Localization of a cruciform cutting endonuclease to yeast mitochondria

We have found a cruciform cutting endonuclease in the yeast, Saccharomyces cerevisiae, which localizes to the mitochondria. This activity apparently is associated with the mitochondrial inner membrane since the activity is not released into solution by osmolysis, in contrast to the matrix enzyme, isocitrate dehydrogenase. The cruciform cutting activity appears to be encoded by CCE1. This gene has been shown to encode one of the major cruciform cutting endonucleases present in a yeast cell. In ccel strains, which lack CCE1 endonuclease activity, the mitochondrial cruciform cutting endonucleolytic activity is also absent. Since CCE1 is allelic to MGT1, a gene required for the highly biased transmission of petite mitochondrial DNA in crosses between ⁺ and hypersuppressive ^– cells, it seems likely that the CCE1 endonuclease functions within mitochondria.     

Stable association of 70-kDa heat shock protein induces latent multisite specificity of a unisite-specific endonuclease in yeast mitochondria.

The multisite-specific endonuclease Endo.SceI of yeast mitochondria is unique among endonucleases because its 50-kDa subunit forms a stable dimer with the mitochondrial 70-kDa heat shock protein (mtHSP70), which otherwise fulfills a chaperone function by binding transiently to unfolded proteins. Here we show that the mtHSP70 subunit confers broader sequence specificity, greater stability, and higher activity on the 50-kDa subunit. The 50-kDa subunit alone displayed weaker activity and highly sequence-specific endonuclease activity. The 50-kDa protein exists as a heterodimer with mtHSP70 in vivo, allowing Endo.SceI to cleave specifically at multiple sites on mitochondrial DNA. Endo.SceI may have evolved from a highly specific endonuclease that gained broader sequence specificity after becoming a stable partner of mtHSP70.     

Multi-site-specific endonucleases and the initiation of homologous genetic recombination in yeast

The notion that homologous recombination is a regulated biological process is not a familiar one. In yeasts, homologous recombination and most site-specific ones are initiated by site-specific double-stranded breaks that are introduced within cis-acting elements for the recombination. On the other hand, yeasts have a group of site-specific endonucleases (multi-site-specific endonucleases) that have a number of cleavage sites on each DNA. One of them, Endo.SceI of S. cerevisiae, was shown to introduce double-stranded breaks at a number of welldefined sites on the mitochondrial DNA in vivo. An Endo.SceI-induced double-stranded break was demonstrated to induce homologous recombination in mitochondria. Like the case of homologous recombination of nuclear chromosomes, the double-stranded break induces gene conversion of both genetic markers flanking and in the proximity of the cleavage site, and the cleaved DNA acts as a recipient of genetic information from the uncleaved partner DNA. The 70 kDa-heat-shock protein (HSP70)-subunit of Endo.SceI and a general role of the HSP70 in the regulation of protein-folding suggest the regulation of nucleolytic activity of Endo.SceI.     

A maturase-like subunit of the sequence-specific endonuclease endo.SceI from yeast mitochondria

Some yeast strains possess a sequence-specific endonuclease, Endo.SceI, which is a heterodimeric enzyme localized in mitochondria. The larger subunit (75 kDa) of Endo.SceI, encoded by a nuclear gene (ENS1), is transported from the cytosol into the mitochondria. In this study, we determined the partial amino acid sequence of the smaller subunit (50 kDa) of Endo.SceI. The determined sequence matched well the partial sequence deduced from a mitochondrial open reading frame (RF3). The RF3 locus is known to exhibit polymorphism since this reading frame in some yeast strains is supposed to encode a maturase-like protein, whereas in other strains, the frame is interrupted by GC clusters, which thus break the frame. Southern blot analysis of various yeast strains showed that the continuity of RF3 is correlated with the presence of Endo.SceI activity. These data indicate that the continuous RF3 sequence is a functional gene (ENS2) coding for the smaller subunit of Endo.SceI. The results of cytoduction, by which the continuous RF3 sequence was transferred into a yeast strain lacking mitochondrial DNA, confirmed this conclusion. This study suggests the involvement of Endo.SceI in genetic recombination of mitochondrial DNA.     

A subunit of yeast site-specific endonuclease SceI is a mitochondrial version of the 70-kDa heat shock protein

Endo.SceI of Saccharomyces cerevisiae is a heterodimeric site-specific endonuclease, which is distinguishable from prokaryotic restriction endonucleases in the mode of recognition of its cleavage site. We have used monoclonal antibodies specific to the larger subunit (75 kDa) of Endo.SceI to isolate the gene for the subunit (ENS1) from S. cerevisiae. Unexpectedly, ENS1 was found to encode a 70-kDa heat shock protein-related polypeptide and to be identical to recently cloned SSC1. Subcellular fractionation experiments on yeast cells revealed that the primary target site of the larger subunit is mitochondria, where almost all the Endo.SceI activity is localized. Molecular genetic analysis of ENS1 demonstrated its indispensability for growth and the requirement of a high level of its expression at the sporulation and germination stages. The data suggest that ENS1 plays an important role, especially at these differentiation stages.     

Purification of a eukaryotic site-specific endonuclease, Endo.Sce I, from Saccharomyces cerevisiae and effectors on its specificity and activity

A site-specific endonuclease (Endo.Sce I) which caused double-strand scission of DNA was highly purified from a eukaryote, Saccharomyces cerevisiae IAM4274. The molecular weight of the active form of Endo.Sce I was estimated to be 120,000 and 110,000 by sedimentation analysis on a glycerol density gradient and gel filtration on Ultrogel AcA34, respectively. Analysis of the fractions from the last column chromatography by polyacrylamide gel-electrophoresis in the presence of sodium dodecyl sulfate and by an assay of the endonucleolytic activities suggested that Endo.Sce I consists of two non-identical subunits with molecular weights of 75,000 and 50,000. Unlike restriction endonucleases, Endo.Sce I was active on chromosomal DNA of the cells which produced Endo.Sce I. Single-stranded DNA was not cleaved by Endo.Sce I, but inhibited the endonucleolytic activity of the enzyme on double-stranded DNA. The endonucleolytic activity of Endo.Sce I required the magnesium ions (Mg2+) as a sole cofactor; Mg2+ could not be replaced by Ca2+ or Zn2+. When Mg2+ was replaced by manganese ions (Mn2+), extensively purified Endo.Sce I cleaved double-stranded DNA at many other sites in addition to the sites at which DNA was cleaved in the presence of Mg2+. Experiments indicated that this is not the activation of contaminating endonuclease in the preparation of Endo.Sce I, but the result of relaxation in the site-specificity of cleavage.     

Two new site-specific endonucleases from Staphylococcus species strain D5

Staphylococcus species strain D5 containing two site-specific endonucleases, SspD5 I and SspD5 II, was found during screening of a bacterial strain collection from soil. These endonucleases were purified to functional homogeneity by sequential chromatography. Site-specific endonuclease SspD5 I recognizes sequence 5;-GGTGA(8N/8N) downward arrow-3; on DNA. Unlike Hph I, it cleaves DNA at a distance of 8 nucleotides from the recognized sequence on both chains producing blunt-end DNA fragments, while endonuclease Hph I cleaves DNA forming mononucleotide 3;-OH protruding ends. Thus, endonuclease SspD5 I is a new type II site-specific endonuclease and a neoschizomer of endonuclease Hph I. The advantage of this new endonuclease is that the blunt-end DNA products of this enzyme can be inserted without additional treatment into vector DNAs cleaved with endonucleases yielding DNA blunt-ends. Endonuclease SspD5 II recognizes site 5'-ATGCA T-3' and thus is an isoschizomer of endonuclease Nsi I. The molecular mass of SspD5 I is about 35 kD and that of SspD5 II is 40 kD. The enzymes exhibit maximal activity at 37 degrees C. The optimal buffer for the reaction is HRB (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM NaCl, and 1 mM dithiothreitol).     

A new specific DNA endonuclease activity in yeast mitochondria

Summary Two group I intron-encoded proteins from the yeast mitochondrial genome have already been shown to have a specific DNA endonuclease activity. This activity mediates intron insertion by cleaving the DNA sequence corresponding to the splice junction of an intronless strain. We have discovered in mitochondrial extracts from the yeast strain 777-3A a new DNA endonuclease activity which cleaves the fused exon A3-exon A4 junction sequence of the COXI gene.     

An endonuclease with multiple cutting sites, Endo.SceI, initiates genetic recombination at its cutting site in yeast mitochondria.

Endo.SceI is a mitochondrial sequence-specific endonuclease which has multiple cutting sites. In order to examine the possible role of Endo.SceI in homologous recombination, we analyzed the mode of recombination upon mating using antibiotic resistance markers on the mitochondrial genome. The segregation of a marker located very close to one of the Endo.SceI cutting sites showed a disparity (polarized segregation, i.e. gene conversion). This gene conversion depended on the presence of the functional Endo.SceI gene. In vivo cutting of mitochondrial DNA upon mating was detected at the cutting site in the antibiotic marker region, which also depended on the Endo.SceI activity. These results suggest that mitochondrial recombination is induced by cleavage of mitochondrial DNA by this sequence-specific endonuclease. This is the first demonstration that a sequence-specific endonuclease with multiple cutting sites induces genetic recombination.     

Functional screening of a soil metagenome for DNA endonucleases by acquired resistance to bacteriophage infection

Endonucleases play a crucial role as reagents in laboratory research and diagnostics. Here, metagenomics was used to functionally screen a fosmid library for endonucleases. A fosmid library was constructed using metagenomic DNA isolated from soil sampled from the unique environment of the Kogelberg Nature Reserve in the Western Cape of South Africa. The principle of acquired immunity against phage infection was used to develop a plate-based screening technique for the isolation of restriction endonucleases from the library. Using next-generation sequencing and bioinformatics tools, sequence data were generated and analysed, revealing 113 novel open reading frames (ORFs) encoding putative endonuclease genes and ORFs of unknown identity and function. One endonuclease designated Endo52 was selected from the putative endonuclease ORFs and was recombinantly produced in Escherichia coli Rosetta? (DE3) pLysS. Endo52 was purified by immobilised metal affinity chromatography and yielded 0.437 g per litre of cultivation volume. Its enzyme activity was monitored by cleaving lambda DNA and pUC19 plasmid as substrates, and it demonstrated non-specific endonuclease activity. In addition to endonuclease-like genes, the screen identified several unknown genes. These could present new phage resistance mechanisms and are an opportunity for future investigations.

     

Isolation and partial purification of a novel type II restriction endonuclease Bsu121 I,from Bacillus subtilis – Bsu121I,a type II restriction endonuclease from Bacillus subtilis

A new type II restriction endonuclease which we designated as Bsu121I has been isolated from gram-positive bacterium Bacillus subtilis strain 121 and partially purified. The restriction endonuclease was isolated from cell extracts using step-wise purification through ammonium sulfate precipitation, followed by phosphocellulose column chromatography. SDS-PAGE profile showed denatured molecular weights (23 and 67 kDa) of the endonuclease. The partially purified enzyme restricted pBR322 DNA into two fragments of 3200 and 1700 bp. The endonuclease activity required Mg⁺² as cofactor like other type II endonucleases.     

A mutation in a mitochondrial ABC transporter results in mitochondrial dysfunction through oxidative damage of mitochondrial DNA

We have isolated a Saccharomyces cerevisiae mutant that shows an increased tendency to form cytoplasmic petites (respiration-deficient ρ⁻ or ρ⁰ mutants) in response to treatment of cells growing on a solid medium with the DNA-damaging agent methyl methanesulfonate or ultraviolet light. The mutation in this strain, atm1-1, was found to cause a single amino acid substitution in ATM1, a nuclear gene that encodes the mitochondrial ATP-binding cassette (ABC) transporter. When the mutant cells were grown in liquid glucose medium, they accumulated free iron within the mitochondria and at the same time gave rise to spontaneous cytoplasmic petite mutants, as seen previously in cells carrying a mutation in a gene homologous to the human gene responsible for Friedreich's ataxia. Analysis of the effects of free iron and malonic acid (an inhibitor of oxidative respiration in mitochondria) on the incidence of petites among the mutant cells indicated that spontaneous induction of petites was a consequence of oxidative stress rather than a direct effect of either a defect in the ATM1 gene or the accumulation of free iron. We observed an increase in the incidence of strand breaks in the mitochondrial DNA of the atm1-1 mutant cells. Furthermore, we found that rates of induction of petites and accumulation of strand breaks in mitochondrial DNA were enhanced in the atm1-1 mutant by the introduction of another mutation, mhr1-1, which results in a deficiency in mitochondrial DNA repair. These observations indicate that spontaneous induction of petites in the atm1-1 mutant is a consequence of oxidative damage to mitochondrial DNA mediated by enhanced accumulation of mitochondrial iron. Received: 26 March 1999 / Accepted: 29 June 1999     

A new class of site-specific endodeoxyribonucleases. Endo.Sce I isolated from a eukaryote, Saccharomyces cerevisiae

We had found that yeasts had intracellular endodeoxyribonucleases that cut phage DNA into a set of double-stranded fragments with discrete chain lengths. We purified one of them to apparent homogeneity from Saccharomyces cerevisiae and designated it Endo.Sce I. Sequence analysis around 5 cleavage sites in plasmid DNA and phage DNA revealed that Endo.Sce I cuts a defined phosphodiester bond in each strand of double helix at the cleavage sites and produces free cohesive ends consisting of 4 nucleotides protruding at 3'-termini. However, unlike in the case of prokaryotic type II-restriction endonucleases, (i) Endo.Sce I seems to consist of two nonidentical subunits, (ii) no common palindrome or consensus sequence including more than 5 base pairs is detected at or near these cleavage sites, and (iii) Endo.Sce I can cut the DNA isolated from the cells that produced Endo.Sce I. All of the 5 cleavage sites are included in inverted repeats, but these inverted repeats are variable in size, nucleotide sequence, and distance between repeating units. An inverted repeat itself is not a structure recognized by Endo.Sce I. This study shows that Endo.Sce I is the first example of eukaryotic site-specific endonuclease and has properties, as described above, which distinguish it from prokaryotic restriction endonucleases.     

Characterization of the I-Spom I endonuclease from fission yeast: insights into the evolution of a group I intron-encoded homing endonuclease

The first group I intron in the cox1 gene (cox1I1b ) of the mitochondrial genome of the fission yeast Schizosaccharomyces pombe is a mobile DNA element. The mobility is dependent on an endonuclease protein that is encoded by an intronic open reading frame (ORF). The intron-encoded endonuclease is a typical member of the LAGLIDADG protein family of endonucleases with two consensus motifs. In addition to this, analysis of several intron mutants revealed that this protein is required for intron splicing. However, this protein is one of the few group I intron-encoded proteins that functions in RNA splicing simultaneously with its DNA endonuclease activity. We report here on the biochemical characterization of the endonuclease activity of this protein artificially expressed in Escherichia coli. Although the intronic ORF is expressed as a fusion protein with the upstream exon in vivo, the experiments showed that a truncated translation product consisting of the C-terminal 304 codons of the cox1I1b ORF restricted to loop 8 of the intron RNA secondary structure is sufficient for the specific endonuclease activity in vitro. Based on the results, we speculate on the evolution of site-specific homing endonucleases encoded by group I introns in eukaryotes.     

Mammalian mitochondrial endonuclease activities specific for ultraviolet-irradiated DNA.

Mitochondrial forms of uracil DNA glycosylase and UV endonuclease have been purified and characterized from the mouse plasmacytoma cell line, MPC-11. As in other cell types, the mitochondrial uracil DNA glycosylase has properties very similar to those of the nuclear enzyme, although in this case the mitochondrial activity was also distinguishable by extreme sensitivity to dilution. Three mitochondrial UV endonuclease activities are also similar to nuclear enzymes; however, the relative amounts of these enzyme activities in the mitochondria is significantly different from that in the nucleus. In particular, mitochondria contain a much higher proportion of an activity analogous to UV endonuclease III. Nuclear UV endonuclease III activity is absent from XP group D fibroblasts and XP group D lymphoblasts have reduced, but detectable levels of the mitochondrial form of this enzyme. This residual activity differs in its properties from the normal mitochondrial form of UV endonuclease III, however. The presence of these enzyme activities which function in base excision repair suggests that such DNA repair occurs in mitochondria. Alternatively, these enzymes might act to mark damaged mitochondrial genomes for subsequent degradation.     

Fractionation and purification of DNA methylases and specific endonucleases from cells of Escherichia coli SK]

Fractionation and purification of DNA methylases and specific endonucleases from E. coli SK responsible for DNA specificity to host prokaryotic cells were studied. The most efficient purification was achieved by precipitation of proteins by 0.6 saturated ammonium sulfate with subsequent chromatography on KM-cellulose and concentration of fractions by dialysis against glycerol. Under these conditions the methylase activity produced 4 discrete fractions. Due to purification the specific activity of methylases increased 11--20-fold in various fractions. Methylase from the first (A) and fourth (BII) peaks catalyzed the methylation of cytosine to produce 5-methylcytosine; methylase from the third peak (BI) methylated adenine to produce 6-methylaminopurine. The chemical specificity of the second peak (B) methylase could not be established due to very high lability of the enzyme in this fraction. Specific endonuclease was found in the gradient zones eluted by 0.1--0.2 M and 0.65--0.75 M NaCl. It is assumed that those enzymes providing for DNA hydrolysis up to the formation of high--molecular discrete fragments, are restricting endonucleases of the SK system. The results obtained strongly suggest the existence of several types of methylases and restricting endonucleases in E. coli SK cells.     

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.     

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.     

Abasic site recognition by two apurinic/apyrimidinic endonuclease families in DNA base excision repair: the 3' ends justify the means

DNA damage occurs unceasingly in all cells. Spontaneous DNA base loss, as well as the removal of damaged DNA bases by specific enzymes targeted to distinct base lesions, creates non-coding and lethal apurinic/apyrimidinic (AP) sites. AP sites are the central intermediate in DNA base excision repair (BER) and must be processed by 5' AP endonucleases. These pivotal enzymes detect, recognize, and cleave the DNA phosphodiester backbone 5' of, AP sites to create a free 3'-OH end for DNA polymerase repair synthesis. In humans, AP sites are processed by APE1, whereas in yeast the primary AP endonuclease is termed APN1, and these enzymes are the major constitutively expressed AP endonucleases in these organisms and are homologous to the Escherichia coli enzymes Exonuclease III (Exo III) and Endonuclease IV (Endo IV), respectively. These enzymes represent both of the conserved 5' AP endonuclease enzyme families that exist in biology. Crystal structures of APE1 and Endo IV, both bound to AP site-containing DNA reveal how abasic sites are recognized and the DNA phosphodiester backbone cleaved by these two structurally unrelated enzymes with distinct chemical mechanisms. Both enzymes orient the AP-DNA via positively charged complementary surfaces and insert loops into the DNA base stack, bending and kinking the DNA to promote flipping of the AP site into a sequestered enzyme pocket that excludes undamaged nucleotides. Each enzyme-DNA complex exhibits distinctly different DNA conformations, which may impact upon the biological functions of each enzyme within BER signal-transduction pathways.     

Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells

Summary The molecular size of mitochondrial DNA (mtDNA) molecules and the number of copies of mtDNA per mitochondrion were evaluated from cultured cells of the tobacco BY-2 line derived fromNicotiana tabacum L. cv. Bright Yellow-2. To determine the DNA content per mitochondrion, protoplasts of cultured cells were stained with 4,6-diamidino-2-phenylindole (DAPI), and the intensity of the fluorescence emitted from the mitochondrial nuclei (mt-nuclei) was measured with a video-intensified photon counting microscope system (VIM system). Each mitochondrion except for those undergoing a division contained one mt-nucleus. The most frequently measured size of the DNA in the mitochondria was between 120 and 200 kilobase pairs (kbp) throughout the course of culture of the tobacco cells. Mitochondria containing more than 200 kbp of DNA increased significantly in number 24 h after transfer of the cells into fresh medium but their number fell as the culture continued. Because division of mitochondria began soon after transfer of the cells into fresh medium and continued for 3 days, the change of the DNA content per mitochondrion during the culture must correspond to DNA synthesis of mitochondria in the course of mitochondrial division. By contrast, the analyses of products of digestion by restriction endonucleases indicated that the genome size of the mtDNA was at least 270 kbp. Electron microscopy revealed that mtDNAs were circular molecules and their length ranged from 1 to 35 m, and 60% of them ranged from 7 to 11 rn. These results indicate that the mitochondrial genome in tobacco cells consists of multiple species of mtDNA molecules, and mitochondria do not contain all the mtDNA species. Therefore, mitochondria are heterogeneous in mtDNA composition.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - mtDNA mitochondrial DNA - mt-genome mitochondrial genome - mt-nucleus mitochondrial nucleus - ptDNA proplastid DNA - pt-nucleus proplastid nucleus - VIM system video-intensified photon counting microscope system