SSB facilitates fork substrate discrimination by PriA

PriA is a member of the SuperFamily 2 helicase family. Its role in vivo is to reload the primosome onto stalled replication forks resulting in the restart of the previously stalled DNA replication process. SSB is known to play key roles in mediating activities at replication forks and it is known to bind to PriA. To gain mechanistic insight into the PriA-SSB interaction, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of PriA in vitro in the presence of fork substrates. The results demonstrate that SSB enhances the ability of PriA to discriminate between fork substrates 140-fold. This is due to a significant increase in the catalytic efficiency of the helicase induced by DNA-bound SSB. This interaction is species-specific as bacteriophage gene 32 protein cannot substitute for the E.coli protein. SSB, while enhancing the activity of PriA on its preferred fork, both decreases the affinity of the helicase for other forks and decreases catalytic efficiency. Central to the stimulation afforded by SSB is the unique ability of PriA to bind with high affinity to the 3-OH placed at the end of the nascent leading strand at the fork. When both the 3-OH and SSB are present, the maximum effect is observed. This ensures that PriA will only load onto the correct fork, in the right orientation, thereby ensuring that replication restart is directed to only the template lagging strand.