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

The beta sliding clamp encircles DNA and enables processive replication of the Escherichia coli genome by DNA polymerase III holoenzyme. The clamp loa der, gamma complex, assembles beta around DNA in an ATP-fueled reaction. Pr evious studies have shown that gamma complex opens the beta ring and also i nteracts with DNA on binding ATP, Here, a rapid kinetic analysis demonstrat es that gamma complex hydrolyzes two ATP molecules sequentially when placin g beta 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) Th is step-wise reaction depends on both primed DNA and beta, DNA alone promot es rapid hydrolysis of two ATP molecules, while beta alone permits hydrolys is of only one ATP, These results suggest that beta inserts a slow step bet ween the two ATP hydrolysis events in clamp assembly, during which the clam p loader may perform work on the clamp. Moreover, one ATP hydrolysis is suf ficient for release of beta from the gamma complex. This implies that DNA-d ependent hydrolysis of the other ATP is coupled to a separate function, per haps involving work on DNA, A model is presented in which sequential ATP hy drolysis drives distinct events in the clamp-assembly pathway. We also disc uss underlying principles of this step-wise mechanism that may apply to the workings of other ATP-fueled biological machines.