The progression of replication forks at natural replication barriers in live bacteria

Abstract

Protein–DNA complexes are one of the principal barriers the replisome encounters during replication. One such barrier is the Tus–ter complex, which is a direction dependent barrier for replication fork progression. The details concerning the dynamics of the replisome when encountering these Tus–ter barriers in the cell are poorly understood. By performing quantitative fluorescence microscopy with microfuidics, we investigate the effect on the replisome when encountering these barriers in live Escherichia coli cells. We make use of an E. coli variant that includes only an ectopic origin of replication that is positioned such that one of the two replisomes encounters a Tus–ter barrier before the other replisome. This enables us to single out the effect of encountering a Tus–ter roadblock on an individual replisome. We demonstrate that the replisome remains stably bound after encountering a Tus–ter complex from the non-permissive direction. Furthermore, the replisome is only transiently blocked, and continues replication beyond the barrier. Additionally, we demonstrate that these barriers affect sister chromosome segregation by visualizing specific chromosomal loci in the presence and absence of the Tus protein. These observations demonstrate the resilience of the replication fork to natural barriers and the sensitivity of chromosome alignment to fork progression.

27nt-RNAs guide histone variant deposition via ‘RNA-induced DNA replication interference’ and thus transmit parental genome partitioning in Stylonychia

“…Remarkably, a unidirectional sterical DNA barrier function by Tus-Ter affects replication fork progression in E. coli . Interestingly, it was shown that the Tus-Ter only transiently blocks the replisome in the non-permissive direction [ 43 ], which is reminiscent of our hypothesis for Stylonychia . We thus believe that a transient DNA replication ‘road block’ is the most plausible explanation for 27nt-RNA/PIWI1 complexes.…”

Chromosomal over-replication in Escherichia coli recG cells is triggered by replication fork fusion and amplified if replichore symmetry is disturbed

“…It was recently reported that replisomes remain stably bound at ter /Tus complexes (56). However, both in vitro and in vivo measurements of fork stability at obstacles such as supercoiling and repressor-operator complexes suggest a limited half-life of 4–6 min (57–59).…”

Replication Termination: Containing Fork Fusion-Mediated Pathologies in Escherichia coli

“…This would then trigger recombination-driven replication restart [35,36,37]. Thus, while forks blocked at a ter /Tus complex can be restarted eventually, a single replisome will be stably arrested at a ter /Tus complex for a significant period of time, as recently demonstrated [38], with RecA being important for maintaining stability at forks [39]. In line with this idea, it was shown that the inversion of one ter site, which blocked replication of ~1 kb of the chromosome in the presence of Tus protein, caused severe filamentation [40].…”