ATP Binding to the Escherichia coli Clamp Loader Powers Opening of the Ring-shaped Clamp of DNA Polymerase III Holoenzyme

Manju M Hingorani,Mike O'Donnell

doi:10.1074/jbc.273.38.24550

Manju M Hingorani, Mike O'Donnell

Open Access

https://doi.org/10.1074/jbc.273.38.24550

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Abstract

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 (gamma2 delta delta'chi psi) binds two ATP molecules (one per gamma subunit in the complex) with high affinity (Kd = 1-2. 5 x 10(-6) M) or two adenosine 5'-O-(3-thiotriphosphate)(ATPgammaS) molecules with slightly lower affinity (Kd = 5-6.5 x 10(-6) M). Experiments performed prior to the first ATP turnover (kcat = 4 x 10(-3) s-1 at 4 degreesC), or in the presence of ATPgammaS (kcat = 1 x 10(-4) s-1 at 37 degreesC), 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.

Highlights

Rapid and efficient duplication of genomic DNA depends on biomolecular machines known as DNA replicases
Earlier studies have demonstrated that ␥ complex loads the circular clamp ␤ onto a primed DNA template, and ␤ tethers the polymerase to DNA, facilitating highly processive DNA synthesis
According to data from UV cross-linking experiments, the ␦ subunit may interact with ATP [41]; mutation of Lys-225 to Ala in the nearconsensus nucleotide-binding site in the ␦ subunit did not inhibit the ATPase activity or the clamp loading activity of ␥ complex reconstituted with mutant ␦ [40]

Summary

Introduction

Rapid and efficient duplication of genomic DNA depends on biomolecular machines known as DNA replicases In diverse organisms these multicomponent biological machines exhibit varying degrees of complexity, including substantial differences in subunit composition. In the current model for E. coli DNA replication, after the assembly of an initiation complex in which the polymerase III holoenzyme is tethered to a primed DNA template by circular protein clamps, the two core polymerases in the holoenzyme synthesize leading and lagging strand DNA synchronously (20 –23). It has been postulated that in the presence of ATP a conformational change in ␥ complex presents ␦ for interaction with ␤ [27] Once this interaction is established, the clamp loader must assemble the ␤ ring around DNA, and it must release the ␤1⁄7DNA complex to complete its job as a molecular matchmaker. The ATP-bound ␥ complex1⁄7␤ complex binds primer-template DNA leading to formation of a stable ␥ complex1⁄7open ␤ ring1⁄7DNA composite that is an important intermediate in the clamp loading pathway (Fig. 10)

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