An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase.

Helicases couple ATP hydrolysis to nucleic acid binding and unwinding via molecular mechanisms that remain poorly defined for most enzyme subfamilies within the superfamily 2 (SF2) helicase group. A crystal structure of the PriA SF2 DNA helicase, which governs restart of prematurely terminated replication processes in bacteria, revealed the presence of an aromatic-rich loop (ARL) on the presumptive DNA-binding surface of the enzyme. The position and sequence of the ARL was similar to loops known to couple ATP hydrolysis with DNA binding in a subset of other SF2 enzymes, however, the roles of the ARL in PriA had not been investigated. Here, we show that changes within the ARL sequence uncouple PriA ATPase activity from DNA binding. In vitro protein-DNA crosslinking experiments define a residue-and nucleotide-specific interaction map for PriA, showing that the ARL binds replication fork junctions whereas other sites bind the leading or lagging strands. We propose that DNA binding to the ARL allosterically triggers ATP hydrolysis in PriA. Additional SF2 helicases with similarly positioned loops may also couple DNA binding to ATP hydrolysis using related mechanisms.