ASSEMBLY OF A CHROMOSOMAL REPLICATION MACHINE - 2 DNA-POLYMERASES, A CLAMP LOADER, AND SLIDING CLAMPS IN ONE HOLOENZYME PARTICLE .2. INTERMEDIATE COMPLEX BETWEEN THE CLAMP LOADER AND ITS CLAMP

The Escherichia coli replicase, DNA polymerase III holoenzyme, derives its processivity from the beta subunit sliding clamp that encircles D NA and tethers the replicase to the template. The beta dimer is assemb led around DNA by the gamma complex clamp loader in an ATP-dependent r eaction. In this report, the essential contact between the clamp loade r and beta is identified as mediated through the delta subunit of the gamma complex. The delta subunit appears to contact the face of the be ta dimer ring that contains the two C termini. Surprisingly, ATP is re quired for the gamma complex to bind beta, but not for delta to bind p . This indicates that delta is buried in the gamma complex and suggest s a role for ATP in exposing delta for interaction with p. A protease protection assay has been developed to specifically probe the delta su bunit within the gamma complex. The results of the assay are consisten t with an ATP-induced conformational change in the gamma complex that alters the state of the delta subunit within it. The implication of th ese key features to the clamp loading mechanism of the gamma complex i s discussed.