Biochemical activities of the transposase TnpA in transposition and target immunity mediated by the replicative transposon Tn4430

(eng) Transposons are mobile genetic elements that move by transposition from one location to another within genomes. The present work is focused on the characterization of the activities mediated by the transposase protein (TnpA) of the bacterial transposon Tn4430 belonging to the Tn3 family. TnpA catalyzes replicative transposition of Tn4430 and target immunity that prevents multiple insertions of the transposon into the same DNA molecule. Very little is known regarding the molecular mechanisms that control the transposition and target immunity reactions catalyzed by TnpA of the Tn3 family. In this study, we have achieved decisive steps toward the understanding of the replicative transposition mechanism by developing in vitro reactions with TnpA reproducing specific steps of the transposition process. Additional observations have also revealed an unexpected link between the mechanism of DNA targeting by Tn4430 and DNA replication, suggesting a mechanism for recruiting replication machinery during the replicative transposition. This work also provides important findings toward the understanding of target immunity. We first showed that formation of a specific complex between TnpA and the immunizing transposon is necessary to generate immunity. In addition, the biochemical assays revealed that TnpA mutants affected in their immunity function had promiscuous activities when compared to wild type TnpA, suggesting that the activity of the protein is normally tightly regulated. Finally, we were interested in the biological significance of target immunity. We tested the involvement of this process in avoiding intra-molecular replicative transposition leading to deleterious events within the DNA molecule. The results showed that the immunity mutants exhibited a higher intra-molecular transposition frequency than wild type TnpA, which was in accordance with the hypothesis.