Visualization of the complete preprimosome reveals the structural mechanisms governing DNA replication restart

Abstract Replication restart pathways reinitiate DNA replication processes following their premature termination. In Escherichia coli , this essential process begins with regulated assembly of the preprimosome complex, comprising the PriA, PriB, and DnaT proteins, onto an abandoned replication fork. Here, we present two distinct preprimosome structures. One represents an intermediate stage in preprimosome assembly with a single DnaT C-terminal domain (DnaT CTD ) bound to PriA/PriB/DNA. The second captures the mature preprimosome, in which filamentation of multiple DnaT CTD molecules catalyzes the handoff of the single-stranded lagging-strand DNA from PriB to DnaT. The DnaT N-terminal domain forms a separate, independent oligomer in the mature structure. Taken together, our results detail the molecular mechanisms underlying replication restart initiation and regulation and suggest mechanistic similarities between DnaT and the canonical initiator protein DnaA.