Tn4430 transposase activity on Mini-Tn-containing DNA molecules in vitro

Details

Supervisors

Hallet, Bernard

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family. This family poses a threat to human health because it contributes to the spread of antibiotic resistance among pathogens. Recently, new discoveries have been made in understanding the transposition mechanism of Tn4430, making this transposon a new paradigm for the study of Tn3 family transposons. Biochemical analysis of the initial steps of Tn4430 transposition has unveiled a unique pathway leading to the assembly and activation of the transposition complex. This pathway is now supported by high-resolution cryo-EM structures depicting the transposition complex at different stages of the reaction. In addition to catalyse transposition, TnpA is responsible for a mechanism called "target immunity" which prevents the transposon from being inserted several times in the same locus. The molecular details of this mechanism are not known, but TnpA mutants that are affected in target immunity have been isolated. These mutants showed hyperactivity in vitro, being more prone than wild-type TnpA to catalyse DNA cleavage and strand transfer reactions. Finally, complementary in vivo and in vitro data converge to a new "replication hijacking" mechanism according to which Tn3-family transposons integrate into replication intermediates to recruit the host replication machinery and produce a new copy of themselves during transposition. The aim of this work is to set up a biochemical assay to determine the activity of TnpA and its hyperactive mutants (TnpA3X and TnpAS911R) in vitro. Through this test, the impact of DNA topology on transposase activity will be determined as well as the requirements of transposon end orientation. Other factors such as the role of the cofactor or the role of glycerol will be tested. The results of this work showed that the topology of the DNA can influence the activity of TnpA such as the presence of both ends of the transposon, the type of cofactor used and the presence of glycerol. In addition, additional bands appear on gels and hypotheses are put forward as to the origin of these bands.