In vitro characterization of the Tn4430 transposase activity on DNA molecules containing Mini-Tn4430 element

Details

Supervisors

Hallet, Bernard

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité spécialisée: biotechnologie

Abstract

Transposons are defined DNA sequences capable of moving from one location to another within and between genomes. This process is known as transposition and it happens in a nucleoproteic complex, the transpososome. This complex contains the protein allowing the transposition: the transposase, the target DNA molecule, and the transposon ends. The Tn4430 transposon is a member of a widespread family of transposons, the Tn3 family, and is used as a representative model for the study of this family. The Tn3 family significantly impacts human health because it is implicated in the dissemination and persistence of antibiotic resistance among pathogens. Despite this biological significance, there are still many unknown molecular aspects of the transposition mechanism of this family. In previous works, the initial steps of the Tn4430 transposition have been studied and they have shown that this pathway leads to the assembly and activation of the transposition complex. Thanks to cryo-EM microscopy, different conformations of the transposase (TnpA) have been obtained and have allowed us to characterize the transposition complex at different steps of the reaction. Moreover, in vivo and in vitro data have converged into a new transposition mechanism called “replication hijacking”. During this mechanism, Tn3 family transposons can integrate into intermediates of replication allowing them to recruit the host’s replication machinery and then create a new copy of themselves. During transposition, TnpA mediates another mechanism called “immunity” where it prevents the insertion of a new transposon copy into a target DNA molecule that already presents a copy of the same transposon. Its mechanism is still not completely understood, but we have been able to isolate TnpA mutants that have lost this “immunity” mechanism which makes them more prone than the wild-type transposase to catalyze the DNA cleavage and strand transfer reactions. In this work, we use the hyperactive mutants of the TnpA (TnpA3X and TnpAS911R) to continue the in vitro study of their activity. To this end, a biochemical assay was used to determine how the DNA and transposon topology on the donor molecule impacts the TnpA activity. A supercoiled, open circular, or linear version of a plasmid containing a Mini-Tn4430 was used as a substrate. Furthermore, other important factors for transposition were studied. The temperature to further optimize the Tn4430 transposition reaction, and the donor and protein concentrations to test the hypothesis of assembly site occlusion (ASO) control of Tn4430 transposition. This thesis shows that the DNA topology of the donor molecule (supercoiled, open circular, or linear) does not seem to impact the TnpA activity. Additionally, the transposon topology does not seem to affect the TnpA activity as the presence of either end of the Tn4430 is sufficient to reach an activity similar to when both ends are present on the same molecule. Finally, we started an analysis of unknown cleavage products that appeared in different reactions. Although this analysis will have to be expanded and completed in further works, we proposed some hypotheses concerning the potential topologies and origins of these products which could be the result of strand transfer.