Structural visualisation of the dynamics of DNA unwinding by a replicative helicase

Ring-shaped hexameric helicases are nucleotide hydrolases that unwind double-stranded DNA into single strands, a necessary step in DNA replication. Two main questions are critical to understanding their function: the location of DNA strand separation within the helicase, and the precise dynamic linkage between nucleotide hydrolysis and DNA translocation. We explore these questions by employing cryo-EM to visualize the translocation mechanics of a AAA+ helicase, the SV40 Large Tumor Antigen (LTag), on forked DNA. We find the DNA fork nexus is positioned deep within the core helicase domain, with each DNA-binding loop from the six subunits securing the tracking strand by forming a paired staircase spiral that substitutes the passive strand. This structure forges an internal separation wedge, channeling the passive strand through a gap in the subunit C-tiers at the back of the helicase. Via cryo-EM continuous heterogeneity analysis we capture, and exhaustively model, a seamless spectrum of conformations of translocating LTag at high resolution. ATP hydrolysis at the tightest inter-subunit interface operates like an ‘entropy switch’, triggering coordinated rigid-body rotations of the subunit C-tiers and DNA-binding loops, resulting in directional escorting of the tracking strand through the central channel, concomitantly setting preparatory steps for cycle restart. Overall, we demonstrate a dynamic model for hydrolysis-coupled translocation and DNA unwinding by a model helicase active in replication, with implications for origin unwinding. High structural conservation of core helicase regions suggests this mechanism is applicable to hexameric helicases across domains.