| Encoded in certain mobile group I introns are highly specific enzymes that cleave intronless DNA near the site of intron insertion. The break in the DNA induces an efficient type of homologous recombination called intron homing. Homing endonucleases are classified based on the presence of one of four conserved motifs. The ORF in the fourth intron of the Chlamydomonas reinhardtii chloroplast psbA gene (Cr.psbA-4 ) appears to be unique in containing two of the conserved motifs, H-N-H, and a putative G1Y-Y1G motif, respectively. This ORF was expressed in E. coli, and shown to be a site-specific endonuclease that cleaves intronless psbA DNA very near the exon 4-exon 5 junction. Efficient expression in E. coli in a soluble form required the co-expression of E. coli chaperonins, and a stringently controlled T7 system. This protein, named I-CreII, was purified and its cleavage activity characterized. Divalent cations (Mg 2+, Mn2+, or Ca2+) were required for cleavage; other conditions for optimum cleavage were: pH 8, 37°C, and low concentrations of (or no) monovalent cations. The exact cleavage sites on each strand were mapped; I-CreII cleaved 4-nt upstream of the intron insertion site on the sense strand, and 6-nt upstream on the anti-sense strand. Thus, the cleavage pattern of I-CreII resembles G1Y-Y1G proteins in leaving 2-nt 3'-OH overhangs. The overall size of the recognition sequence was estimated by cleaving sequence ladders. It was found to be ∼21 bp, and spanned the intron-insertion and cleavage sites.; I also partially characterized another homing endonuclease, I- CreI, which was under study in the laboratory, and is encoded in the chloroplast 23S rRNA intron Cr.LSU. I-CreI is a member of the LAGLI-DADG family. I-CreI, expressed in E. coli, was purified and used for biochemical characterization. Chemical cross-linking showed that the functional unit of the enzyme was a dimer. Endonuclease activity required divalent cations such as Mg2+ or Mn2+, and monovalent cations were inhibitory. I- CreI had an usually high temperature optimum (50--70°C). Interestingly, a protein denaturant was needed to dissociate I-Cre I from the DNA products. |
