DNA sequence recognition by a eukaryotic sequence-specific endonuclease, Endo.SceI, from Saccharomyces cerevisiae

A eukaryotic sequence-specific endonuclease, Endo.SceI, causes sequence-specific double-stranded scission of double-stranded DNA to produce cohesive ends with four bases protruding at the 3' termini. Unlike in the case of restriction enzymes, an asymmetric 26-base pair consensus sequence was found around the cleavage site for Endo.SceI instead of a common sequence. We analyzed the base pairs that interacted with Endo.SceI on the recognition of its cleavage sites. A region comprising -10 through +16 base pairs from the center of the cleavage site was shown to be essential and sufficient for the sequence-specific cutting with Endo.SceI by experiments involving synthesized DNAs. Methylation interference experiments indicate that bases in the region comprising the +7 through +14 base pairs is involved in close contact with Endo.SceI in its recognition of the cleavage site. This +7 through +14-base pair region overlaps the most stringently conserved sequence in the consensus sequence for the cleavage site, suggesting that this region constitutes the core for the recognition by Endo.SceI.     

Sequence-specific complex formation of DNA and a eukaryotic sequence-specific endonuclease, SceI.

Endo.SceI is a eukaryotic sequence-specific endonuclease of 120 kDa that causes sequence-specific double-stranded scission of DNA. Unlike results with restriction enzymes, we found a consensus sequence around the cleavage sites for Endo.SceI instead of a common sequence. We searched for conditions for studying the binding of Endo.SceI to DNA other than cutting. Under optimized conditions including gel mobility shift assay, Endo.SceI exhibited sequence-specific binding to a short double-stranded DNA (41 base pairs) containing a cleavage site and the DNA reisolated from the protein-DNA complex was not cleaved. The analysis of the complex of Endo.SceI and DNA isolated by the gel mobility shift experiments showed that the DNA-binding entity in the Endo.SceI preparation does have Endo.SceI activity and consists of an equal amount of 75-kDa and 50-kDa polypeptides. Based on this observation and those from previous studies, we conclude that Endo.SceI is a heterodimer of the 75-kDa and 50-kDa subunits. Under the present assay conditions, Endo.SceI did not show binding to single-stranded DNA having the same sequence of either plus or minus strand of the double-stranded DNA containing the cleavage site (the 41-bp DNA). Endo.SceI showed significantly higher affinity for the consensus sequence than the major cleavage site in pBR322 DNA. Unlike the cleavage of DNA by Endo.SceI which requires Mg2+, this sequence-specific binding is independent of but stimulated by Mg2+.     

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.     

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.     

Endo.SK1: an inducible site-specific endonuclease from yeast mitochondria

Site-specific endonucleases have been found in various eukaryotic organelles such as mitochondria, chloroplasts and nuclei. These endonucleases initiate site-specific or homologous gene conversion in mitochondrial and nuclear DNA. Here, we report a new site-specific endonuclease activity, Endo.SK1, identified in mitochondria of strain SK1, a homothallic diploid strain ofSaccharomyces cerevisiae. Nucleotide sequences around the Endo.SK1-cleavage sites are different from those of known yeast site-specific endonucleases. The Endo.SK1 activity is, at least partly, specified by a gene in the SK1-derived mitochondria. A novel feature of the Endo.SK1 activity is its inducibility: the endonuclease activity was induced by ca. 40-fold by transfer of cells from a glucose medium into an acetate medium, and was then repressed. This transient induction was independent of the ploidy level of the cells, and coincided with induction of fumarase, a mitochondrial enzyme involved in the TCA cycle. Co-induction and co-repression of the mitochondrial site-specific endonuclease activity and a respiration-related enzyme indicate that the endonuclease activity is regulated in response to physiological conditions, and suggest a possible role for the endonuclease in mitochondrial DNA metabolism.     

Roles of the HSP70-subunit in a eukaryotic multi-site-specific endonuclease, Endo.SceI: autophosphorylation and heat stability

The 70 kDa heat shock proteins (HSP70) are a family of molecular chaperones that bind transiently to unfolded proteins in an ATP/ADP dependent manner. Endo.SceI comprises a unique example for mitochondrial HSP70, which exists in a stable complex with a nucleolytic subunit as a multi-site specific DNase. The HSP70-subunit in Endo.SceI was autophosphorylated by ATP in vitro. The autophosphorylation was higher in the Endo.SceI complex form than in the free form. Although the autophosphorylation had no significant effect on the endonucleolytic activity of Endo.SceI, the factors favoring autophosphorylation protected the endonucleolytic activity of Endo.SceI against heat inactivation. ATP, adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), and ADP not only protected the endonucleolytic activity against heat inactivation in the presence of Ca(2+) ions, but also reduced the labeling of the HSP70-subunit by [gamma-(32)P]ATP in Endo.SceI. These findings suggest that the HSP70-subunit shields Endo.SceI from heat inactivation through ATP/ADP binding.     

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.     

Double strand break-induced recombination in Chlamydomonas reinhardtii chloroplasts.

The mechanisms of chloroplast recombination are largely unknown. Using the chloroplast-encoded homing endonuclease I-CreI from Chlamydomonas reinhardtii, an experimental system is described that allows the study of double strand break (DSB)-induced recombination in chloroplasts. The I-CreI endonuclease is encoded by the chloroplast ribosomal group I intron of C.reinhardtii and cleaves specifically intronless copies of the large ribosomal RNA (23S) gene. To study DSB-induced recombination in chloroplast DNA, the genes encoding the I-CreI endonuclease were deleted and a target site for I-CreI, embedded in a cDNA of the 23S gene, was integrated at an ectopic location. Endonuclease function was transiently provided by mating the strains containing the recombination substrate to a wild-type strain. The outcome of DSB repair was analyzed in haploid progeny of these crosses. Interestingly, resolution of DSB repair strictly depended upon the relative orientation of the ectopic ribosomal cDNA and the adjacent copy of the 23S gene. Gene conversion was observed when the 23S cDNA and the neighbouring copy of the 23S gene were in opposite orientation, leading to mobilization of the intron to the 23S cDNA. In contrast, arrangement of the 23S cDNA in direct repeat orientation relative to the proximal 23S gene resulted in a deletion between the 23S cDNA and the 23S gene. These results demonstrate that C.reinhardtii chloroplasts have an efficient system for DSB repair and that homologous recombination is strongly stimulated by DSBs in chloroplast DNA.     

DNA packaging and cutting by phage terminases: Control in phage T4 by a synaptic mechanism

Phage DNA packaging occurs by DNA translocation into a prohead. Terminases are enzymes which initiate DNA packaging by cutting the DNA concatemer, and they are closely fitted structurally to the portal vertex of the prohead to form a ‘packasome’. Analysis among a number of phages supports an active role of the terminases in coupling ATP hydrolysis to DNA translocation through the portal. In phage T4 the small terminase subunit promotes a sequence-specific terminase gene amplification within the chromosome. This link between recombination and packaging suggests a DNA synapsis mechanism by the terminase to control packaging initiation, formally homologous to eukaryotic chromosome segregation.     

Identification and characterization of yeast mutants and the gene for a cruciform cutting endonuclease.

An assay was developed that detected DNA cruciform cutting endonuclease activity in crude extracts of Saccharomyces cerevisiae. A collection of temperature-sensitive strains was screened using this assay, and a mutant lacking the activity was found. The mutation leading to the enzymatic defect was mapped to the left arm of chromosome XI within 3 cM of the centromere. Cloning of the gene for this endonuclease was achieved by chromosome walking from the nearby PUT3 locus. The gene, called CCE1 (cruciform cutting endonuclease), was sequenced and found to have an open reading frame encoding a 41 kDa protein. The amino acid sequence of this eukaryotic endonuclease shows homology neither to its prokaryotic counterparts nor to other proteins in available databases. A cce1 null mutant has no obvious growth defect, and despite the ability of the CCE1 enzyme to cleave Holliday junction analogs, the mutant shows no defect in meiotic or mitotic recombination. A second cruciform cutting activity was detected in extracts from a cce1 null mutant, indicating that yeast has at least two such enzymes. The only phenotype observed for cce1 mutants is a higher than normal frequency of appearance of petite cells, suggesting that the CCE1 protein is important for the maintenance of mitochondrial DNA.     

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.     

Effect of lig-7 on strand joining in repair of damaged DNA and on cutting of intact homologous DNA (cutting in trans) in Escherichia coli

Genetic recombination in Escherichia coli depends on the recA⁺ gene and can be increased in frequency by certain treatments that damage DNA. In previous studies (Ross &; Howard-Flanders, 1977a,b), E. coli (λ) cells were infected with undamaged λ phages and then with λ phages that were either undamaged, or had interstrand crosslinks produced in their DNA by treatment with psoralen and light. When the superinfecting DNA contained psoralen crosslinks, the intact DNA was cut. This cutting, referred to as cutting in trans, occurred only in DNA genetically homologous to the damaged DNA, required recA⁺ and behaved as expected of a step in damage-induced genetic recombination.In the present studies, we investigated the effect on cutting in trans of lig-7, a thermosensitive allele of the structural gene for E. coli polynucleotide ligase and also of uvrA, which controls the excision of damaged bases from DNA. The ligase deficiency caused gaps due to the action of the uvrA⁺ endonuclease on damaged DNA to remain open for at least 25 minutes. For low levels of damage, cutting in trans was also enhanced in the lig-7 cells at non-permissive temperatures but was not increased in wild-type cells. The enhanced cutting in trans depended upon genetic homology, as expected if it reflected elevated levels of damage-induced genetic recombination. Presumably, the unrepaired gaps in the damaged DNA made it a good substrate for the enzymes that promote cutting in trans of its homologs.     

A nuclear mutation defective in mitochondrial recombination in yeast.

Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.     

I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila.

As a step toward the development of a homologous recombination system in Drosophila, we have developed a methodology to target double-strand breaks (DSBs) to a specific position in the Drosophila genome. This method uses the mitochondrial endonuclease I-SceI that recognizes and cuts an 18-bp restriction site. We find that >6% of the progeny derived from males that carry a marker gene bordered by two I-SceI sites and that express I-SceI in their germ line lose the marker gene. Southern blot analysis and sequencing of the regions surrounding the I-SceI sites revealed that in the majority of the cases, the introduction of DSBs at the I-SceI sites resulted in the complete deletion of the marker gene; the other events were associated with partial deletion of the marker gene. We discuss a number of applications for this novel technique, in particular its use to study DSB repair mechanisms.     

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.     

Subunit structure of a yeast site-specific endodeoxyribonuclease, endo SceI. A study using monoclonal antibodies

Endo SceI is a eucaryotic site-specific endoDNase of 120 kDa that causes double-stranded scission at well-defined sites, but is distinguishable from procaryotic restriction endonucleases by its mode of sequence recognition and lack of related specific DNA modification. In purified preparations of endoSceI, only two polypeptide species of 75 kDa (75-kDa peptide) and 50 kDa (50-kDa peptide) are detected in apparently equal amounts. We prepared mouse monoclonal IgGs that bound specifically to the 75-kDa peptide (but not the 50-kDa peptide) without inhibiting the endoSceI activity. Immunoprecipitation experiments with these IgGs revealed that the 75-kDa peptide and the 50-kDa peptide are physically associated with each other and with the endonucleolytic activity. Full endoSceI activity was recovered by mixing the purified 75-kDa peptide and the partially purified 50-kDa peptide, each of which exhibited little or no endonuclease activity alone. These observations indicate that endoSceI consists of two non-identical subunits of 75 kDa and 50 kDa, and that both subunits are required for full enzyme activity.     

The evolutionarily conserved repetitive sequence d(TG.AC)n promotes reciprocal exchange and generates unusual recombinant tetrads during yeast meiosis.

We have studied the genetic behavior of the alternating copolymer d(TG.AC)n inserted into a defined position in the genome of the yeast Saccharomyces cerevisiae. When d(TG.AC)n sequences were present at the HIS3 locus on homologous chromosomes, diploid cells undergoing meiosis generated an excess of tetrads containing reciprocally recombined products with crossover points close to the repetitive DNA insert. Most of these tetrads exhibited gene conversion of a d(TG.AC)n insert. However, the insertion of d(TG.AC)n sequences had no effect on the frequency of gene conversion of closely linked marker genes. Surprisingly, when d(TG.AC)n sequences were present on only one homolog at the HIS3 locus, one-half of the tetrads exhibiting nonparental segregation for marker genes that flanked the repetitive DNA insert were very unusual and appeared to have arisen by multiple recombination events in the vicinity of the d(TG.AC)n insert. Similar multiply recombinant tetrads were seen in crosses in which d(TG.AC)n sequences were present on both homologs. Combined, the data strongly suggest that d(TG.AC)n sequences significantly enhance reciprocal meiotic recombination and may be important in causing multiple recombination events to occur within a relatively small region of the yeast chromosome. Molecular evidence is presented that clearly documents the postmeiotic segregation of an 80-base stretch of d(TG.AC)n.     

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.     

A latent intron-encoded maturase is also an endonuclease needed for intron mobility

Some yeast mitochondrial introns encode proteins that promote either splicing (maturases) or intron propagation via gene conversion (the fit1 endonuclease). We surveyed introns in the coxl gene for their ability to engage in gene conversion and found that the group I intron, al4 alpha, was efficiently transmitted to genes lacking it. An endonucleolytic cleavage is detectable in recipient DNA molecules near the site of intron insertion in vivo and in vitro. Conversion is dependent on an intact al4 alpha open reading frame. This intron product is a latent maturase, but these data show that it is also a potent endonuclease involved in recombination. Dual function proteins that cleave DNA and facilitate RNA splicing may have played a pivotal role in the propagation and tolerance of introns.     

Preferential strand transfer and hybrid DNA formation at the recombination hotspot ade6-M26 of Schizosaccharomyces pombe.

The ade6-M26 mutation of Schizosaccharomyces pombe stimulates intragenic and intergenic meiotic recombination. M26 is a single base pair change creating a specific heptanucleotide sequence that is crucial for recombination hotspot activity. This sequence is recognized by proteins that may facilitate rate-limiting steps of recombination at the ade6 locus. To start the elucidation of the intermediate DNA structures formed during M26 recombination, we have analyzed the aberrant segregation patterns of two G to C transversion mutations flanking the heptanucleotide sequence in crosses homozygous for M26. At both sites the level of post-meiotic segregation is typical for G to C transversion mutations in S. pombe in general. Quantitative treatment of the data provides strong evidence for heteroduplex DNA being the major recombination intermediate at the M26 site. We can now exclude a double-strand gap repair mechanism to account for gene conversion across the recombination hotspot. Furthermore, the vast majority (> 95%) of the heteroduplexes covering either of the G to C transversion sites are produced by transfer of the transcribed DNA strand. These results are consistent with ade6-M26 creating an initiation site for gene conversion by the introduction of a single-strand or a double-strand break in its vicinity, followed by transfer of the transcribed DNA strands for heteroduplex DNA formation.