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.
Highlights
PriC can reload the DnaB helicase onto single-stranded DNA-binding protein (SSB)-coated DNA, which is the first step in DNA replication restart
Plasmids expressing the Gal4 DNA binding domain fused to the N terminus of E. coli PriC and the Gal4 activation domain fused to the N terminus of E. coli SSB were co-transformed into an S. cerevisiae strain in which HIS3 and ADE2 expression were regulated by the GAL1 and GAL2 promoters, respectively
We have examined the mechanisms by which the PriC DNA replication restart protein facilitates DnaB helicase loading onto SSB-coated DNA substrates
Summary
PriC can reload the DnaB helicase onto single-stranded DNA-binding protein (SSB)-coated DNA, which is the first step in DNA replication restart. Singlemolecule 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. Single-molecule (sm) fluorescence resonance energy transfer (FRET) studies demonstrate that PriC binding to SSB alters SSB/DNA complexes in a manner that preferentially stabilizes a highly cooperative binding mode of SSB over that of a less cooperative mode and in the process exposes ssDNA, making it available for additional protein binding These data support a model in which PriC binding to SSB helps to exclude SSB from a region of ssDNA onto which DnaB can be loaded
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