ASYMMETRICAL RECOGNITION AND ACTIVITY OF THE I-SCEI ENDONUCLEASE ON ITS SITE AND ON INTRON-EXON JUNCTIONS

Group I intron-encoded endonucleases represent a new class of double s trand cutting endonucleases whose function is to initiate the homing o f introns by generating double strand breaks in site-specific sequence s. We have studied the mechanism of interaction of the I-SceI endonucl ease with different DNA substrates derived from its natural site in th e intron-less gene or from intron-exon junctions in the gene with an i ntron. We show that the enzyme recognizes its asymmetrical site with h igh affinity binding to the sequence corresponding to the downstream e xon followed by binding to the upstream exon and catalysis of phosphod iester bond hydrolysis. Asymmetrical nicking activity is observed as a n intermediate of the cleavage reaction. In the intron-containing gene , the enzyme recognizes the downstream intron-exon junction without an y cleavage activity. This binding raises the possibility of a specific function of homing endonucleases in either gene expression or intron homing steps subsequent to DNA cleavage.