Physical Basis for the Loading of a Bacterial Replicative Helicase onto DNA

Abstract

In cells, dedicated AAA+ ATPases deposit hexameric, ring-shaped helicases onto DNA to initiate chromosomal replication. To better understand the mechanisms by which helicase loading can occur, we used cryo-EM to determine sub-4-Å-resolution structures of the E. coli DnaB⋅DnaC helicase⋅loader complex with nucleotide in pre- and post-DNA engagement states. In the absence of DNA, six DnaC protomers latch onto and crack open a DnaB hexamer using an extended N-terminal domain, stabilizing this conformation through nucleotide-dependent ATPase interactions. Upon binding DNA, DnaC hydrolyzes ATP, allowing DnaB to isomerize into a topologically closed, pre-translocation state competent to bind primase. Our data show how DnaC opens the DnaB ring and represses the helicase prior to DNA binding and how DnaC ATPase activity is reciprocally regulated by DnaB and DNA. Comparative analyses reveal how the helicase loading mechanism of DnaC parallels and diverges from homologous AAA+ systems involved in DNA replication and transposition.

Figure 1.. Structure of DNA-free E. Coli DnaBC. See also Figure S1 and S2.

(A) Primary structure of E. coli DnaC and DnaB. Color scheme is maintained throughout all figures. (B) Dilated conformation of DnaB (PDB 2R6A (Bailey et al., 2007)). (C) Constricted conformation of DnaB (PDB 4NMN (Strycharska et al., 2013)). (D) Two orthogonal views of E. coli DnaBC. Insets depict the unsharpened EM density to highlight the gap in the structure. In the upper panel, an entry pathway for ssDNA has been highlighted. (E) View of E. coli DnaBC highlighting domain structure and organization. (F) Detailed view of the boxed area in (E) showing the interaction network between DnaB and DnaC. Density contoured at a threshold value of 0.05.

Figure 2.. DnaC is a functional AAA+ ATPase. See also Figure S3.

(A) DnaC ATPase activity requires both DnaB and ssDNA. Rates for ATP-hydrolysis were determined for WT and K112R Walker-A mutant using a coupled ATP-hydrolysis assay. Data are represented as mean ± SEM. (B) Kinetics of ATP-hydrolysis by DnaC. A coupled ATP-hydrolysis assay was performed using wild type DnaC to calculate hydrolysis rates at various ATP concentrations. Rates were fitted to the Michaelis-Menten equation. Error bars represent the variation over three independent experiments (mean ± SEM). (C) The helical pitch and curvature of the DnaC hexamer in the complex with the helicase is more expanded than that observed for the isolated DnaC AAA+ domains (PDB 3ECC (Mott et al., 2008)). (D)The loop adjacent to the Walker-B motif in E. coli DnaC adopts a different configuration from that of A. aeolicus DnaC.

Figure 3.. Molecular architecture of E. coli DnaBC bound to ssDNA. See also Figure S4.

(A) Top view of the E. coli DnaBC complex bound to dT36. The nucleic acid (red) can be seen in the central pore. (B) Side view of the DnaBC structure. (C) Density for ssDNA bound to the central pore of the helicase and loader hexamers. The most frontal DnaB and DnaC monomers have been removed to visualize the nucleid acid. Threshold value of 0.05 used for rendering.

Figure 4.. Molecular basis for ssDNA recognition by and activation of DnaC.

(A) DNA-bound DnaC associates with ADP. The Arg finger and sensor-II residues are disengaged from the nucleotide. Density contoured at a threshold value of 0.05. (B) The DnaC ATPase hexamer constricts around DNA. (C) DnaC NTDs pivot between apo and ssDNA-bound states. (D) The junction between the DnaC NTD and AAA+ domain is fulcrum that accommodates ATPase movements upon ssDNA binding. (E) Detailed view of DnaC residues involved in engaging bound ssDNA. Density contoured at a threshold value of 0.05. (F) ssDNA binding triggers a conformational change in the extended Walker-B loop.

Figure 5.. ssDNA binding by DnaC is essential for ATP hydrolysis and helicase loading. See also Figure S5.

(A) Coupled ATP-hydrolysis assays show that mutations to residues involved in binding ssDNA impair ATPase activity. Data are represented as mean ± SEM. (B) Fluorescence anisotropy assays show that the mutation of residues seen to interact with ssDNA lead to binding defects. Binding reactions contained 10 nM fluorescein-labeled dT25 oligonucleotide titrated against either WT or specified DnaC mutant protein. Error bars represent the variation over three independent experiments (mean ± SEM). (C) DnaC-DNA interactions are critical for DnaB loading. Data presented are from three independent experiments (mean ± SEM).

Figure 6.. DNA remodels the molecular organization of DnaB.

(A) Close-up view DnaB residues involved in DNA binding. Density contoured at a threshold value of 0.05. (Inset) View of ssDNA bound to the RecA domains of DnaB. (B) The DnaB RecA domains adopt a more planar, crack-ring configuration in the presence of nucleic acid. (C) ADP•BeF₃ is associated with the active sites of ssDNA-bound DnaB. Density contoured at a threshold value of 0.05. (D) A terminal DnaB subunit relocates from one end of the cracked helicase ring to the other during a conformation transition induced by ssDNA binding. The open crack in the DnaB ring in absence of DNA is sealed off by the linker helix following DNA engagement, topologically linking the helicase around DNA. (Inset) Top-down views showing the conformational transition in the DnaB collar and the shift in the position of the crack in the helicase ring following the transition. Asterisks denote the position of the crack in the DnaB ring. (E) The configuration of the helicase in the ssDNA-bound DnaBC complex is highly reminiscent to that seen for the free helicase bound to nucleic acid and GDP•AlF_x (PDB 4ESV (Itsathitphaisarn et al., 2012)).

Figure 7.. Comparison of ring-loading reactions occurring during replication (see text for details). See also Figure S6 and S7.

(A) Diagram of the DNA polymerase clamp loader reaction. (B) Schematic of the helicase loading reaction catalyzed by DnaC. (C) Overview of MCM2–7 loading by ORC and Cdc6. Although DNA is encircled by ORC (Li et al., 2018), the complex is not topologically closed and readily dissociates from DNA (Duzdevich et al., 2015); Cdc6 binding to ORC topologically entraps DNA (Bleichert et al., 2018; Yuan et al., 2017). Recent studies have shown that DNA is bent substantially upon both ORC and ORC•Cdc6 binding (Bleichert et al., 2018; Li et al., 2018).