Bacterial DnaB helicase interacts with the excluded strand to regulate unwinding [Molecular Biophysics]

Replicative hexameric helicases are thought to unwind duplex DNA by steric exclusion (SE), where one DNA strand is encircled by the hexamer and the other is excluded from the central channel. However, interactions with the excluded strand on the exterior surface of hexameric helicases have also been shown to be important for DNA unwinding, giving rise to the steric exclusion and wrapping (SEW) model. For example, the archaeal SsoMCM helicase has been shown to unwind DNA via a SEW mode to enhance unwinding efficiency. Using single molecule FRET (smFRET), we now show that the analogous E. coli DnaB helicase also interacts specifically with the excluded DNA strand during unwinding. Mutation of several conserved and positively-charged residues on the exterior surface of EcDnaB resulted in increased interaction dynamics and states compared to wild-type. Surprisingly, these mutations also increased the DNA unwinding rate, suggesting that electrostatic contacts with the excluded strand act as a regulator for unwinding activity. In support, experiments neutralizing charge of the excluded strand with a morpholino substrate instead of DNA also dramatically increased the unwinding rate. Of note, although the stability of the excluded strand was nearly identical for EcDnaB and SsoMCM, these enzymes are from different superfamilies and unwind DNA with opposite polarities. These results support the SEW model of unwinding for EcDnaB that expands on the existing SE model of hexameric helicase unwinding to include contributions from the excluded strand to regulate the DNA unwinding rate.

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