title: Strand separation establishes a sustained lock at the Tus-Ter replication fork barrier.
abstract: The bidirectional replication of a circular chromosome by many bacteria necessitates proper termination to avoid the head-on collision of the opposing replisomes. In Escherichia coli, replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand separation on the blocking (nonpermissive) side of Tus-Ter triggers roadblock formation, but biochemical evidence also suggests roles for protein-protein interactions. Here DNA unzipping experiments demonstrate that nonpermissively oriented Tus-Ter forms a tight lock in the absence of replicative proteins, whereas permissively oriented Tus-Ter allows nearly unhindered strand separation. Quantifying the lock strength reveals the existence of several intermediate lock states that are impacted by mutations in the lock domain but not by mutations in the DNA-binding domain. Lock formation is highly specific and exceeds reported in vivo efficiencies. We postulate that protein-protein interactions may actually hinder, rather than promote, proper lock formation.
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands.
authors.forename: Bojk A
authors.initials: BA
authors.lastname: Berghuis
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands.
authors.forename: David
authors.initials: D
authors.lastname: Dulin
authors.affiliation: Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia.
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Xu
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands.
authors.forename: Theo
authors.initials: T
authors.lastname: van Laar
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands.
authors.forename: Bronwen
authors.initials: B
authors.lastname: Cross
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands.
authors.forename: Richard
authors.initials: R
authors.lastname: Janissen
authors.affiliation: Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia.
authors.forename: Slobodan
authors.initials: S
authors.lastname: Jergic
authors.affiliation: Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia.
authors.forename: Nicholas E
authors.identifier: 0000000259586945
authors.initials: NE
authors.lastname: Dixon
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands.
authors.forename: Martin
authors.initials: M
authors.lastname: Depken
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands.
authors.forename: Nynke H
authors.initials: NH
authors.lastname: Dekker
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002876:Chromosomes, Bacterial / Q000737:chemistry / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004270:DNA, Circular / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: C062011:DNA replication terminus site-binding protein, E coli; D004269:DNA, Bacterial; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C489236:tus protein, E coli