title: PriC-mediated DNA replication restart requires PriC complex formation with the single-stranded DNA-binding protein.
abstract: Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated with DNA replication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step of DNA replication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Single-molecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading.
authors: Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, USA.
authors: Sarah R
authors: SR
authors: Wessel
authors: Aimee H
authors: AH
authors: Marceau
authors: Shawn C
authors: SC
authors: Massoni
authors: Ruobo
authors: R
authors: Zhou
authors: Taekjip
authors: T
authors: Ha
authors: Steven J
authors: SJ
authors: Sandler
authors: James L
authors: JL
authors: Keck
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D053488:DnaB Helicases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D020798:Two-Hybrid System Techniques
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C476430:SSB protein, E coli; C069995:PriC protein, E coli; C506730:dnaB protein, E coli; D053488:DnaB Helicases
keywords: DNA Repair; DNA Replication; Fluorescence Resonance Energy Transfer (FRET); Protein Complexes; Protein-DNA Interaction; Protein-Protein Interactions; Single Molecule Biophysics
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