Replication termination without a replication fork trap

Abstract

Bacterial chromosomes harbour a unique origin of bidirectional replication, oriC . They are almost always circular, with replication terminating in a region diametrically opposite to oriC , the terminus. The oriC -terminus organisation is reflected by the orientation of the genes and by the disposition of DNA-binding protein motifs implicated in the coordination of chromosome replication and segregation with cell division. Correspondingly, the E. coli and B. subtilis model bacteria possess a replication fork trap system, Tus/ ter and RTP/ ter , respectively, which enforces replication termination in the terminus region. Here, we show that tus and rtp are restricted to four clades of bacteria, suggesting that tus was recently domesticated from a plasmid gene. We further demonstrate that there is no replication fork system in Vibrio cholerae , a bacterium closely related to E. coli . Marker frequency analysis showed that replication forks originating from ectopic origins were not blocked in the terminus region of either of the two V. cholerae chromosomes, but progressed normally until they encountered an opposite fork. As expected, termination synchrony of the two chromosomes is disrupted by these ectopic origins. Finally, we show that premature completion of the primary chromosome replication did not modify the choreography of segregation of its terminus region.

High-resolution whole-genome analysis of sister-chromatid cohesion

“…The extensive cohesion of the dif1-terrirory fits with previous fluorescence microscopy observations, which suggested that sister dif1 copies remained associated until the initiation of septum constriction Galli et al, 2017). However, sister copies of dif2, which is replicated at the same time of the cell cycle as dif1, were found to split ahead of the initiation of septum constriction, in apparent contradiction with the Hi-SC2 data Galli et al, 2019). To resolve this issue, we analysed sister-chromatid contacts at the time of cell division using a reporter based on a site-specific recombination machinery whose activity is restricted to the time of constriction, Xer Kennedy et al, 2008;Val et al, 2008).…”

“…Chr1 and Chr2 harbour a single origin of bi-directional replication, ori1 and ori2, respectively. Replication terminates in a region opposite of their origin, next to a site dedicated to the resolution of topological problems due to their circularity, dif1 and dif2, respectively (Galli et al, 2019;Val et al, 2008). Most strains of V. cholerae are not pathogenic or only cause local outbreaks of gastroenteritis.…”

Too Much of a Good Thing: How Ectopic DNA Replication Affects Bacterial Replication Dynamics

“…In oriC + oriX + tus cells the fork fusion point was close to the arithmetic mid-point between oriC and oriX and only slightly shifted toward the termination area (∼20 kb) (Dimude et al, 2018b), while for oriC + oriZ + tus cells forks terminated 60 kb in the direction of oriC (Ivanova et al, 2015;Dimude et al, 2016). We do not have any direct information about the speed of individual forks, but these results suggest that the forks leaving the termination area and traveling in the wrong orientation have, on average, a similar speed to the forks coming from oriC (oriX) or are even slightly faster (oriZ) (Ivanova et al, 2015;Dimude et al, 2016Dimude et al, , 2018b), similar to the situation observed in Vibrio cholerae where replication forks simply fused opposite the origin even when the origin was moved to an ectopic location (Galli et al, 2019).…”

“…Indeed, sequence analysis suggests that a replication fork trap is absent in many bacterial species. This was experimentally demonstrated for the two circular Vibrio cholerae chromosomes (Galli et al, 2019). Similarly, no specific termination-related pause sites have been identified in eukaryotes and archaea, even though multiple replication origins per chromosome result in a much higher number of fork fusions.…”

“…A recent analysis from Galli et al (2019) has shown that Tus-related sequences are found in most Enterobacteriales, in the Pseudoalteromonas, and in most Aeromonadales. In contrast, RTP-related sequences are restricted to a subgroup of the Bacillales (Galli et al, 2019). Indeed, sequence analysis suggests that a replication fork trap is absent in many bacterial species.…”