| This dissertation presents research on Integrase-DNA interactions in the Tn916 conjugative transposon. Conjugative transposons are mobile genetic elements, which have been implicated in the spread of antibiotic resistance in human pathogens. Tn916 is one of the most closely studied conjugative transposons, and it transposes in a manner analogous to bacteriophage λ recombination. Tn916 encodes all of the necessary factors for its excision from a donor bacterial chromosome, transfer to a recipient bacteria, and insertion into the recipient bacterial chromosome. Tn916 recombination reactions are catalyzed by integrase (Int), a bivalent DNA binding protein which recognizes two different types of DNA sites within the transposon. The N-terminal DNA binding domain of Int (INT-DBD) specifically binds direct repeat (DR2) sites near the ends of the transposon, and the C-terminal catalytic domain binds the transposon-chromosome junctions and executes cleavage and strand exchange reactions. A second factor, the transposon-encoded excisionase (Xis) protein, may influence the efficiency and/or direction of the Int-catalysed recombination reactions. The research presented in this dissertation focuses on the interactions of INT-DBD and Xis with their cognate DNA binding sites. It follows these interactions from the atomic level (with structural studies and mutagenesis) to the broader context of recombination in vivo. This dissertation will present: (1) the solution structure of the Tn916 INT-DBD; (2) a thermodynamic study of INT-DBD-DNA binding and the determinants of DNA recognition; and (3) the role of the specific INT-DBD-DNA, Xis-DNA, and host factor interactions in vivo, in the context of Tn916 transposition. I have determined that the INT-DBD-DNA interaction is mediated by a unique 3-stranded β-sheet DNA binding interface, and have characterized the nucleoprotein contacts within the interface. I have also determined that in Tn916 excisive recombination, there is flexibility in the number, orientation, and placement of DR2 sites at the right arm of the transposon. Furthermore, I have shown that Xis binding at the right arm is not required for efficient excision, and that Tn916 and bacteriophage λ utilize different host-encoded factors for excision in vivo. |
