Bacterial DnaB helicase interacts with the excluded strand to regulate unwinding

Abstract

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 minichromosome maintenance (MCM) 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 () 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 DnaB 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 of DNA also dramatically increased the unwinding rate. Of note, although the stability of the excluded strand was nearly identical forDnaB and MCM, these enzymes are from different superfamilies and unwind DNA with opposite polarities. These results support the SEW model of unwinding forDnaB that expands on the existing SE model of hexameric helicase unwinding to include contributions from the excluded strand to regulate the DNA unwinding rate.

Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase

“…Control of DNA unwinding by replicative helicases through their interaction with the excluded strand has been demonstrated in different organisms. Although wrapping of the displaced strand around an archaeal MCM was proposed to increase its helicase activity 28 , interaction of DnaB with the displaced strand through its exterior surface adversely affects DNA unwinding 29 . While it is not clear whether CMG makes contacts with the lagging-strand template via specific residues on its outer surface, the presence of the excluded strand is important for unwinding of dsDNA by CMG.…”

Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

“…While one possible reason for the observed inhibition is the partial entrapment of the displaced strand (hence the parental duplex) in the helicase central pore, the other consideration is the interaction of the displaced strand with the outer surface of CMG. If a physical contact between the exterior of CMG and the laggingstrand template adversely affects helicase activity, as suggested for DnaB (Carney et al, 2017), hybridization of a complementary oligonucleotide or binding of RPA to the lagging-strand arm of a fork substrate may stimulate DNA unwinding by disrupting this interaction. To determine whether the inhibition of CMG helicase activity stems from its interaction with the displaced strand through the helicase exterior residues or interior channel, we introduced a biotinstreptavidin complex on the lagging-strand arm of a fork substrate.…”

“…Control of DNA unwinding by replicative helicases through their interaction with the excluded strand has been demonstrated in different organisms. For example, while wrapping of the displaced strand around an archaeal MCM was proposed to increase its helicase activity (Graham et al, 2011), interaction of DnaB with the displaced strand through its exterior surface adversely affects DNA unwinding (Carney et al, 2017). Although, it is not clear whether CMG makes contacts with the lagging-strand template via specific residues on its outer surface, the presence of the excluded strand is important for unwinding of dsDNA by CMG.…”

The Macromolecular Machines that Duplicate the Escherichia coli Chromosome as Targets for Drug Discovery

“…By comparison, the eukaryotic replicative DNA helicase named MCM2-7 is a member of Superfamily 6. In the process of unwinding DNA, independent biochemical studies support the model that DnaB is bound at the replication fork to one of the two parental DNA strands [ 145 , 146 , 147 ]. Cryoelectron microscopy of E. coli DnaB [ 148 , 149 ], and X-ray crystallography of Geobacillus kaustophilus , Geobacillus stearothermophilus and Bacillus subtilis DnaB showed that the native structure of DnaB is a hexamer of identical subunits assembled as a toroid [ 81 , 150 , 151 ].…”

“…Evidence indicates that DnaB and its homologues in other organisms translocate in the 5’-to-3’ direction on the single-stranded DNA to which they are bound [ 157 , 159 , 160 , 161 ]. Like other DNA helicases that are toroids [ 140 , 141 , 162 , 163 , 164 ], this DNA strand passes through the central cavity of DnaB, apparently interacting with specific residues that line the cavity during movement while the other parental DNA strand is excluded [ 146 , 147 ].…”