I-SCE-III AN INTRON-ENCODED DNA ENDONUCLEASE FROM YEAST MITOCHONDRIA - ASYMMETRICAL DNA-BINDING PROPERTIES AND CLEAVAGE REACTION

We have previously discovered the new intron-encoded endonuclease I-Sc e III by expressing, in E.coli, the ORF contained in the third intron of the yeast mitochondrial COX I gene. In this work, we analyzed the i n vitro properties of partially purified I-Sce III and found that it i s a very specific DNA endonuclease, tolerating relatively few base cha nges in its 20 base pair long target site. I-Sce III should be a usefu l molecular tool to analyze the structure of large genomes. Interestin gly, I-Sce III is the first P1-P2 DNA endonuclease for which DNA bindi ng properties could be analyzed by band-shift experiments. Clearly, th e cleavage products corresponding to the upstream A3 exon and to the d ownstream A4 exon could compete with the substrate A3-A4 in forming a DNA-protein complex. However, the A3 exon competes more efficiently th an the downstream A4 product. The cleavage of the two DNA strands is a lso asymmetric the top strand (non-transcribed strand) is cleaved fast er than the bottom strand, a property found under various experimental conditions. These findings suggest that this intron-encoded DNA endon uclease may have a role in the RNA splicing process of the intron.