The goal of this application is to study the mechanism of action and the substrate recognition properties of two intron-encoded endonucleases, I-DmoI and I-TevI, that catalyze double-strand DNA cleavage to initiate mobility of their introns. These endonucleases are remarkable in that they interact with lengthy recognition sequences. They possess exquisite cleavage site specificities and distort their DNA substrates. I-DmoI and I-TevI represent the two major subclasses of homing endonucleases. I-DmoI is a member of the majority class, containing two copies of a conserved motif, LAGLI-DADG. This enzyme recognizes a 14 base pair binding site in a sequence-specific manner, cleaves close to the intron insertion site and leaves a 4-nucleotide 3'-overhang. I- TevI, a member of the GIY-YIG family, recognizes a 35 base pair DNA target in a sequence-tolerant manner, cleaves 23 to 25 nucleotides upstream of the intron insertion site and leaves 2-nucleotide 3'- overhangs. While LAGLI-DADG enzymes interact with their substrates mainly through the major groove of the DNA, I-TevI, the best characterized of the GIY-YIG enzymes, interacts predominantly through the minor groove. The specific aims of the project are to determine the three-dimensional structures of the enzymes, of isolated domains and of complexes with substrates by a combination of X-ray crystallography and NMR spectroscopy. This will allow the comparison of representative examples of the two classes of enzymes that, although otherwise unrelated, carry out the same biological function.