US EPA

1744776 

Journal Article 

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

Hingorani, MM; O'Donnell, M 

1998 

Yes 

Journal of Biological Chemistry
ISSN: 0021-9258
EISSN: 1083-351X 

273 

38 

24550-24563 

9733750 

10.1074/jbc.273.38.24550 

WOS:000076007300042 

The Escherichia coli gamma complex serves as a clamp loader, catalyzing ATP-dependent assembly of beta 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 gamma complex during clamp loading. We show that the energy from ATP binding to gamma complex powers several initial events in the clamp loading pathway. The gamma complex ((gamma 2)delta delta'(chi)psi) binds two ATP molecules (one per gamma subunit in the complex) with high affinity (K-d = 1-2.5 x 10(-6) M) or two adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) molecules with slightly lower affinity (K-d = 5-6.5 x 10(-6) nr). Experiments performed prior to the first ATP turnover (k(cat) = 4 x 10(-3) s(-1) at 4 degrees C), or in the presence of ATP gamma S (k(cat) = 1 x 10(-4) s(-1) at 37 degrees C), demonstrate that upon interaction with ATP the gamma complex undergoes a change in conformation. This ATP-bound gamma complex binds beta and opens the ring at the dimer interface. Still prior to ATP hydrolysis, the composite of gamma complex and the open beta ring binds with high affinity to primer-template DNA. Thus ATP binding powers all the steps in the clamp loading pathway leading up to the assembly of a gamma complex open beta 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.