Dynamics of loading the Escherichia coli sliding clamp

Sliding clamps are ring‐shaped protein complexes that encircle DNA to tether DNA polymerases to the template being copied and increase the processivity of synthesis. Clamp loaders assemble these ring‐shaped sliding clamps onto DNA in an ATP‐dependent reaction. Binding and hydrolysis of ATP promote conformational changes in the clamp loaders that modulate their affinities for clamps and DNA so that the clamp loaders can catalyze this mechanical reaction. Our laboratory has been investigating the mechanism by which the Escherichia coli γ complex clamp loader assembles the β‐sliding clamp onto DNA. We are using real time fluorescence‐based techniques to measure the kinetics of DNA binding, clamp loader·clamp binding, and ATP hydrolysis directly in solution to correlate the timing of these events. Our results demonstrated that primed template DNA specifically triggers the γ complex to hydrolyze ATP and release the clamp on DNA. All molecules of ATP are hydrolyzed in the same global step, releasing the clamp onto DNA, but ATP binding and hydrolysis at individual sites modulates the affinity of different interactions. ATP binding and hydrolysis at one subset of sites regulates interactions with the clamp whereas binding and hydrolysis at another modulates interactions with DNA. Our current efforts are aimed at defining the number of molecules of ATP hydrolyzed by different forms of the clamp loader and in the presence of SSB to further delineate the mechanism by which ATP binding and hydrolysis are coupled to mechanical work.