Dissecting the Hermes Transposase: Residues Important for Target DNA Binding and Phosphorylation

The developing fields of gene therapy and genetic transformation are constantly searching for new autonomously replicating transposable elements with the ability to stably integrate into the genome of a wide range of diverse hosts. The Hermes transposase has become a particularly attractive element because of its ability to transform a wide range of hosts, the diverse distribution of hAT elements in nature, and the extensive genetic, molecular, and biochemical data available. In order to develop Hermes as an efficient molecular biology tool this dissertation explored (1) how the transposase chooses it's target DNA for integration and (2) regulatory mechanisms which may effect transposition activity. The work presented in this dissertation identifies, for the first time, residues in Hermes that are important for target DNA binding. These residues are part of a model that I call the "rim of the wheel" based on the Hermes crystal structure. Data collected from a comprehensive analysis designed to tease apart different steps of transposition suggests that these residues may contribute to a bi-functional domain responsible for both excising the transposon and binding to target DNA. The work presented here adds to our understanding of transposase integration biochemistry and can serve as a model for other elements. In order for Hermes to be used as a biological tool for gene therapy or genetic studies in medically and agriculturally important insects it is important that Hermes has its greatest chance for successful integration into its host. The second project presented here goal of explored whether Hermes was subject to phosphorylation and if so whether this affected transposition. Here I report for the first time, direct evidence of phosphorylation of a eukaryotic class II transposase. Indirectly two other residues in Hermes were found to be important for transposition, as mutating them to residues with different chemistries decreased Hermes transposition frequency. This work has expanded our still growing understanding of essential residues that are functionally important for Hermes transposition