Ends-synapsis and target integration during replicative transposition of the Tn3-family transposon Tn4430
(2018)
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- Authors
- Supervisors
- Hallet, Bernard
- Abstract
- Transposons are mobile genetic elements encountered in all domains of life. They are characterized by their ability to displace or copy themselves between distant genomic sites, a process that is catalyzed by a specific enzyme called transposase. The present thesis focused on the study of the transposase (TnpA) from the transposon Tn4430. This element belongs to the Tn3 family, a major transposon family that is widespread among bacteria. TnpA is a versatile protein that recognizes the transposon terminal inverted repeats (IR), cuts and merge them with an acceptor molecule. The overall mechanism is said to be replicative because it requires the host replication machinery to duplicate the element. In addition to mediate transposition, TnpA is also responsible for target immunity, an intriguing phenomenon that precludes multiple insertions of the transposon in the same DNA molecule. Previous studies had identified deregulated TnpA mutants that are deficient in immunity, but remain proficient in transposition. In the first part of the thesis, biochemical characterization of the TnpA immunity mutants unraveled the assembly of a “paired end complex”, a transposition intermediate in which both extremities of the transposon are brought together by the transposase. Formation of this transposition intermediate turned out to be a key regulatory step required prior to catalysis of DNA cleavage and strand transfer. In the second part of the work, Tn4430 transposition was shown to be biased toward replication stalling sites in vivo and to substrates mimicking a replication fork in vitro. Biochemical analysis of the strand transfer reaction demonstrated that TnpA joins the transposon ends specifically at the branch point of the fork-like substrate, immediately downstream of the 3' end of leading strand synthesis. Also, experiment with conditionally-replicating plasmids showed that replication of the target is absolutely required for transposition. Together, the results led us to propose a new model of replicative transposition according to which replication forks are hijacked by the transposon in order to recruit the replisome and to promote its duplication. Finally, we present preliminary data aiming at establishing the 3D structure of the TnpA by Cryo-Electron Microscopy in the framework of a collaboration with the group of Rouslan Efremov (VUB-VIB, Belgium).
- Affiliations
Citations
Oger, C. (2018). Ends-synapsis and target integration during replicative transposition of the Tn3-family transposon Tn4430. https://hdl.handle.net/2078.5/123551