title: 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 uponin vitroreconstitution andin vivoensemble 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 processin vivodue to pervasive replication-transcription conflicts.
authors.affiliation: Department of Physics, University of Washington, Seattle, United States.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Mangiameli
authors.affiliation: Department of Microbiology, University of Washington, Seattle, United States.
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Merrikh
authors.affiliation: Department of Physics, University of Washington, Seattle, United States.|Department of Microbiology, University of Washington, Seattle, United States.|Department of Bioengineering, University of Washington, Seattle, United States.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Wiggins
authors.affiliation: Department of Microbiology, University of Washington, Seattle, United States.|Department of Genome Sciences, University of Washington, Seattle, United States.
authors.forename: Houra
authors.identifier: 0000-0001-9956-9640
authors.initials: H
authors.lastname: Merrikh
mesh_terms: D001419:Bacteria / Q000201:enzymology* / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004261:DNA Replication; D009097:Multienzyme Complexes / Q000378:metabolism*; D055550:Protein Stability; D014158:Transcription, Genetic
chemical_list: D018797:Cell Cycle Proteins; D009097:Multienzyme Complexes
keywords: B. subtilis; DNA replication; E. coli; biophysics; cell biology; replication rates; replication-transcription conflicts; replisome; structural biology