Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum.

Abstract

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 ...

Sp1 Facilitates DNA Double-Strand Break Repair through a Nontranscriptional Mechanism

“…I-PpoI's recognition sequence is found in the coding region of the 28S ribosomal DNA (rDNA) sequence, which is tandemly repeated at 5 chromosomal loci (30), as well as in intron 1 of the DAB1 gene on chromosome 1 (7). The enzyme produces about 20 to 30 DSBs per cell (20,60). Using this system and others, ␥H2Ax has been shown by chromatin immunoprecipitation (ChIP) to be present up to a megabase away from the break site but absent from the actual break site and regions in close proximity to the break.…”

Telomeric organization of a variable and inducible toxin gene family in the ancient eukaryote Giardia duodenalis.

“…We have no evidence for internal sites of telomeric sequences as found in chromosome III of yeast (Oliver et al 1992). However, cleavage of intact Giardia chromosomes with the intron-specific endonuclease, I-PpoI (Muscarella et al 1990), which also cleaves a 15-bp recognition site in the Saccharomyces cerevisiae rDNA sequence (Lowery et al 1992) and a number of other rDNA sequences, including Giardia (P. Upcroft, unpubl. ), removes all rDNA arrays.…”

Expression of the Naegleria intron endonuclease is dependent on a functional group I self-cleaving ribozyme

“…Group I introns are phylogenetically widespread autocatalytic RNA elements sharing a common secondary and tertiary structure (Cech et al+, 1994)+ Many group I introns are capable of self-splicing in vitro through a series of transesterification reactions (Cech, 1990), although proteins may aid splicing in vivo (Lambowitz & Perlman, 1990;Shaw & Lewin, 1997)+ About one third of group I introns harbor open reading frames (ORFs) coding for either structural proteins, maturases, or DNA endonucleases (Johansen et al+, 1997a)+ The DNA endonucleases, which are the most common intron-encoded proteins, are usually involved in intron mobility at the DNA level (reviewed in Belfort & Roberts, 1997)+ The endonuclease creates a doublestrand break at the intron-lacking allele, which is subsequently repaired in a gene-conversion event, using the intron-containing allele as a template+ The resulting unidirectional transfer of intron sequences is termed intron homing because of its high specificity at a homologous site+ Only 5% of the ;300 reported nuclear rDNA group I introns contain ORFs, and among these are the optional PpLSU3 and DiSSU1 of the myxomycetes Physarum polycephalum and Didymium iridis, respectively (Muscarella & Vogt, 1989;Johansen & Vogt, 1994), and NaSSU1 in a few species of the free-living amoebaflagellate Naegleria (De Jonckheere, 1994)+ These introns interrupt rRNA genes on extrachromosomal rDNA molecules (see Einvik et al+, 1998a), and encode the functional site-specific His-Cys box homing endonucleases named I-PpoI, I-Dir I, and I-NjaI, respectively (Muscarella et al+, 1990;Johansen et al+, 1997b;Elde et al+, 1999)+ I-PpoI and I-Dir I are involved in homing in their natural hosts after mating (Muscarella & Vogt, 1989;Johansen et al+, 1997b)+ NaSSU1 and DiSSU1 constitute a unique class of group I introns referred to as twin-ribozyme group I introns (Decatur et al+, 1995;Einvik et al+, 1997Einvik et al+, , 1998a)+ In these introns, the endonuclease ORF is found downstream of a small group I self-cleaving ribozyme (GIR1) that catalyzes hydrolytic cleavage of the RNA just upstream of the endonuclease ORF (see Fig+ 1)+ These two elements are both embedded in a loop of a more regular group I self-splicing ribozyme (GIR2)+ Strikingly, some species of Naegleria contain a shorter version of NaSSU1 that lacks both the ORF and GIR1 (De Jonckheere & Brown, 1994;Einvik et al+, 1997), suggesting that these two elements form a functional genetic unit, acquired or lost at the same time in evolution+ Despite their small si...…”