Collisions between cellular DNA replication machinery (replisomes) and damaged DNA or immovable protein complexes can dissociate replisomes before the completion of replication. This potentially lethal problem is resolved by cellular "replication restart" reactions that recognize the structures of prematurely abandoned replication forks and mediate replisomal reloading. In bacteria, this essential activity is orchestrated by the PriA DNA helicase, which identifies replication forks via structure-specific DNA binding and interactions with fork-associated ssDNA-binding proteins (SSBs). However, the mechanisms by which PriA binds replication fork DNA and coordinates subsequent replication restart reactions have remained unclear due to the dearth of high-resolution structural information available for the protein. Here, we describe the crystal structures of full-length PriA and PriA bound to SSB. The structures reveal a modular arrangement for PriA in which several DNA-binding domains surround its helicase core in a manner that appears to be poised for binding to branched replication fork DNA structures while simultaneously allowing complex formation with SSB. PriA interaction with SSB is shown to modulate SSB/DNA complexes in a manner that exposes a potential replication initiation site. From these observations, a model emerges to explain how PriA links recognition of diverse replication forks to replication restart.X-ray crystal structure | single-molecule FRET
DNA replication restart is an essential genome maintenance process by which replication complexes (replisomes) are reloaded onto abandoned DNA replication forks. In Escherichia coli and related bacteria this process is orchestrated by the PriA DNA helicase, which binds to replication forks in a structure‐specific manner. PriA also binds to single‐stranded DNA‐binding protein (SSB), a protein that coats the lagging‐strand template at DNA replication forks. Complex formation with SSB stimulates PriA DNA unwinding DNA and, as we show here, this interaction is required for PriA remodeling of SSB‐DNA nucleoprotein complexes. Proteins that interact with SSB typically do so by binding directly to the evolutionarily conserved amphipathic C‐terminal tail of SSB (SSB‐CT). Although previous studies indicated that PriA also binds to SSB via the SSB‐CT, a lack of structural information on PriA has left the SSB binding site on PriA unknown. We determined the 4 Å resolution structure of PriA bound to the SSB‐CT. The electrostatic characteristics of the PriA SSB‐Ct binding site resemble those of other known SSB‐interacting proteins. Single‐molecule FRET studies show that a PriA variant that has lost the ability to bind the SSB‐CT fails to modulate SSB‐DNA complexes. Our studies point to the importance of the PriA/SSB interaction in allowing PriA to reload replisomes at damaged replication forks where SSB is bound to the lagging‐strand template.
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