External Seminar "Hot in front! Hunting host replication by integrating ahead of replication forks during replicative DNA transposition" by Bernard Hallet

Bernard HALLET, UCLouvain

Title: "Hot in front! Hunting host replication by integrating ahead of replication forks during replicative DNA transposition"

Abstract: 
Bacterial transposons of the Tn3 family are known for their prevalence in the dissemination of antimicrobial resistance among pathogens. The efficiency of these transposons relies on their replicative mode of transposition, generating a new copy of themselves every time they move. Our recent work on the Tn3-family transposon Tn4430 has provided new insights into how the transposition complex assembles and activates to catalyse DNA breakage and re-joining reactions. This include high-resolution CryoEM structures of the transposases bound to the DNA substrates at different stages of transposition. However, the mechanism by which host DNA replication is recruited during transposition remains unknown.
Transposition of Tn4430 only occurs in target DNA molecules that undergo replication, suggesting that ongoing target replication is crucial for replisome recruitment. Furthermore, a genome-wide analysis of Tn4430 insertion sites revealed that integration occurs preferentially in regions where replication dynamics is altered. In the chromosome, this corresponds to the terminus region where replisomes are stalled by Tus/ter complexes, or to regions where converging replication forks meet and collide. Strong integration hotspots are also observed at other replication roadblocks, such as head-on collisions between DNA and RNA polymerases downstream of highly transcribed genes. Interestingly, these hotspots overlap with the preferred binding regions of GapR, a Caulobacter crecentus protein that specifically binds to positively supercoiled DNA. Consistently, alignment of Tn4430 integration sites reveals a signature sequence composed of a 7-bp 5'-CTNNNAG-3' core consensus flanked by periodically alternating G/C- and A/T-rich stretches. This sequence is predicted to adopt a right-handed superhelical twist such as is found at the apex of positive supercoils. 
Together, these results support the hypothesis of a replication hijacking mechanism by which Tn3-family transposons specifically integrate into transient positive DNA supercoils that form downstream of replication forks, in order to recruit the host replication machinery during transposition.