A Single Subunit Directs the Assembly of the Escherichia coli DNA Sliding Clamp Loader

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

Graphical AbstractFigure optionsDownload high-quality image (286 K)Download as PowerPoint slideHighlights
► γ forms more a stable homotetramer than τ
► δ′ plays a direct role in the assembly as a τ/γ oligomer breaker
► Both δ and ψχ with δ′ stabilize the τ/γ trimer, thus completing the assembly
► Replisomes with fewer than two copies of τ are not used in replication