Conjugative transposons are extremely promiscuous genetic elements that are clinically significant in the dissemination of antibiotic resistance amongst Gram-positive and -negative bacteria. This proposal outlines a comprehensive study of molecular recognition and protein assembly in the transposition mechanism of conjugative transposon Tn916. It also proposes complementary studies of the site-specific integrative and excisive reactions of the lambda bacteriophage, which are mechanistically related to Tn916 transposition and prototypical of a large number of biologically significant recombination processes that promote genetic diversity in both prokaryotes and eukaryotes. Both mobile genetic elements move using functionally related heterobivalent tyrosine recombinase (Int) and excisionase (Xis) proteins. This research will study how these factors assemble into higher-order nucleoprotein structures that recombine DNA. Combining structural, biochemical, and genetic methods, we aim to determine the molecular mechanism of excision stimulation by the transposon-encoded Xis protein, and to map the complex set ofprotein-DNA interactions required for Tn916 excisive recombination. In synergistic research, we will study the structural basis of protein assembly in lambda phage recombination. Specifically, we will elucidate how the phage encoded excisionase protein acts as a molecular switch between the excisive and integrative pathways of recombination. This will be accomplished by determining how it assembles with integrase to form the Int-Xis-Xis:DNA ternary complex that regulates phage excision. Combined, this research will provide molecular level insights into the recombination reactions of lambda bacteriophage, and will begin to reveal how this system has been hamessed to disseminate antibiotic resistance in pathogenic bacteria.
