A novel and only recently recognized class of enzymes is composed of the site-specific endonucleases encoded by some group I introns. We have characterized several aspects of I-Ppo, the endonuclease that mediates the mobility of intron 3 in the ribosomal DNA of Physarum polycephalum. This intron is unique among mobile group I introns in that it is located in nuclear DNA. We found that I-Ppo is encoded by an open reading frame in the 5' half of intron 3, upstream of the sequences required for self-splicing of group I introns. Either of two AUG initiation codons could start this reading frame, one near the beginning of the intron and the other in the upstream exon, leading to predicted polypeptides of 138 and 160 amino acid residues. The longer polypeptide was the major form translated in vitro in a reticulocyte extract. From nuclease assays of proteins synthesized in vitro with partially deleted DNAs, we conclude that both polypeptides possess endonuclease activity. We also have expressed I-Ppo in Escherichia coli, using a bacteriophage T7 RNA polymerase expression system. The longer polypeptide also was the predominant form made in this system. It showed enzymatic activity in bacteria in vivo, as demonstrated by the cleavage of a plasmid carrying the target site. Like several other intron-encoded endonucleases, I-Ppo makes a four-base staggered cut in its ribosomal DNA target sequence, very near the site where intron 3 becomes integrated in crosses of intron 3-containing and intron 3-lacking Physarum strains.Group I introns are defined by the presence of conserved sequences and structural elements (reviewed in reference 2). Many group I introns can undergo autocatalytic splicing (self-splicing) in vitro, as first described for the prototypic intron of Tetrahymena cells (5). Of the more than 60 group I introns that have been identified to date (4), most are present in the DNA of mitochondria or chloroplasts or in the DNA of T-even bacteriophage. In only three organisms have group I introns been observed in nuclear genes, and in each of these they are located in the DNA coding for ribosomal RNA (rDNA). Pneumocystis carinii contains an intron in the rDNA encoding the small-subunit rRNA (18), but neither it nor the rDNA of this organism has been extensively characterized. Several but not all Tetrahymena species and strains contain the well-known self-splicing intron in extrachromosomal rDNA coding for the large-subunit rRNA (22,23,38). Depending on the strain, Physarum polycephalum contains two or three group I introns in the extrachromosomal rDNA coding for the large-subunit rRNA (28,30,31). Some group I introns have been found to be mobile elements. They rapidly and efficiently spread in vivo from a locus that contains the intron (I+) to the same locus in a homologous gene that lacks the intron (I). This process, which has been termed intron homing (15,16), is initiated by a double-strand break in the I-locus that is introduced by a site-specific endonuclease encoded by the intron itself. The actual homing of the ...

Endonucleases encoded by mobile group I introns are highly specific DNases that induce a double-strand break near the site to which the intron moves. I-PpoI from the acellular slime mold Physarum polycephalum mediates the mobility of intron 3 (Pp LSU 3) in the extrachromosomal nuclear ribosomal DNA of this organism. We showed previously that cleavage by I-PpoI creates a four-base staggered cut near the point of intron insertion. We have now characterized several further properties of the endonuclease. As determined by deletion analysis, the minimal target site recognized by I-PpoI was a sequence of 13 to 15 bp spanning the cleavage site. The purified protein behaved as a globular dimer in sedimentation and gel filtration. In gel mobility shift assays in the presence of EDTA, I-PpoI formed a stable and specific complex with DNA, dissociating with a half-life of 45 min. By footprinting and interference assays with methidiumpropyl-EDTAiron(II), I-PpoI contacted a 22-to 24-bp stretch of DNA. The endonuclease protected most of the purines found in both the major and minor grooves of the DNA helix from modification by dimethyl sulfate (DMS). However, the reactivity to DMS was enhanced at some purines, suggesting that binding leads to a conformational change in the DNA. The pattern of DMS protection differed fundamentally in the two partially symmetrical halves of the recognition sequence.More than 100 group I introns, which are defined by a common RNA secondary structure and mechanism of splicing, have been identified in the organellar genomes of lower eucaryotes (for reviews, see references 4 and 29). Several of these introns also have been found in bacteria, bacteriophage, and extrachromosomal nuclear ribosomal DNA (rDNA). The RNAs of many group I introns are capable of self-splicing in vitro. Some group I introns behave as mobile elements, as first described for the optional omega intron (Sc LSU 1) of Saccharomyces cerevisiae (9). Additional mobile group I introns have been found in yeast mitochondria (32,45,53), bacteriophage (42), Chlamydomonas chloroplasts (11,23), Chlamydomonas mitochondria (8, 26), and nuclear rDNA of slime molds (16,36). In each of these cases, the mobile intron is copied into an intron-allele of the gene in which it resides, converting it to intron+ by gene conversion initiated at a double-strand break near the site of insertion. This process, known as intron homing (10), is mediated by an endonuclease encoded within the intron, which recognizes and cleaves the intron-DNA within a few nucleotides of the insertion site (for reviews, see references 22 and 33). In three cases, the procaryotic enzymes I-TevI, I-TevII, and I-TevIII, the DNA cut is somewhat more removed, 13 to 26 bp from the point of intron insertion (for a review, see reference 33). In any case, the homology between the intron-allele and the DNA sequences flanking the intron in the intron+ allele allows the break to be repaired via a gene conversion event which uses the intron+ allele as a template. Since intron insertion render...

A novel and only recently recognized class of enzymes is composed of the site-specific endonucleases encoded by some group I introns. We have characterized several aspects of I-Ppo, the endonuclease that mediates the mobility of intron 3 in the ribosomal DNA of Physarum polycephalum. This intron is unique among mobile group I introns in that it is located in nuclear DNA. We found that I-Ppo is encoded by an open reading frame in the 5' half of intron 3, upstream of the sequences required for self-splicing of group I introns. Either of two AUG initiation codons could start this reading frame, one near the beginning of the intron and the other in the upstream exon, leading to predicted polypeptides of 138 and 160 amino acid residues. The longer polypeptide was the major form translated in vitro in a reticulocyte extract. From nuclease assays of proteins synthesized in vitro with partially deleted DNAs, we conclude that both polypeptides possess endonuclease activity. We also have expressed I-Ppo in Escherichia coli, using a bacteriophage T7 RNA polymerase expression system. The longer polypeptide also was the predominant form made in this system. It showed enzymatic activity in bacteria in vivo, as demonstrated by the cleavage of a plasmid carrying the target site. Like several other intron-encoded endonucleases, I-Ppo makes a four-base staggered cut in its ribosomal DNA target sequence, very near the site where intron 3 becomes integrated in crosses of intron 3-containing and intron 3-lacking Physarum strains.

Endonucleases encoded by mobile group I introns are highly specific DNases that induce a double-strand break near the site to which the intron moves. I-PpoI from the acellular slime mold Physarum polycephalum mediates the mobility of intron 3 (Pp LSU 3) in the extrachromosomal nuclear ribosomal DNA of this organism. We showed previously that cleavage by I-PpoI creates a four-base staggered cut near the point of intron insertion. We have now characterized several further properties of the endonuclease. As determined by deletion analysis, the minimal target site recognized by I-PopI was a sequence of 13 to 15 bp spanning the cleavage site. The purified protein behaved as a globular dimer in sedimentation and gel filtration. In gel mobility shift assays in the presence of EDTA, I-PpoI formed a stable and specific complex with DNA, dissociating with a half-life of 45 min. By footprinting and interference assays with methidiumpropyl-EDTA-iron(II), I-PpoI contacted a 22- to 24-bp stretch of DNA. The endonuclease protected most of the purines found in both the major and minor grooves of the DNA helix from modification by dimethyl sulfate (DMS). However, the reactivity to DMS was enhanced at some purines, suggesting that binding leads to a conformational change in the DNA. The pattern of DMS protection differed fundamentally in the two partially symmetrical halves of the recognition sequence.