A structural view of bacterial DNA replication

Abstract

DNA replication mechanisms are conserved across all organisms. The proteins required to initiate, coordinate, and complete the replication process are best characterized in model organisms such as Escherichia coli. These include nucleotide triphosphate-driven nanomachines such as the DNA-unwinding helicase DnaB and the clamp loader complex that loads DNA-clamps onto primer-template junctions. DNA-clamps are required for the processivity of the DNA polymerase III core, a heterotrimer of α, ε, and θ, required for leading- and lagging-strand synthesis. DnaB binds the DnaG primase that synthesizes RNA primers on both strands. Representative structures are available for most classes of DNA replication proteins, although there are gaps in our understanding of their interactions and the structural transitions that occur in nanomachines such as the helicase, clamp loader, and replicase core as they function. Reviewed here is the structural biology of these bacterial DNA replication proteins and prospects for future research.

Keywords: DNA clamp; DNA polymerase; DNA replication; antimicrobials; clamp loader complex; helicase; macromolecular structure; primase; protein-DNA interaction; single-stranded DNA binding protein.

Figure 1

A schematic view of the replisome. Protein assemblies are modeled using representative structures from the PDB. Parent DNA strands are represented by continuous black and red lines for parental and nascent DNA strands, respectively. RNA primers are represented by dashed red lines. Disordered regions in proteins are indicated by dotted lines (lengths not to scale). On the lagging strand is DnaB helicase, which uses the energy of NTP hydrolysis to unwind dsDNA. DnaG primases bind to DnaB and synthesizes RNA primers, required by the polymerase on the lagging strand to initiate Okazaki fragment synthesis. The lagging strand ssDNA is protected by SSB. The clamp loader complex (CLC), with subunit composition δ(γ/τ)₃δ'ψχ, uses ATP hydrolysis to load β₂ clamps onto RNA‐primed templates DNA. The accessory ψ and χ subunits stimulate the CLC and bridge the CLC to SSB, respectively. The polymerase III core, commencing at primed‐templates, uses ssDNA as a template to synthesize new DNA on both the leading and lagging strands. The β₂ sliding clamp acts as a mobile tether and is essential for the processivity of the polymerase. The C‐terminal domains of τ subunits are coupled to Pol III cores and (weakly) to DnaB.

Figure 2

Cartoon representations of DnaG primase. (a) Arrangement of domains. The subdomains of RPD are indicated in orange (N‐terminal segment), blue (TOPRIM), and pink (C‐terminal segment). (b) ZBD from G. stearothermophilus (PDB ID 1D0Q) with Zn²⁺ (gray sphere) and zinc‐binding residues in stick form. (c) Staphylococcus aureus RPD domain with bound Mn²⁺ ions (magenta spheres) and ATP (PDB ID 4EDG). Bound ATP (black carbon atoms) and interacting side‐chains (carbon atoms yellow) are shown in stick form. (d) Staphylococcus aureus RPD domain with bound Mn²⁺ ions (magenta spheres) and alarmone ppGpp (PDB ID 4EDT). Bound ppGpp (black carbon atoms) and interacting residues (yellow carbon atoms) in stick form. (e) Aquifex aeolicus ZBD and RPD domains (PDB ID 2AU3). Zn²⁺ bound to ZBD is shown as a white sphere. Catalytic‐ and Zn²⁺‐binding residues shown in stick form. (f) Escherichia coli RPD domain with ssDNA (PDB ID 3B39). (g) DnaGC from E. coli (PDB ID 2HAJ). (h) DnaB‐NTD from G. stearothermophilus (PDB ID 2R6A).

Figure 3

Cartoon representations of DnaB helicase. (a) Arrangement and color coding of DnaB domains and associated proteins. (b) Orthogonal views of G. stearothermophilus DnaB₆.DnaGC₃ complex (PDB ID 2R6A). (c) Orthogonal views of G. stearothermophilus DnaB₆ in complex with ssDNA (VDW spheres) (PDB ID 4ESV). (d) Orthogonal views of the DnaB₆.DnaC₆ complex (E. coli) (PDB ID 6QEL). (e) Orthogonal views of the DnaB₆.DnaC₆ complex with ssDNA (E. coli) (PDB ID 6QEM). Bound nucleoside phosphates are represented as green VDW spheres.

Figure 4

Cartoon representations of SSB. (a) Escherichia coli tetramer (PDB ID 4MZ9). Each monomer is shown in a different color. Disordered C‐terminal region indicated by dashed line. (b) Mode of (SSB)₆₅ complex based on E. coli SSB‐DNA complex (PDB ID 1EYG). DNA is shown as a black trace. (c) Bacillus subtilis SSBA octamer with bridging DNA (black trace) (PDB ID 6BHX).

Figure 5

Cartoon representations of bacterial DNA polymerases. (a) PolC from G. kaustophilus with arrangement and color coding of domains shown below (PDB ID 3F2B). (b) PolC catalytic site. Template and nascent DNA strands shown in stick form with carbon atoms black and pink, respectively. (c) Thermus aquaticus DnaE with arrangement and color coding of domains shown below (PDB ID 2HPI). (d) Thermus aquaticus PolIIIα in complex with DNA (PDB ID 3E0D).

Figure 6

Cartoon representations of polIIIε NTD in complex with HOT (PDB ID 2IDO). Superposed on HOT is the NMR structure of polIIIθ from the polIIIεθ complex (PDB ID 2AXD). The disordered C‐terminal region of ε is indicated by a dashed line. Catalytic residues and bound dTMP are shown (yellow and black carbon atoms, respectively). Catalytic Mn²⁺ ions are shown as magenta spheres.

Figure 7

Cartoon representations of complexes of the β‐sliding clamp. β‐subunits are shown in green and cyan. Domains I–III are labeled. (a) Superposed are β‐sliding clamp in complex with polIIIδ (blue) (PDB ID 1JQJ), Pol IV little‐finger domain (red) (PDB ID 1UNN), and DNA (black) (PDB ID 3BEP). (b) Close‐up of the common binding site of β‐clamp binding partners.

Figure 8

Cartoon representations of the polIII replicase cores in different complexes. Color coding of different subunits in the complexes is indicated. (a) Without DNA (PDB ID 5FKU). (b) With DNA (PDB ID 5FKV). (c) With DNA in proofreading mode (PDB ID 5M1S).

Figure 9

Representations of clamp loader complex proteins. (a) Organization of τ domains; the truncated version γ comprises domains I–III. (b) Cartoon representations of E. coli CLC in complex with primed template DNA (PDB ID 3GLI). Views perpendicular and parallel to the axis of DNA are shown. The view on the right shows one of the γ‐subunits (domains I–III) with others in gray. (c) Cartoon representation of the ψ:χ complex with SSB‐Ct peptide (black carbon atoms) (PDB ID 3SXU).