Transcription leads to pervasive replisome instability in bacteria

Abstract

The canonical model of DNA replication describes a highly-processive and largely continuous process by which the genome is duplicated. This continuous model is based upon in vitro reconstitution and in vivo ensemble experiments. Here, we characterize the replisome-complex stoichiometry and dynamics with single-molecule resolution in bacterial cells. Strikingly, the stoichiometries of the replicative helicase, DNA polymerase, and clamp loader complexes are consistent with the presence of only one active replisome in a significant fraction of cells (>40%). Furthermore, many of the observed complexes have short lifetimes (<8 min), suggesting that replisome disassembly is quite prevalent, possibly occurring several times per cell cycle. The instability of the replisome complex is conflict-induced: transcription inhibition stabilizes these complexes, restoring the second replisome in many of the cells. Our results suggest that, in contrast to the canonical model, DNA replication is a largely discontinuous process in vivo due to pervasive replication-transcription conflicts.DOI: http://dx.doi.org/10.7554/eLife.19848.001

Replication Restart in Bacteria

“…The reasons for the discrepancy between these population and single-cell studies are unknown. In the same single-molecule microscopy study, measurements of the lifetimes of DNA complexes suggest that restart could be as frequent as five times per generation in Bacillus subtilis, as in E. coli (22). This result highlights the importance of replication restart and shows clearly that replication restart most often does not involve homologous recombination, as this frequency would then be incompatible with the viability of recombination mutants.…”

“…That study suggested that the range could be around 18 to 25% of the E. coli cells in a population (21). In contrast, direct measurements of helicase stability in the same dnaC2(Ts) mutant by single-molecule microscopy showed that both helicase complexes disassembled within 20 min in 86% of the cells studied (22). The reasons for the discrepancy between these population and single-cell studies are unknown.…”

The Clash of Macromolecular Titans: Replication-Transcription Conflicts in Bacteria

“…However, recent findings suggest that the most common cause of replisome stalling in vivo is RNAP (28, 35, 65). The majority of endogenous replication fork stalling detected by various methods is caused by transcription (28, 35).…”

“…However, recent findings suggest that the most common cause of replisome stalling in vivo is RNAP (28, 35, 65). The majority of endogenous replication fork stalling detected by various methods is caused by transcription (28, 35). However, regardless of the cause, stalled replication forks, in general, are a major problem for growing cells and must be dissolved and restarted in order for replication to be completed and the chromosomes to be segregated into daughter cells.…”

“…A single RNAP moving head-on can dislodge the replisome in vitro (26, 50). In vivo, in a head-on collision, the replisome disassembles, the DNA breaks, and local mutation rates more than double (7, 14, 24, 28, 38, 39, 41, 48, 55, 65). Therefore, despite the common assumption in the field, the consequences of head-on conflicts are not necessarily linked to transcription levels.…”