Functional analysis of Sleeping Beauty transposase regulation

Transposable elements are segments of DNA that are able to move around from one genomic location to another. They are found virtually in every organism in nature, and through their use as a genetic tool, have done much to further our understanding of biology. As transposable elements become increasingly used as a means for gene delivery and discovery, it grows more important that we understand their molecular mechanism and the factors regulating their mobilization. The work presented in this thesis examined both element- and host-encoded factors that affect the efficiency of the vertebrate transposase Sleeping Beauty (SB). Through mutagenic analysis of the N-terminal region of SB by a quantitative cell-culture based transposition assay, we were able to confirm that predicted secondary structures in the DNA binding domain are necessary for enzyme function. By electromobility shift and yeast one-hybrid DNA-binding analysis we also found that binding is a critical rate-limiting step of transposition and, in certain instances, changes in binding activity directly affect transposition levels. Further transposon affinity studies of SB deletion and missense mutants led to the identification of a novel transposase functional domain in the region between the N-terminal DNA binding and the C-terminal catalytic domains. Our results from gel mobility, yeast one-hybrid, and quantitative transposition assays indicate that this linker domain is involved in inhibiting DNA binding as well as contributing to overall regulation of transposition. We also examined the possibility of cellular host factors influencing the efficiency of SB activity. Transposition rates were determined in a variety of cell types and a subsequent yeast two-hybrid screen was conducted to identify host proteins that could enhance or inhibit transposition. Although we were unable to identify any SB:host factor interactions by this method, it does not preclude the existence of said interactions and further investigation into this subject is needed.