Prereplicative complexes of components of DNA polymerase III holoenzyme of Escherichia coli.

Stepwise reconstitution of the subunits of DNA polymerase III holoenzyme of Escherichia coli offers insights into the organization and function of this multisubunit assembly. A highly processive, holoenzyme-like activity can be generated when the gamma complex, in the presence of ATP and a primed template, activates the beta subunit to form a preinitiation complex, and this is then followed by addition of the core polymerase. Further analysis of early replicative complexes has now revealed: 1) that the gamma complex can stably bind a single-stranded DNA binding protein (SSB)-coated template, 2) that neither SSB coating of the template nor a proper primer terminus is required to form the preinitiation complex, and 3) that the gamma complex stabilizes the preinitiation complex in the presence of ATP and destabilizes it in the presence of adenosine 5'-O-(thiotriphosphate). Based on these findings, a sequence of stages can be formulated for an activation of the beta subunit that enables it to bind the template-primer and thereby interact with the core to create a processive polymerase.