DMA SUBSTRATE-SPECIFICITY AND CLEAVAGE KINETICS OF AN ARCHAEAL HOMING-TYPE ENDONUCLEASE FROM PYROBACULUM-ORGANOTROPHUM

The protein encoded by intron 1 of the single 23S rRNA gene of the archaeal hyperthermophile Pyrobaculum organotrophum was isolated and shown to constitute a homing-type DNA endonuclease, I-Porl. It cleaves the intron- 23S rDNA of the closely related organism Pyrobaculum islandicum near the site of intron insertion in Pb.organotrophum. In contrast, no endonuclease activity was detected for the protein product of intron 2 of the same gene of Pb.organotrophum which, like I-Porl, carries the LAGLI-DADG motif, common to group I intron-encoded homing enzymes. I-Porl cleaves optimally at 80 degrees C, with k(cat) and K-m values of about 2 min(-1) and 4 nM, respectively, and generates four nucleotide 3'-overhangs and 5'-phosphates. It can cleave a 25 base pair DNA fragment encompassing the intron insertion site. A mutation-selection study established the base pair specificity of the endonuclease within al 17 bp region, from positions -6 to +11 with respect to the intron-insertion site. Four of the essential base pairs encode the sequence involved in the intron-exon interaction in the pre-rRNA that is required for recognition by the RNA splicing enzymes. Properties of the enzyme are compared and contrasted with thesis of eucaryotic homing endonucleases.