GENETIC-ANALYSIS OF THE CHLAMYDOMONAS-REINHARDTII I-CREI MOBILE INTRON HOMING SYSTEM IN ESCHERICHIA-COLI

We have developed and used a genetic selection system in Escherichia c oli to study functional requirements for homing site recognition and c leavage by a representative eukaryotic mobile intron endonuclease. The homing endonuclease, I-CreI, was originally isolated from the chlorop last of the unicellular green alga Chlamydomonas reinhardtii. I-CreI h oming site mutants contained base pair substitutions or single base de letions that altered the rate of homing site cleavage and/or product r elease. I-CreI endonuclease mutants fell into six phenotypic classes t hat differed in in vivo activity, toxicity or genetic dominance. Inact ivating mutations clustered in the N-terminal 60% of the I-CreI amino acid sequence, and two frameshift mutations were isolated that resulte d in premature translation termination though retained partial activit y. These mutations indicate that the N-terminal two-thirds of the I-Cr eI endonuclease is sufficient for homing site recognition and cleavage . Substitution mutations altered in four potential active site residue s were examined: D20N, Q47H or R70A substitutions inactivated endonucl ease activity, whereas S22A did not. The genetic approach we have take n complements phylogenetic and structural studies of mobile intron end onucleases and has provided new information on the mechanistic basis o f I-CreI homing site recognition and cleavage.