Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase

Abstract

The clamp-loader complex plays a crucial role in DNA replication by loading the β-clamp onto primed DNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry, structure and assembly pathway of this complex, and how it interacts with the replicative helicase, in Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealed that a hetero-pentameric τ3-δ-δ' Bacillus subtilis clamp-loader assembles via multiple pathways, which differ from those exhibited by the Gram-negative model Escherichia coli. Based on this information, a homology model of the B. subtilis τ3-δ-δ' complex was constructed, which revealed the spatial positioning of the full C-terminal τ domain. The structure of the δ subunit was determined by X-ray crystallography and shown to differ from that of E. coli in the nature of the amino acids comprising the τ and δ' binding regions. Most notably, the τ-δ interaction appears to be hydrophilic in nature compared with the hydrophobic interaction in E. coli. Finally, the interaction between τ3 and the replicative helicase DnaB was driven by ATP/Mg(2+) conformational changes in DnaB, and evidence is provided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilize its interaction with τ3.

Figure 1.

Electrospray ionization-mass spectra of different combinations of the clamp-loader subunits from B. subtilis sprayed from 1 M ammonium acetate, pH7.5. The τ subunits are represented by red spheres, while the δ and δ′ subunits are represented by green and blue spheres, respectively. A, τ subunit in isolation; B, equimolar mixture of τ and δ subunits; C, equimolar mixture of τ and δ′ subunits; D, equimolar mixture of τ₃, δ and δ′ subunits, resulting in the formation of the clamp-loader complex. E, The clamp-loader complex analysed under high collisional energy, resulting in a partial dissociation of δ and δ′ subunits (peaks corresponding to the low charged τ₃-δ and τ₃-δ′ after dissociation are magnified in the shaded region of the spectrum).

Figure 2.

A homology model of the B. subtilis clamp-loader complex, with δ, δ′ and τ subunits represented in green, blue and red, respectively. The Cτ domain of the τ subunit is represented in pink.

Figure 3.

The crystal structure of B. subtilis δ (yqeN). (A) Cartoon representation of the overall structure of δ. Domain I (residues 1–143), Domain II (residues 144–214) and Domain III (residues 215–339) are coloured magenta, green and blue, respectively. (B) Cartoon representation of δ, with α-helices, β-strands and loops shown as cyan cylinders, red arrows and magenta lines, respectively. (C) Topology diagram of δ with individual elements and residues labelled for α-helices (cyan cylinders) and β-sheets (red arrows) with connecting loops (magenta lines). Produced using TOPDRAW from the CCP4 suite.

Figure 4.

Mass spectra of the B. subtilis τ₃ subunit interaction with the B. stearothermophilus DnaB helicase. Proteins were mixed in a τ₃-DnaB₆ stoichiometry and sprayed from different buffer compositions. Signal for the τ₃-DnaB₆ complex is located in the pink region of the spectra, while unbound DnaB₆ is located in the blue region. (A) Proteins sprayed from 1 M ammonium acetate containing 0.5 mM Mg²⁺ and 0.1 mM ATP. The τ subunit is represented by red spheres, while the DnaB hexameric ring is represented in green. (B) Proteins sprayed from 1 M ammonium acetate containing 0.1 mM ATP. (C) Proteins sprayed from 1 M ammonium acetate containing 0.5 mM Mg²⁺. (D) Proteins sprayed from 1 M ammonium acetate only.