Mechanism of Loading the Escherichia coli DNA Polymerase III Sliding Clamp

Abstract

The Escherichia coli DNA polymerase III γ complex clamp loader assembles the ring-shaped β sliding clamp onto DNA. The core polymerase is tethered to the template by β, enabling processive replication of the genome. Here we investigate the DNA substrate specificity of the clamp-loading reaction by measuring the pre-steady-state kinetics of DNA binding and ATP hydrolysis using elongation-proficient and deficient primer/template DNA. The ATP-bound clamp loader binds both elongation-proficient and deficient DNA substrates either in the presence or absence of β. However, elongation-proficient DNA preferentially triggers γ complex to release β onto DNA with concomitant hydrolysis of ATP. Binding to elongation-proficient DNA converts the γ complex from a high affinity ATP-bound state to an ADP-bound state having a 10 5 -fold lower affinity for DNA. Steady-state binding assays are misleading, suggesting that γ complex binds much more avidly to non-extendable primer/template DNA because recycling to the high affinity binding state is rate-limiting. Pre-steady-state rotational anisotropy data reveal a dynamic association-dissociation of γ complex with extendable primer/templates leading to the diametrically opposite conclusion. The strongly favored dynamic recognition of extendable DNA does not require the presence of β. Thus, the γ complex uses ATP binding and hydrolysis as a mechanism for modulating its interaction with DNA in which the ATP-bound form binds with high affinity to DNA but elongation-proficient DNA substrates preferentially trigger hydrolysis of ATP and conversion to a low affinity state.