<p>The <i>E. coli</i> γ-clamp loader is a 200&#xa0;kDa pentameric AAA + ATPase comprised of γ, δ and δ′ subunits in a 3:1:1 ratio, which opens the ring shaped β-clamp homodimer and loads it onto DNA in a process essential for DNA replication. The clamp loading is initiated by ATP binding, which induces conformational changes in the clamp loader allowing it to bind and open the β-clamp. This is followed by DNA primer-template binding, ATP hydrolysis, and clamp release onto DNA. Despite a wealth of structural and functional data, dynamics and interactions of the γ-clamp loader and the β-clamp underlying elementary steps of this process remain elusive. Here we employed a “divide-and-conquer” strategy for the initial NMR characterization of the γ-clamp loader. A new protocol for the clamp loader assembly was proposed allowing selective incorporation of the isotope-labeled δ and δ′ subunits for NMR studies. The nearly complete ¹H, ¹⁵N and ¹³C NMR resonance assignments were obtained for the isolated modular domains of the δ and δ′ subunits, which facilitated the assignments of the full-length subunits, and side-chain methyl assignments of the subunits in the context of pentameric γ-clamp loader. NMR chemical shift analysis using the random coil index approach revealed increased flexibility in the ATP, DNA, and β-clamp binding interfaces of the isolated subunits, highlighting a potential significance of conformational dynamics for the clamp loading process. The reported clamp loader assembly protocol and resonance assignments enable the detailed NMR studies of protein dynamics and mechanochemistry of the clamp loading cycle.</p>