The impact of DNA replication on insertion site selection of the bacterial transposon TN4430

Details

Supervisors

Hallet, Bernard

Faculty

Faculté des sciences

Degree label

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

Abstract

enTn4430 is a 4.1 kb Tn3-family transposon from Bacillus thuringiensis that functions in E. coli. It encodes TnpA (a transposase) and TnpI (a site-specific recombinase) and appears to transpose through a unique mechanism termed replication hijacking, in which the transposon targets stalled or slowed replication intermediates and simultaneously recruits the host’s replication machinery for its own duplication during integration. Tn4430 transposition mapping in E. coli chromosome showed preferred insertion hotspots near the replication terminus, indicating a strong influence of DNA replication dynamics on target site selection. In contrast, insertion into the ColE-1 derived plasmid pRS415 revealed a completely different insertion pattern with high- and low-frequency integrations sites distributed throughout the target DNA. In this master’s thesis, I investigated Tn4430 insertion pattern in pRS415 by recreating specific chromosomal features, such as a bidirectional origin of replication and a highly transcribed operon, within the plasmid. I also tested the hypothesis that insertion pattern was linked to Okazaki fragment maturation, which could transiently slow the replisome and create a window of vulnerability for transposon integration. My results refuted the Okazaki fragment hypothesis and identified new factors influencing target site selection, thereby refining the replication hijacking model.