This proposal is focused on dissection at the molecular level of the mechanism by which the bacterial transposable element TnF moves from place to place. As transposable elements are widespread, having been found in virtually all organisms, our findings with Tn7 will be relevant to many systems. One prominent role of transposition is the rapid dispersal of elements, often carrying several antibiotic resistance determinants from place to place. Tn7 actually transposes preferentially to conjugating DNAs that are readily moved between bacteria, thus facilitating the rapid spread of this element. Tn7 is also a highly evolved element such that when inserting into the host genome Tn7 inserts specifically into a particular site called att Tn7 downstream of an essential gene such that this insertion is not deleterious to its host. Much of this proposal focuses on the dissection of these different targeting pathways using both genetic and biochemical methods. Another interesting aspect of transposition is the contribution of host proteins. Although the actual act of transposition can be accomplished with only Tn7-encoded proteins, these product DNAs actually contain gaps that must be repaired by host functions. We propose to dissect using genetic and biochemical methods how these molecules are converted to intact DNA duplex. Tn7 can participate in two different kinds of recombination reactions that require different kinds of repair. In the pathway by which Tn7 forms simple insertions, only gap repair between the newly inserted transposon and the target DNA is required; Tn7 can also form co-integrates that require extensive DNA replication. Comparing and contrasting these Tn7 pathways should provide powerful insights into these repair reactions. We now know that there are biochemical similarities between the mechanisms by which Tn7 and other bacterial elements and retroviruses such as HIV transpose. Moreover, the recombinases that execute these reactions are similar. Thus what we learn about Tn7 should lead to a deeper understanding of the movement of other elements such as HIV. Like many complex nucleic acids transactions, Tn7 transposition occurs within an elaborate protein-DNA complex. This understanding in more detail how the Tn7 machinery works should provide insight into other reactions such as DNA replication and transcription.
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