Mechanism of loading the Escherichia coli DNA polymerase III sliding clamp: I. Two distinct activities for individual ATP sites in the gamma complex.


The Escherichia coli DNA polymerase III gamma complex loads the beta clamp onto DNA, and the clamp tethers the core polymerase to DNA to increase the processivity of synthesis. ATP binding and hydrolysis promote conformational changes within the gamma complex that modulate its affinity for the clamp and DNA, allowing it to accomplish the mechanical task of assembling clamps on DNA. This is the first of two reports (Snyder, A. K., Williams, C. R., Johnson, A., O'Donnell, M., and Bloom, L. B. (2004) J. Biol. Chem. 279, 4386-4393) addressing the question of how ATP binding and hydrolysis modulate specific interactions with DNA and beta. Pre-steady-state rates of ATP hydrolysis were slower when reactions were initiated by addition of ATP than when the gamma complex was equilibrated with ATP and were limited by the rate of an intramolecular reaction, possibly ATP-induced conformational changes. Kinetic modeling of assays in which the gamma complex was incubated with ATP for different periods of time prior to adding DNA to trigger hydrolysis suggests a mechanism in which a relatively slow conformational change step (kforward = 6.5 s(-1)) produces a species of the gamma complex that is activated for DNA (and beta) binding. In the absence of beta, 2 of the 3 molecules of ATP are hydrolyzed rapidly prior to releasing DNA, and the 3rd molecule is hydrolyzed slowly. In the presence of beta, all 3 molecules of ATP are hydrolyzed rapidly. These results suggest that hydrolysis of 2 molecules of ATP may be coupled to conformational changes that reduce interactions with DNA, whereas hydrolysis of the 3rd is coupled to changes that result in release of beta.




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