DNA transposons are discrete DNA sequences that can move (transpose) from one location in the genome to another. They are present in virtually all organisms and contribute to both genome structure and function. DNA transposons are also natural gene delivery vehicles that are being developed as tools for genome engineering, such as insertional mutagenesis and transgenesis, and for human gene therapy. The reaction of DNA transposition occurs within a protein?DNA complex (transpososome), which contains two or more transposase enzymes and the ends of the transposon DNA. Despite the significant progress in understanding the biochemistry of DNA transposition, fundamental questions about the functional and structural mechanisms underpinning transpososome assembly and operation remain unaddressed. The long-term objective of this research is to elucidate these mechanisms and how they can be exploited for different genetic applications. In this project we propose to investigate a Sleeping Beauty (SB) DNA transposon, the most widely used transposon in research genetic applications and the first and only DNA transposon in clinical trials for human gene therapy. The specific objective of this proposal is to obtain the critically needed structural details on the SB transposase interactions with the transposon DNA and the model of the SB pre-cleavage nucleoprotein complex. Our experimental approach integrates solution scattering techniques (SANS with contrast variation and SAXS), advanced solution NMR techniques, Frster resonance energy transfer (FRET), mutagenesis, and biochemical and transposition functional assays.
The specific aims of this proposal are directed at (1) building a structural model of the full-length SB transposase and its nucleoprotein complex, and (2) elucidating structural properties of several hyperactive mutations in the primary DNA-recognition subdomain of SB transposase. The results will have broad applications to understanding the functional structural features of DNA transposases and significant particular applications to human gene therapy and gene delivery to animal cells using the SB transposon.
DNA transposons contribute to both genome structure and function in virtually all organisms and have become powerful tools in functional genomics and gene therapy; although the molecular mechanism by which they rearrange genomes has yet to be fully understood. Proposed structural studies will provide this mechanistic insight that is essential to progress for DNA transposon application to genetic research and therapy, particularly for the Sleeping Beauty DNA transposon. This specific transposon is a tool that is widely used for insertional mutagenesis and production of transgenic animals and is currently in the first human clinical trial for the treatment of B-lymphoid malignancies, and for expanding its applications to treat monogenic diseases in humans.
