The long-range goal of this project is to understand the mechanism and control of a recombination reaction, the transposition of the bacterial transposition Tn7 which encodes resistances to trimethoprim and to streptomycin and spectinomycin. In contrast to most other transposable elements which move at low frequency and show little target site selectivity, Tn7 inserts at high frequency into a specific site called attTn7. When attTn7 is unavailable, Tn7 inserts at low frequency into many other sites. Tn7 is an elaborate element: it encodes five transposition genes which mediate several distinct recombination reactions and utilizes large DNA segments at the ends of Tn7 and at its target sites as recombination substrates.
A specific aim of the studies proposed here is to use biochemical methods develop an in vitro Tn7 transposition system in which purified proteins direct the recombination of defined DNA substrates. This will allow the identification of both Tn7- and host-encoded transposition recombination proteins. Another specific aim is to use this purified system to dissect the transposition reaction mechanism at the molecular level. Genetic methods will be used to achieve two other related specific aims: 1) identification of host genes encoding proteins either directly involved in Tn7 recombination or which regulate this process, and 2) production of mutant transposition proteins whose altered activities will allow identification of key steps in recombination. Genetic methods will also be used to pursue another specific aims, understanding cellular responses to Tn7 transposition. The result of these studies will provide important insights into protein- DNA interaction and DNA structure. They will also contribute to a deeper understanding of the interactions between mobile elements and their hosts and of the rapid dispersal of antibiotic resistance determinants.
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