Internal unwinding mechanism of a replicative helicase

Ring-shaped hexameric helicases are nucleotide hydrolases that unwind double-stranded DNA into single strands, a necessary step in DNA replication. Critical to understanding their function are two outstanding questions: the location of DNA strand separation within the helicase, particularly at the onset of unwinding, and the precise dynamic linkage between nucleotide hydrolysis and DNA translocation. We explored 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 which 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 with near-atomic precision. 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 within the central channel, concomitantly setting preparatory steps for cycle restart. Overall, we demonstrate a dynamic model for hydrolysis-coupled translocation and internal DNA unwinding by a model helicase active in replication, and potentially during initial origin melting. Structural conservation of core helicase domains suggests the mechanism may be applicable to hexameric helicases across species.