DnaB helicase dynamics in bacterial DNA replication resolved by single-molecule studies

Abstract

In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment. However, due to technical limitations, accurate assessments of DnaB stability in the context of replication are lacking. Using in vitro fluorescence single-molecule imaging, we visualise DnaB loaded on forked DNA templates. That these helicases are highly stable at replication forks, indicated by their observed dwell time of ∼30 min. Addition of the remaining replication factors results in a single DnaB helicase integrated as part of an active replisome. In contrast to the dynamic behaviour of other replisome components, DnaB is maintained within the replisome for the entirety of the replication process. Interestingly, we observe a transient interaction of additional helicases with the replication fork. This interaction is dependent on the τ subunit of the clamp-loader complex. Collectively, our single-molecule observations solidify the role of the DnaB helicase as the stable anchor of the replisome, but also reveal its capacity for dynamic interactions.

Graphical Abstract

Single-molecule observation of the DnaB helicase in E. coli DNA replication shows a single, stably-integrated helicase supports replication, with additional helicases able to interact via available τ subunits.

Figure 1.

Schematic representation of the architecture of the E. coli replisome. The DnaB helicase enables the progression of the replisome as it unwinds double-stranded DNA. DnaG primase synthesises short RNA primers (shown in red) on the single-stranded DNA template. The clamp-loader complex (CLC; consisting of δ, δ', ψ, χ and three τ) loads the β₂ sliding clamp and the αϵθ polymerase III core onto newly primed sites. The core then synthesises new DNA on both template strands. DNA synthesis occurs continuously on the leading strand and discontinuously on the lagging strand. The CLC tethers the polymerase to the helicase via one τ subunit. Single-stranded DNA-binding protein (SSB) coats and protects the transiently exposed DNA on the lagging strand.

Figure 2.

Visualisation of loaded DnaB helicases at the single-molecule level. (A) Illustration of the singlemolecule helicase-loading assay. DnaB₆(red)DnaC₆, a 2030 bp rolling-circle DNA template and the nucleotides required for loading are mixed and applied to a microfluidic flow channel. The 5′-biotinylated DNA couples to the streptavidin-functionalised surface and immobilises the complex. (B) Loaded DnaB helicases appear as colocalised foci (white) of DnaB₆(red) and SYTOX Orange-stained DNA (green). The table indicates the number of foci, the degree of colocalisation and the degree of coincidental colocalisation by chance. (C) Distribution of DnaB₆(red) stoichiometry loaded onto the 59-nt single-stranded DNA tail (n = 606). The black line represents the sum of three Gaussian distribution functions fit to the data. The dashed grey lines represent the individual Gaussian distributions. (D) The average binding lifetime of loaded DnaB₆(red) molecules (magenta; n = 123). A single-exponential fit to the data (black) gives a binding lifetime of 34.4 ± 0.4 min. Photobleaching time is measured to be ∼60 min (Supplementary Figure S4) and therefore does not significantly impact on the observed kinetics.

Figure 3.

More than one DnaB helicase are frequently present at the replication fork. (A) Illustration of the rolling-circle replication assay with DnaB₆(red) preloaded on the DNA prior to introducing the replication solution. Replication is monitored in real time by flow stretching the replicating DNA products by hydrodynamic force. (B) (Top) Representative kymograph of preloaded DnaB₆(red) moving with the fork during rolling-circle replication. (Bottom) Overlay of the DnaB₆(red) and SYTOX Orange-stained DNA (green) kymographs shows the helicase molecule moving with the replication fork at the tip of the DNA product. (C) Distributions of DnaB helicase stoichiometry at the fork in the absence of DnaB₆(red) in solution (magenta; n = 32) and in the presence of DnaB₆(red) in solution (purple; n = 33). The black lines represent Gaussian fits to the data. (D) Illustration of the in-solution assay, where DnaB₆(red) is preloaded and also included at 2 nM in the replication solution. (E) Representative kymograph showing the DnaB₆(red) signal at the fork when DnaB₆(red) is present in solution. (F) Number of DnaB₆(red) as a function of time for the kymograph in (E) showing the fluctuation in DnaB helicase stoichiometry during the course of replication, where steps are detected by change-point analysis (47–49).

Figure 4.

DnaB helicases are both stable and dynamic during replication. (A) Illustration of the WT DnaB chase assay, where preloaded DnaB₆(red) was ‘chased’ with a relatively high concentration of WT DnaB (30 nM) in the replication solution. Like the standard rolling-circle assay, DNA products are stretched out by hydrodynamic force. (B) Representative kymograph of DnaB₆(red) moving with the fork during rolling-circle replication in the WT DnaB chase assay. (C) The average intensity over time from replicating DnaB₆(red) molecules in the WT DnaB chase assay (magenta; n = 29), compared to the photobleaching lifetime of DnaB₆(red) (grey; n = 667). The curve from each condition is fit with a single-exponential decay to provide the characteristic lifetime. (D) Illustration of the DnaB₆(blue) chase assay, where preloaded DnaB₆(red) is ‘chased’ with DnaB₆(blue) (2 nM) in the replication solution. Again, the DNA products are stretched out by hydrodynamic force. (E) (Top) Representative kymographs of DnaB₆(red) moving with the fork during rolling-circle replication in the DnaB₆(blue) chase assay. (Bottom) The DnaB₆(blue) kymograph from the same replication event shows the frequent association of additional helicases with the replication fork. (F) The stoichiometry over time from both the DnaB₆(red) and DnaB₆(blue) signal corresponding to the kymograph in (E), where steps are detected by change-point analysis (47–49).

Figure 5.

Additional helicases are able to interact with the replisome through the τ subunit of the clamploader complex. (A) Illustration of the stationary replisome-association assay. The replisome (including DnaB₆(blue)) is assembled during the ‘association’ phase after preloading DnaB₆(red) onto DNA. Including only dGTP and dCTP in this reaction permits the replisome to assemble but precludes net DNA synthesis. (B) Example kymographs from different experiments where the CLC has a varying composition of τ subunits. Comparing the DnaB₆(blue) kymographs shows how changing the reaction composition affects the frequency at which free DnaB₆(blue) binds to assembled replisomes. More example kymographs can be found in the Supplementary Figure S9. (C) The detection of DnaB₆(blue) binding from the example kymographs from the τ₃ replisome chase and γ₃ replisome chase experiments in (B). A binding event is recorded (dark blue) when the intensity of the DnaB₆(blue) signal (light blue) passes the threshold level (red). (D) Comparison of the binding frequency of DnaB₆(blue) from different experiments: τ₃ replisome, n = 123; τ₁γ₂ replisome, n = 70; γ₃ replisome, n = 43; DnaB₆(blue) only, n = 74; DnaB₆(blue) and SSB only, n = 36. Tabulation of these values can be found in Supplementary Figure S11.