The hexameric helicase DnaB adopts a nonplanar conformation during translocation

DNA polymerases can only synthesize nascent DNA from single-stranded DNA (ssDNA) templates. In bacteria, the unwinding of parental duplex DNA is carried out by the replicative DNA helicase (DnaB) that couples NTP hydrolysis to 50 to 30 translocation. The crystal structure of the DnaB hexamer in complex with GDP-AlF4 and ssDNA reported here reveals that DnaB adopts a closed spiral staircase quaternary structure around an A-form ssDNA with each C-terminal domain coordinating two nucleotides of ssDNA. The structure not only provides structural insights into the translocation mechanism of superfamily IV helicases but also suggests that members of this superfamily employ a translocation mechanismthat is distinct from other helicase superfamilies. We propose a hand-over-hand mechanism in which sequential hydrolysis of NTP causes a sequential 50 to 30 movement of the subunits along the helical axis of the staircase, resulting in the unwinding of two nucleotides per subunit.