Temporal order of events in the assembly of a ternary clamp loader‐clamp‐DNA complex

Abstract

Sliding clamps are ring‐shaped protein complexes that increase the processivity of DNA synthesis by tethering DNA polymerases to DNA. Clamp loaders catalyze the assembly of sliding clamps onto primed templates. Binding and hydrolysis of ATP promote conformational changes in the clamp loader that alter interactions with the clamp and DNA to facilitate the mechanical clamp loading reaction. Our laboratory developed two independent fluorescence‐based assays to measure clamp loader▸clamp binding and clamp opening for the Escherichia coli γ‐complex clamp loader and β‐sliding clamp. Real time binding measurements revealed that when the γ complex is equilibrated with ATP, clamp binding is rapid and followed by a slower clamp opening reaction. This result demonstrates that the clamp loader binds and opens the clamp rather than capturing clamps that are transiently open in solution. When the β‐clamp and ATP are added to the γ complex simultaneously, a slow ATP‐dependent conformational change in the γ complex precedes clamp opening but not clamp binding. Kinetic assays with wild‐type γ complex and experiments with Arg finger mutants of γ complex were done to define ATP requirements for clamp binding and opening. A model will be presented in which a defined temporal order of events in assembling a ternary clamp loader▸clamp▸DNA complex is established by the rates of a series of ATP binding events and conformational changes.