title: 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.
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: M Charl
authors.initials: MC
authors.lastname: Moolman
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: Sriram
authors.initials: S
authors.lastname: Tiruvadi Krishnan
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: Jacob W J
authors.initials: JW
authors.lastname: Kerssemakers
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
authors.forename: Roy
authors.initials: R
authors.lastname: de Leeuw
authors.affiliation: Université Paris 13, Sorbonne Paris Cité, Laboratoire de Physique des Lasers, CNRS, (UMR 7538), F-93430 Villetaneuse, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Lorent
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Sherratt
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands n.h.dekker@tudelft.nl.
authors.forename: Nynke H
authors.initials: NH
authors.lastname: Dekker
mesh_terms: D020090:Chromosome Segregation / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000235:genetics; D004265:DNA Helicases / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D046911:Macromolecular Substances / Q000378:metabolism
chemical_list: C062011:DNA replication terminus site-binding protein, E coli; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C489236:tus protein, E coli; D004265:DNA Helicases