[unreadable] Homing endonucleases are a unique family of proteins capable of functioning autonomously to recognize and cleave specific long DNA sequences. The proposed studies focus on engineering homing endonucleases that recognize and cleave novel DNA sequences. Ultimately, it should be possible to design endonucleases to target any DNA sequence of interest, such as those implicated in causing specific human diseases. Such engineered endonucleases hold tremendous potential as gene therapy reagents and as anti-microbial agents. I-Crel is arguably the best-characterized homing endonuclease, having been extensively studied at the structural, biochemical and genetic levels. The proposed work builds on those studies, taking a systematic approach to isolate and characterize a family of altered specificity I-Crel derivatives: Specific aim 1 is to identify more I-Crel derivatives with novel protein-DNA contacts. I-Crel derivatives containing amino acid substitutions at or near residues predicted to contact DNA will be generated and assayed against appropriate DNA target sequences. Specific aim 2 is to analyze I-Crel derivatives with combinations of novel protein-DNA contacts. I-Crel substitutions shown to result in novel DNA recognition will be combined and assayed against DNA target sequences with appropriate combinations of base changes. Specific aim 3 is to engineer intramolecular I-Crel dimers, and use these to target asymmetric DNA sequences. This work should provide a great deal of information about this highly specific protein-DNA interaction, and in the process identify a large number of potentially useful novel homing endonucleases. The proposed studies will integrally and actively involve undergraduate scientists. [unreadable] [unreadable]