ATP binding to the Escherichia coli clamp loader powers opening of the ring-shaped clamp of DNA polymerase III holoenzyme

Abstract

The Escherichia coli γ complex serves as a clamp loader, catalyzing ATP-dependent assembly of β protein clamps onto primed DNA templates during DNA replication. These ring-shaped clamps tether DNA polymerase III holoenzyme to the template, facilitating rapid and processive DNA synthesis. This report focuses on the role of ATP binding and hydrolysis catalyzed by the complex during clamp loading. We show that the energy from ATP binding to γ complex powers several initial events in the clamp loading pathway. The γ complex (γ2δδ'χψ) binds two ATP molecules (one per γ sub-unit in the complex) with high affinity (K(d) = 1-2.5 x 10-6 M) or two adenosine 5'-O- (3-thiotriphosphate)(ATPγS) molecules with slightly lower affinity (K(d) = 5-6.5 x 10-6 M). Experiments performed prior to the first ATP turnover (k(cat) = 4 x 10- 3 s-1 at 4 °C), or in the presence of ATPγS (k(cat) = 1 x 10-4 s-1 at 37 °C), demonstrate that upon interaction with ATP the γ complex undergoes a change in conformation. This ATP-bound γ complex binds β and opens the ring at the dimer interface. Still prior to ATP hydrolysis, the composite of γ complex and the open β ring binds with high affinity to primer-term-plate DNA. Thus ATP binding powers all the steps in the clamp loading pathway leading up to the assembly of a γ complex·open β ring·DNA intermediate, setting the stage for ring closing and turnover of the clamp loader, steps that may be linked to subsequent hydrolysis of ATP.