I-Sce III an intron-encoded DNA endonuclease from yeast mitochondria. Asymmetrical DNA binding properties and cleavage reaction

Abstract

We have previously discovered the new intron-encoded endonuclease I-Sce 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 in vitro properties of partially purified I-Sce III and found that it is a very specific DNA endonuclease, tolerating relatively few base changes in its 20 base pair long target site. I-Sce III should be a useful molecular tool to analyze the structure of large genomes. Interestingly, I-Sce III is the first P1-P2 DNA endonuclease for which DNA binding properties could be analyzed by band-shift experiments. Clearly, the cleavage products corresponding to the upstream A3 exon and to the downstream A4 exon could compete with the substrate A3-A4 in forming a DNA-protein complex. However, the A3 exon competes more efficiently than the downstream A4 product. The cleavage of the two DNA strands is also asymmetric the top strand (non-transcribed strand) is cleaved faster than the bottom strand, a property found under various experimental conditions. These findings suggest that this intron-encoded DNA endonuclease may have role in the RNA splicing process of the intron.