Ordered ATP Hydrolysis in the γ Complex Clamp Loader AAA+ Machine

Aaron Johnson,Mike O'Donnell

doi:10.1074/jbc.m212708200

Aaron Johnson, Mike O'Donnell

Open Access

https://doi.org/10.1074/jbc.m212708200

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Abstract

The gamma complex couples ATP hydrolysis to the loading of beta sliding clamps onto DNA for processive replication. The gamma complex structure shows that the clamp loader subunits are arranged as a circular heteropentamer. The three gamma motor subunits bind ATP, the delta wrench opens the beta ring, and the delta' stator modulates the delta-beta interaction. Neither delta nor delta' bind ATP. This report demonstrates that the delta' stator contributes a catalytic arginine for hydrolysis of ATP bound to the adjacent gamma(1) subunit. Thus, the delta' stator contributes to the motor function of the gamma trimer. Mutation of arginine 169 of gamma, which removes the catalytic arginines from only the gamma(2) and gamma(3) ATP sites, abolishes ATPase activity even though ATP site 1 is intact and all three sites are filled. This result implies that hydrolysis of the three ATP molecules occurs in a particular order, the reverse of ATP binding, where ATP in site 1 is not hydrolyzed until ATP in sites 2 and/or 3 is hydrolyzed. Implications of these results to clamp loaders of other systems are discussed.

Highlights

The ␥ complex clamp loader consists of several different subunits, i.e. three ␥ (␶) subunits and one each of ␦, ␦Ј, ␹, and ␺ [2,3,4]
This report demonstrates that the ␦؅ stator contributes a catalytic arginine for hydrolysis of ATP bound to the adjacent ␥1 subunit
The ␦Ј Stator Contributes a Catalytic Arginine to the Clamp Loader Motor—The ␥ complex clamp loader has been proposed to consist of three main components [4, 6], i.e. the ␦ wrench, the ␥ trimer motor, and the ␦Ј stator

Summary

Introduction

The ␥ complex clamp loader consists of several different subunits, i.e. three ␥ (␶) subunits and one each of ␦, ␦Ј, ␹, and ␺ [2,3,4]. At 37 °C either alone or with mutant or wild-type ␥ complex (10.7 nM) in 75 ␮l of reaction buffer containing primed M13mp18 ssDNA (13.3 nM), SSB (3.2 ␮M as tetramer), 1 mM ATP, and 10 mM MgCl2.

Results

Conclusion