Domain dynamics within the PriA DNA helicase regulate DNA replication restart

Prematurely terminated DNA replication processes in bacteria must be restarted for successful genome duplication. In Escherichia coli, the PriA DNA helicase orchestrates replication restart by assembling the PriA/PriB/DnaT preprimosome complex onto abandoned DNA replication forks and reloading the replicative machinery. We show that the structure of the replication fork lagging strand-whether single- or double-stranded-influences the position of the cysteine-rich region in bound PriA (PriACRR). When PriA binds to synthetic replication forks with a single-stranded lagging strand, the PriACRR is found either in a state similar to free PriA or in a rotated position that encircles the lagging strand and allows PriB recruitment. Binding to a synthetic replication fork with a duplex lagging strand neither alters the PriACRR position nor promotes PriB binding, suggesting PriA must unwind duplex lagging-strand DNA to trigger PriACRR movement and subsequent preprimosome formation. PriA variants designed to destabilize the two PriACRR positions differentially affect ATPase activity, helicase function, PriB binding in vitro, and PriA activity in vivo. These results support a regulatory switch model in which the lagging-strand DNA structure modulates the PriACRR position, thereby governing PriA's biochemical and cellular activities.