Project Summary/Abstract Among the several ongoing revolutions in biological research, two (the ability to visualize increasingly complex biomolecular machines and assemblages and the ability to create novel protein structures and corresponding functions) represent the foundation of our laboratory's research program. For many years, we have divided its activities between (i) determining the structures, mechanisms and biological roles of gene targeting proteins and base modifying enzymes, and (ii) employing protein engineering to create new protein folds, assemblages, and functions, and to test our understanding of protein form and function. We now plan to renew and expand upon our research mission by address several questions and problems in the fields of nucleic acid enzymology, molecular recognition, and protein engineering. The first two projects will build upon our experience studying and engineering homing endonucleases (also called `meganucleases'). In doing so, we will answer questions surrounding the evolution, recognition mechanisms and engineerability of (i) sequence-specific microbial RNA endonucleases and (ii) eukaryotic retrotransposons. We plan to leverage our basic studies of these systems to create new tools for transcriptomic analyses and genome engineering, respectively. The third project addresses a current challenge in protein engineering: the accurate design of biomolecular interfaces that rely heavily on stereospecific hydrogen-bond networks to facilitate the balance of affinity, specificity and reversibility that is a hallmark of biomolecular interactions and regulation. To do so, we will create and then couple together protein- protein and protein-small molecule binding functions through the creation of novel synthetic ligand-induced protein multimerization systems. The project will contribute to the field of molecular design and engineering by optimizing strategies for the accurate design of stereospecific hydrogen bond networks for that facilitate protein-protein and protein-ligand recognition and binding.