A single subunit directs the assembly of the Escherichia coli DNA sliding clamp loader

Abstract

Multi-protein clamp loader complexes are required to load sliding clamps onto DNA. In Escherichia coli the clamp loader contains three DnaX (tau/gamma) proteins, delta, and delta', which together form an asymmetric pentameric ring that also interacts with psichi. Here we used mass spectrometry to examine the assembly and dynamics of the clamp loader complex. We find that gamma exists exclusively as a stable homotetramer, while tau is in a monomer-dimer-trimer-tetramer equilibrium. delta' plays a direct role in the assembly as a tau/gamma oligomer breaker, thereby facilitating incorporation of lower oligomers. With delta', both delta and psichi stabilize the trimeric form of DnaX, thus completing the assembly. When tau and gamma are present simultaneously, mimicking the situation in vivo, subunit exchange between tau and gamma tetramers occurs rapidly to form heterocomplexes but is retarded when deltadelta' is present. The implications for intracellular assembly of the DNA polymerase III holoenzyme are discussed.

Figure 1. Mass Spectra of the τ and γ Subunits in Isolation

(A) Mass spectrum of τ in 0.1 M NH₄OAc at pH 7.6 and 1 mM dithiothreitol. The major charge state series corresponds to a tetrameric form of τ. Series of τ in monomeric, dimeric, and trimeric forms are also observed as the minor charge state series. (B) Mass spectrum of γ in 0.1 M NH₄OAc at pH 6.9. Well resolved charge states indicate that γ forms a well defined tetramer. (C) Mass spectrum of all four oligomeric species of γ in 1 M NH₄OAc at pH 6.9. See also Table S1.

Figure 2. Mass Spectra of τ in the Presence of Various Subunits

(A) In the presence of δ′, τ forms three subcomplexes, τ₃δ′, τ₂δ′, and τδ′ but not τ₄δ′. (B) None of the oligomeric species of τ associates with δ. (C) The ψχ complex binds to τ, forming τ₄ψχ and τ₃ψχ. (D) δ does not associate with τ in the presence of ψχ. See also Figure S1 for the γ subunit interaction and Table S1.

Figure 3. Assembly Pathway of the Clamp Loader and Mass Spectra of Complexes Formed Along the Pathway

First, δ′ initiates the assembly by dissociating tetramers of τ into trimers, the functional unit of the clamp loader. Both δ or ψχ interact with the initial τ₃δ′ subcomplex, resulting in either τ₃δδ′ or τ₃δ′ψχ subcomplexes. Further addition of ψχ or δ leads to the final clamp loader, τ₃δδ′ψχ. Mass spectra are recorded for τ₃δδ′ (A), τ₃δ′ψχ (B), and τ₃δδ′ψχ (C and D), when ψχ was added to (A) and δ to (B). For γ, analogous results were observed in Figure S2, suggesting identical assembly pathways. See also Table S1. Note that although all four species of oligomers of τ are present in solution, only tetramers of τ are shown initially.

Figure 4. Subunit Exchange Dynamics between τ and γ

(A) τ and γ undergo rapid subunit exchange at 0°C and form all five possible tetrameric species, τ₄, τ₃γ, τ₂γ₂, τγ₃, and γ₄, with the dead time of the experiment <15 s. See also Figure S3. (B) Mass spectra of τ₃δδ′ in the presence of γ₄. At τ = 0, free δδ′ interacts with γ₄ directly and forms the γ complex (bottom spectrum). At τ = 1 hr, low intensity peaks are observed (not labeled, middle spectrum), which increase in intensity after 24 hr and are assigned to all four possible complexes, γ₃δδ′, γτ₂δδ′, γ₂τδδ′, and τ₃δδ′ (top spectrum). See also Table S1.