PriC-mediated DNA replication restart requires PriC complex formation with the single-stranded DNA-binding protein

Sarah R. Wessel, Aimee H. Marceau, Shawn C. Massoni, Ruobo Zhou, Taekjip Ha, Steven J. Sandler, James L. Keck

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)17569-17578
Number of pages10
JournalJournal of Biological Chemistry
Volume288
Issue number24
DOIs
StatePublished - Jun 14 2013

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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