The overall objective of the research proposed in this grant is to elucidate the biochemical and regulatory mechanisms of a eukaryotic transposable element in a higher metazoan using an integrated approach that combines biochemistry, genetics and molecular biology. Our efforts will focus on understanding the biochemical mechanism of P element transposition and how these eukaryotic transposable DNA elements tie into the DNA repair pathways of the fruit fly, Drosophila melanogaster. The experiments in this proposal will provide necessary information for the development of P elements as genetic tools in other organisms. In order to accomplish our overall objective, we will: 1. Analyze the phenotypes of mutant P element transposase proteins using an in vivo excision assay. 2. Analyze the biochemical properties of wild type and mutant transposase proteins; further develop biochemical assays for their activities. 3. Analyze the biochemical effects of phosphorylation of the transposase protein; identify the kinases involved in these phosphorylation events. 4. Determine what role GTP binding and/or hydrolysis play in P element transposition. 5. Analyze the role of the Drosophila proteins IRBP, DmKup70, DmKup80 and DmDNA-PKcs (p470) in P element transposition. 6. Facilitate the development of P element-mediated transposition as a genetic tool in other organisms.
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