| Open reading frames (ORFs) are frequently inserted into group I self-splicing introns. These ORFs encode either maturases that are required for splicing of the intron, or DNA endonucleases that promote intron mobility. A self-splicing intron in the tRNAfMet gene of Synechocystis PCC 6803 contains an ORF that is unrelated to known intron-encoded endonucleases or maturases. Here, using an in vitro transcription-translation system the intronic ORF is shown to encode a double-strand DNA endonuclease, I-Ssp6803I. I-Ssp6803I cleaves each strand of the intronless tRNAfmet gene adjacent to the anticodon triplet leaving 3-bp 3′ extensions, and has no activity at intron-exon boundaries. Using an in vitro cleavage assay and scanning deletion mutants of the intronless target site, the minimal recognition site was determined to be a partially palindromic 20-bp region encompassing the entire anticodon stem and loop of the tRNA fmet gene. I-Ssp6803I represents a novel intron-encoded DNA endonuclease and is the first example of a chromosomally encoded group I intron endonuclease in bacteria. Weak similarity of I-Ssp6803I to gene 5.3 from bacteriophage T7 prompted a screen of this locus in T7-like phages. A group I self-splicing intron was discovered in the DNA polymerase gene (gene 5) of two phages, ΦI and W31, 156 by upstream from the end of the gene. Encoded within the intron is a small, 131 amino acid endonuclease, I-TslI, belonging to the H-N-H family of homing endonucleases. The closest relative to I-TslI is gene 5.3 from phage T3, which is not homologous to its T7 counterpart. T3 gene 5.3, a 101 codon ORF of previously unknown function with similarity to H-N-H homing endonucleases, was shown to encode an endonuclease. I-TslI and T3gp5.3 cleave all intronless targets tested at the identical position, but are inactive on intron-containing substrates with one exception: T3gp5.3 does not cleave a target generated from phage T3. Based on this evidence a rationale is proposed for the origin of mobile group I introns. |
