A model for Escherichia coli DNA polymerase III holoenzyme assembly at primer/template ends - DNA triggers a change in binding specificity of the gamma complex clamp loader

The gamma complex of the Escherichia coli DNA polymerase III holoenzyme ass embles the beta sliding clamp onto DNA in an ATP hydrolysis driven reaction . Interactions between gamma complex and primer/template DNA are investigat ed using fluorescence depolarization to measure binding of gamma complex to different DNA substrates under steady-state and presteady-state conditions . Surprisingly, gamma complex has a much higher affinity for single-strande d DNA (K-d in the nM range) than for a primed template (K-d in the mu M ran ge) under steady-state conditions. However, when examined on a millisecond time scale, we find that gamma complex initially binds very rapidly and wit h high affinity to primer/template DNA but is converted subsequently to a m uch lower affinity DNA binding state. Presteady-state data reveals an effec tive dissociation constant of 1.5 nM for the initial binding of gamma compl ex to DNA and a dissociation constant of 5.7 mu M for the low affinity DNA binding state. Experiments using nonhydrolyzable ATP gamma S show that ATP binding con verts gamma complex hom a low affinity "inactive" to high affin ity "active" DNA binding state while ATP hydrolysis has the reverse effect, thus allowing cycling between active and inactive DNA binding forms at ste ady-state. We propose that a DNA-triggered switch between active and inacti ve states of gamma complex provides a two-tiered mechanism enabling gamma c omplex to recognize primed template sites and load beta, while preventing g amma complex from competing with DNA polymerase III core for binding a newl y loaded beta.DNA complex.