Molecular basis and manipulation of mariner/Tc1 transposon DNA integration

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Transposons are segments of DNA that can move from one place to another in their host's genome. The movement of these DNA segments around genomes is orchestrated by a transposase protein encoded in the transposon itself. This enzyme cuts the DNA out of its original location and pastes it into a new site. This cut-and-paste property is being exploited to develop biotechnology tools for scientific research and medical applications. Learning more about how transposases move DNA will enable scientists to understand how to better control these enzymes and thereby speed the development of transposon tools. The mariner/Tc1 family of transposons is particularly widespread, being found in insects, plants, and animals including humans, and there is evidence that they can move from one host species to another over evolutionary time. This family is unusual in that all the elements are pasted into specific, but very short sequences of DNA comprising just two DNA bases. This proposal is aimed at discovering how transposases from this family choose this sequence and how they insert the transposon DNA into their target. To do this we will obtain a molecular snapshot of the Mos1 transposase in action at the DNA integration step of the transposition process. This will involve solving an X-ray crystal structure of the enzyme bound to both transposon DNA and target DNA. We have already solved structures of the enzyme and transposon DNA at earlier stages in the transposition process. The results of biochemical experiments will support and validate our findings. Having gained this detailed molecular understanding of the transposition machinery, we propose to engineer the transposase so that it can be directed to insert transposon DNA precisely where it is required. The ability to control the enzyme, based on a thorough knowledge of its three-dimensional structure as well as its chemical mechanism, should be of enormous benefit to developing gene therapy applications of this family of transposons, where precise control will be essential.