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 arc haeal hyperthermophile Pyrobaculum organotrophum was isolated and show n to constitute a homing-type DNA endonuclease, I-Porl. It cleaves the intron- 23S rDNA of the closely related organism Pyrobaculum islandic um near the site of intron insertion in Pb.organotrophum. In contrast, no endonuclease activity was detected for the protein product of intr on 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 nucleoti de 3'-overhangs and 5'-phosphates. It can cleave a 25 base pair DNA fr agment encompassing the intron insertion site. A mutation-selection st udy established the base pair specificity of the endonuclease within a l 17 bp region, from positions -6 to +11 with respect to the intron-in sertion site. Four of the essential base pairs encode the sequence inv olved 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 endonucle ases.