Division of labor---sequential ATP hydrolysis drives assembly of a DNA polymerase sliding clamp around DNA

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Abstract
The β sliding clamp encircles DNA and enables processive replication of the Escherichia coli genome by DNA polymerase III holoenzyme. The clamp loader, γ complex, assembles β around DNA in an ATP‐fueled reaction. Previous studies have shown that γ complex opens the β ring and also interacts with DNA on binding ATP. Here, a rapid kinetic analysis demonstrates that γ complex hydrolyzes two ATP molecules sequentially when placing β around DNA. The first ATP is hydrolyzed fast, at 25‐30 s−1, while the second ATP hydrolysis is limited to the steady‐state rate of 2 s−1. This step‐wise reaction depends on both primed DNA and β. DNA alone promotes rapid hydrolysis of two ATP molecules, while β alone permits hydrolysis of only one ATP. These results suggest that β inserts a slow step between the two ATP hydrolysis events in clamp assembly, during which the clamp loader may perform work on the clamp. Moreover, one ATP hydrolysis is sufficient for release of β from the γ complex. This implies that DNA‐dependent hydrolysis of the other ATP is coupled to a separate function, perhaps involving work on DNA. A model is presented in which sequential ATP hydrolysis drives distinct events in the clamp‐assembly pathway. We also discuss underlying principles of this step‐wise mechanism that may apply to the workings of other ATP‐fueled biological machines.