Conserved residues in the delta subunit help the E. coli clamp loader, gamma complex, target primer-template DNA for clamp assembly

The Escherichia coli clamp loader, c complex (c3dd’kc), catalyzes ATP-driven assembly of b clamps onto primer-template DNA (p/tDNA), enabling processive replication. The mechanism by which c complex targets p/tDNA for clamp assembly is not resolved. According to previous studies, charged/polar amino acids inside the clamp loader chamber interact with the double-stranded (ds) portion of p/tDNA. We find that dsDNA, not ssDNA, can trigger a burst of ATP hydrolysis by c complex and clamp assembly, but only at far higher concentrations than p/tDNA. Thus, contact between c complex and dsDNA is necessary and sufficient, but not optimal, for the reaction, and additional contacts with p/tDNA likely facilitate its selection as the optimal substrate for clamp assembly. We investigated whether a conserved sequence— HRVW279QNRR—in d subunit contributes to such interactions, since Tryptophan-279 specifically cross-links to the primer-template junction. Mutation of d-W279 weakens c complex binding to p/tDNA, hampering its ability to load clamps and promote proccessive DNA replication, and additional mutations in the sequence (d-R277, d-R283) worsen the interaction. These data reveal a novel location in the C-terminal domain of the E. coli clamp loader that contributes to DNA binding and helps define p/tDNA as the preferred substrate for