| The Escherichia coli DNA polymerase III holoenzyme is responsible for the accurate and efficient duplication of the bacterial chromosome before cell division. This process is facilitated by accessory proteins, which serve to enhance the functionality of the core polymerase. One of these accessory proteins, the sliding clamp, functions as a physical tether between the polymerase and the template DNA being copied. The sliding clamp is loaded onto the appropriate DNA substrate through the action of the clamp loader, a seven-subunit molecular machine which uses the energy of ATP binding and hydrolysis to modulate protein-protein and protein-DNA interactions to complete the process of clamp assembly on DNA.; Our study characterized the mechanism by which ATP binding and hydrolysis is coupled to conformational changes within the clamp loader. A model is presented in which the filling of the individual ATP binding sites of the clamp loader with nucleotide is regulated in an ordered manner, and the order of filling is dictated by the presence of the Psi subunit of the clamp loader complex. The Psi subunit is also required for the formation of a conformational state with high affinity for DNA. Likewise, evidence is presented regarding the temporal correlation of DNA and clamp release relative to the hydrolysis of ATP by the clamp loader. |
