Bacterial DnaB helicase interacts with the excluded strand to regulate unwinding

Abstract

Replicative hexameric helicases are thought to unwind duplex DNAby steric exclusion (SE) where oneDNAstrand 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 Sulfolobus solfataricus minichromosome maintenance (SsoMCM) helicase has been shown to unwind DNA via a SEW mode to enhance unwinding efficiency. Using single-molecule FRET, we now show that the analogous Escherichia coli (Ec) 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 with 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 of this, experiments neutralizing the charge of the excluded strand with a morpholino substrate instead ofDNAalso 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 unwindDNAwith opposite polarities. These results support the SEWmodel 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.