the complex produces a similar cleavage product after extensive incubation with native T7 DNA or T7 concatemers. Degradation of concatemers occurs by a mechanism in which the DNA is converted to molecules one-half the size of T7. This product is in turn converted to fragments one-fourth the size of mature phage DNA. The endonuclease is not present in membrane complexes from uninfected cells or cells infected with gene 1 mutants. The enzyme activity is, however, present in cells infected with mutants defective in T7 DNA synthesis or maturation.     

New sequence-specific human ribonuclease: purification and properties.

A new sequence-specific RNase was isolated from human colon carcinoma T84 cells. The enzyme was purified to electrophoretical homogeneity by pH precipitation, HiTrapSP and Superdex 200 FPLC. The molecular weight of the new enzyme, which we have named RNase T84, is 19 kDa. RNase T84 is an endonuclease which generates 5'-phosphate-terminated products. The new RNase selectively cleaved the phosphodiester bonds at AU or GU steps at the 3' side of A or G and the 5' side of U. 5'AU3' or 5'GU3' is the minimal sequence required for T84 RNase activity, but the rate of cleavage depends on the sequence and/or structure context. Synthetic ribohomopolymers such as poly(A), poly(G), poly(U) and poly(C) were very poorly hydrolysed by T84 enzyme. In contrast, poly(I) and heteroribopolymers poly(A,U) and poly(A,G,U) were good substrates for the new RNase. The activity towards poly(I) was stronger in two colon carcinoma cell lines than in three other epithelial cell lines. Our results show that RNase T84 is a new sequence-specific enzyme whose gene is abundantly expressed in human colon carcinoma cell lines.     

Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death).

Cell death by apoptosis occurs in a wide range of physiological events including repertoire selection of lymphocytes and during immune responses in vivo. A hallmark of apoptosis is the internucleosomal DNA degradation for which a Ca2+,Mg(2+)-dependent endonuclease has been postulated. This nuclease activity was extracted from both rat thymocyte and lymph node cell nuclei. When incubated with nuclei harbouring only limited amounts of endogenous nuclease activity, the ladder pattern of DNA fragments characteristic of apoptosis was induced. This extractable nucleolytic activity was immunoprecipitated with antibodies specific for rat deoxyribonuclease I (DNase I) and was inhibited by actin in complex with gelsolin segment 1, strongly pointing to the presence of a DNase I-type enzyme in the nuclear extracts. COS cells transiently transfected with the cDNA of rat parotid DNase I expressed the enzyme, and their nuclei were able to degrade their DNA into oligosome-sized fragments. PCR analysis of mRNA isolated from thymus, lymph node cells and kidney yielded a product identical in size to that from rat parotid DNase I. Immunohistochemical staining with antibodies to rat DNase I confirmed the presence of DNase I antigen in thymocytes and lymph node cells. The tissue distribution of DNase I is thus extended to tissues with no digestive function and to cells which are known to be susceptible to apoptosis. We propose that during apoptosis, an endonuclease indistinguishable from DNase I gains access to the nucleus due to the breakdown of the ER and the nuclear membrane.     

RNase H is an exo- and endoribonuclease with asymmetric directionality,depending on the binding mode to the structural variants of RNA:DNA hybrids

RNase H is involved in fundamental cellular processes and is responsible for removing the short stretch of RNA from Okazaki fragments and the long stretch of RNA from R-loops. Defects in RNase H lead to embryo lethality in mice and Aicardi-Goutieres syndrome in humans, suggesting the importance of RNase H. To date, RNase H is known to be a non-sequence-specific endonuclease, but it is not known whether it performs other functions on the structural variants of RNA:DNA hybrids. Here, we used Escherichia coli RNase H as a model, and examined its catalytic mechanism and its substrate recognition modes, using single-molecule FRET. We discovered that RNase H acts as a processive exoribonuclease on the 3′ DNA overhang side but as a distributive non-sequence-specific endonuclease on the 5′ DNA overhang side of RNA:DNA hybrids or on blunt-ended hybrids. The high affinity of previously unidentified double-stranded (ds) and single-stranded (ss) DNA junctions flanking RNA:DNA hybrids may help RNase H find the hybrid substrates in long genomic DNA. Our study provides new insights into the multifunctionality of RNase H, elucidating unprecedented roles of junctions and ssDNA overhang on RNA:DNA hybrids.     

RNase F and 2′,5′-oligoA synthetase activities in mice after poly(I). poly(C) administration

The present study demonstrates the presence of considerable levels of 2′, 5′-oligoA synthetase activity in tissue extracts from mice. The interferon inducer, poly(I) .poly(C) , induced the synthetase activity in all the tissue extractsin vivo. Similarly, a significant amount of endonuclease RNase F activity is found to be present in these tissue extracts. But interferon induction does not seem to have any significant effect on RNase F activity.     

A Bridge Crosses the Active-Site Canyon of the Epstein-Barr Virus Nuclease with DNase and RNase Activities

Epstein-Barr virus, a double-stranded DNA (dsDNA) virus, is a major human pathogen from the herpesvirus family. The nuclease is one of the lytic cycle proteins required for successful viral replication. In addition to the previously described endonuclease and exonuclease activities on single-stranded DNA and dsDNA substrates, we observed an RNase activity for Epstein-Barr virus nuclease in the presence of Mn²⁺, giving a possible explanation for its role in host mRNA degradation. Its crystal structure shows a catalytic core of the D-(D/E)XK nuclease superfamily closely related to the exonuclease from bacteriophage lambda with a bridge across the active-site canyon. This bridge may reduce endonuclease activity, ensure processivity or play a role in strand separation of dsDNA substrates. As the DNA strand that is subject to cleavage is likely to make a sharp turn in front of the bridge, endonuclease activity on single-stranded DNA stretches appears to be possible, explaining the cleavage of circular substrates.     

NucA is required for DNA cleavage during transformation of Bacillus subtilis

We have re-examined the roles of nucA and nin, in the transformation of Bacillus subtilis as conflicting accounts have been presented concerning the importance of these genes for transformation. The present report demonstrates that nucA deficiency lowers the rate of DNA transport and that NucA is needed for the double-strand cleavage of transforming DNA, probably acting directly as an endonuclease. A relative paucity of DNA termini, resulting from the absence of this endonuclease activity, most probably accounts for the decreased transport rate. NucA is a bitopic integral membrane protein, with its C-terminus external to the membrane where it is appropriately located to effect the cleavage of bound transforming DNA. We have also investigated the roles of the known competence genes in the DNA processing that accompanies transformation in B. subtilis. The genes that are required for DNA transport (comEA, comEC and comFA) are also required for the degradation of the non-transforming strand that accompanies internalization, but comEC and comFA are not needed for the double-strand cleavage that occurs external to the cell membrane.     

Membrane location of a deoxyribonuclease implicated in the genetic transformation of Diplococcus pneumoniae.

The cellular localization of enzymes in Diplococcus pneumoniae was examined by fractionation of spheroplasts. A deoxyribonuclease implicated in the entry of deoxyribonucleic acid (DNA) into the cell during genetic transformation was located in the cell membrane. This enzyme, the major endonuclease of the cell (endonuclease I), which is necessary for the conversion of donor DNA to single strands inside the cell and oligonucleotides outside, thus could act at the cell surface. Another enzyme, the cell wall lysin (autolysin), was also found in the membrane fraction. Other enzymes, including amylomaltase, two exonucleases, and adenosine triphosphate-dependent deoxyribonuclease, and a restriction type endonuclease, were located in the cytosol within the cell. None of the enzymes examined were predominantly periplasmic in location. Spheroplasts were obtained spontaneously on incubation of pneumococcal cells in concentrated sugar solutions. The autolytic enzyme appears to be involved in this process. Cells that were physiologically competent to take up DNA formed osmotically sensitive spheroplasts two to three times faster than cells that were not in the competent state. Although some genetically incompetent mutants also formed spheroplasts more slowly, other such mutants formed them at the faster rate.     

Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells

Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the mitochondrial replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation.     

Association of the Folded Chromosome with the Cell Envelope of Escherichia coli: Nature of the Membrane-Associated DNA

Membrane-associated folded chromosomes isolated from Escherichia coli in the presence of spermidine sedimented at about 5,800S. The folded chromosome and the membrane fragment were each stable in the absence of the other; a 1,700S folded chromosome was obtained after removal of the membrane by a Sarkosyl treatment, and a 4,000S membrane fragment remained after digestion of the chromosomal DNA with deoxyribonuclease I. The interaction between the folded chromosome and the membrane fragment was stable, and, even when the DNA was unfolded, both components remained associated and cosedimented. The large frictional effect of the unfolded DNA reduced the sedimentation rate of the complex to about 2,000S. Partial removal of this unfolded DNA with restriction endonucleases caused the membrane fragments and the remaining associated DNA to sediment faster, at about 3,500S. The DNA remaining associated with the membrane fragments after restriction endonuclease treatment, about 4.5% of the total DNA when EcoRI was used, was indistinguishable from the DNA released from the membranes by three criteria: (i) DNA size distribution in agarose gels after electrophoresis, (ii) reassociation kinetics, and (iii) thermal elution from hydroxylapatite. This finding, that random DNA sequences rather than specific ones were responsible for the majority of the DNA-membrane interactions, argues against the folded chromosome's being a static structure with specific DNA sequences interacting with the cell envelope.     

Crystal structure of MutS2 endonuclease domain and the mechanism of homologous recombination suppression

DNA recombination events need to be strictly regulated, because an increase in the recombinational frequency causes unfavorable alteration of genetic information. Recent studies revealed the existence of a novel anti-recombination enzyme, MutS2. However, the mechanism by which MutS2 inhibits homologous recombination has been unknown. Previously, we found that Thermus thermophilus MutS2 (ttMutS2) harbors an endonuclease activity and that this activity is confined to the C-terminal domain, whose amino acid sequence is widely conserved in a variety of proteins with unknown function from almost all organisms ranging from bacteria to man. In this study, we determined the crystal structure of the ttMutS2 endonuclease domain at 1.7-angstroms resolution, which resembles the structure of the DNase I-like catalytic domain of Escherichia coli RNase E, a sequence-nonspecific endonuclease. The N-terminal domain of ttMutS2, however, recognized branched DNA structures, including the Holliday junction and D-loop structure, a primary intermediate in homologous recombination. The full-length of ttMutS2 digested the branched DNA structures at the junction. These results indicate that ttMutS2 suppresses homologous recombination through a novel mechanism involving resolution of early intermediates.