Mechanism of β Clamp Opening by the δ Subunit of Escherichia coli DNA Polymerase III Holoenzyme

Abstract

The β sliding clamp encircles the primer-template and tethers DNA polymerase III holoenzyme to DNA for processive replication of the Escherichia coli genome. The clamp is formed via hydrophobic and ionic interactions between two semicircular β monomers. This report demonstrates that the β dimer is a stable closed ring and is not monomerized when the γ complex clamp loader (γ3δ1δ1χ 1ψ1) assembles the β ring around DNA. δ is the subunit of the γ complex that binds β and opens the ring; it also does not appear to monomerize β. Point mutations were introduced at the β dimer interface to test its structural integrity and gain insight into its interaction with δ. Mutation of two residues at the dimer interface of β, I272A/L273A, yields a stable β monomer. We find that δ binds the β monomer mutant at least 50-fold tighter than the β dimer. These findings suggest that when δ interacts with the β clamp, it binds one β subunit with high affinity and utilizes some of that binding energy to perform work on the dimeric clamp, probably cracking one dimer interface open.