PriA helicase and SSB interact physically and functionally

Abstract

PriA helicase is the major DNA replication restart initiator in Escherichia coli and acts to reload the replicative helicase DnaB back onto the chromosome at repaired replication forks and D-loops formed by recombination. We have discovered that PriA-catalysed unwinding of branched DNA substrates is stimulated specifically by contact with the single-strand DNA binding protein of E.coli, SSB. This stimulation requires binding of SSB to the initial DNA substrate and is effected via a physical interaction between PriA and the C-terminus of SSB. Stimulation of PriA by the SSB C-terminus may act to ensure that efficient PriA-catalysed reloading of DnaB occurs only onto the lagging strand template of repaired forks and D-loops. Correlation between the DNA repair and recombination defects of strains harbouring an SSB C-terminal mutation with inhibition of this SSB-PriA interaction in vitro suggests that SSB plays a critical role in facilitating PriA-directed replication restart. Taken together with previous data, these findings indicate that protein-protein interactions involving SSB may coordinate replication fork reloading from start to finish.

Structural mechanisms of PriA-mediated DNA replication restart

“…In addition to structure-specific DNA binding, PriA interacts with the SSB C terminus (Ct) at replication forks (13)(14)(15). To identify the SSBCt binding site on PriA, we determined the 4.1-Å resolution structure of KpPriA bound to an SSB-Ct peptide (Table S1).…”

“…In addition to structure-specific DNA binding, PriA takes advantage of a direct protein interaction with ssDNAbinding protein (SSB) tetramers to target its activity to replication forks. SSBs coat the lagging strand template when it is single-stranded and form a complex with PriA that stimulates PriA's helicase activity (11)(12)(13)(14)(15). In this interaction, PriA binds to the extreme C terminus (Ct) of SSB, which is a known docking site for numerous genome maintenance proteins that process ssDNA within SSB/ssDNA nucleoprotein complexes (16).…”

The PriA Replication Restart Protein Blocks Replicase Access Prior to Helicase Assembly and Directs Template Specificity through Its ATPase Activity

“…Because it is thought that only the first 6 -10 nts of the lagging strand tail productively interact with the helicase, the remaining length requirement must be for additional protein factors to bind. At least part of the binding site for PriA, PriB and DnaT is near the fork (14), but these factors productively interact with SSB (44,45), and additional lagging strand length is likely required to accommodate that interaction. The requirement for a long duplex ahead of the fork and within the leading strand arm is less explicable.…”

Small-molecule tools for dissecting the roles of SSB/protein interactions in genome maintenance

“…Direct SSB-Ct binding and SSB-stimulated DNA unwinding activity have been described for both RecQ and PriA (8,10,11). However, the fold that binds SSB in ExoI is not conserved in RecQ, nor is it predicted to be conserved in PriA, which leaves open the question of whether the inhibitors can act on these SSB/protein complexes.…”