Transcription leads to pervasive replisome instability in bacteria.

Sarah M Mangiameli,Paul A Wiggins,Houra Merrikh,Christopher N Merrikh

doi:10.7554/elife.19848

Sarah M Mangiameli, Paul A Wiggins + Show 2 more

Open Access

https://doi.org/10.7554/elife.19848

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Journal: eLife	Publication Date: Jan 16, 2017
Citations: 68	License type: CC BY 4.0

Affiliation: Seattle University, University of Washington

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.

Highlights

IntroductionThe cellular machinery responsible for DNA replication, has been extensively studied both in vitro and in vivo (O’Donnell et al, 2013), fundamental questions remain about the dynamics and stability of the replication complex in the context of the living cell, where replication is one of a number of essential cellular processes competing for the genetic material as a template
The rapid and faithful replication of the genome is essential to cell proliferation
The quantitative characterization of the molecular stoichiometry of the replisome in living E. coli cells was recently realized by Single-Molecule Fluorescence Microscopy (SMFM) (Reyes-Lamothe et al, 2010), and this SMFM analysis has been applied in many other contexts (e.g. [Leake et al, 2006] and [Ulbrich and Isacoff, 2007])

Summary

Introduction

The cellular machinery responsible for DNA replication, has been extensively studied both in vitro and in vivo (O’Donnell et al, 2013), fundamental questions remain about the dynamics and stability of the replication complex in the context of the living cell, where replication is one of a number of essential cellular processes competing for the genetic material as a template This competition results in replication conflicts, the stalling or pausing of the replication process in the face of obstacles, including transcription and tightly-bound DNA-binding proteins (Baharoglu et al, 2010; Bidnenko et al, 2006; Boubakri et al, 2010; Brewer, 1988; Dutta et al, 2011; Marsin et al, 2001; Merrikh et al, 2011, 2012; Mirkin and Mirkin, 2005, 2007; Soultanas, 2011). Estimates of less than one event per cell cycle are in conflict with the essentiality of the restart protein PriA in (Bacillus subtilis essentiality is demonstrated in rapid growth [Polard et al, 2002]), and the synthetic lethality of PriB and PriC

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