Abstract

The DnaX complex (DnaX(3)δδ'χ psi) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, β(2), onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. β(2) binding (determined functionally) is diminished 12-30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for β(2). DNA synthesis activity can be restored by increased concentrations of β(2). In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances β(2) loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.

Highlights

  • DNA polymerase III holoenzyme2 exhibits features common to all other cellular replicases

  • A model was presented in which DnaX complex-ATP␥S-␤2 binds primed DNA, with a small percentage of ␤2 closing around DNA spontaneously in equilibrium with a mostly open ␤2 population. pol III was proposed to attach to the closed ␤2 and displace DnaX from ␤2 and the DNA in a reaction that does not necessarily require nucleotide hydrolysis

  • These observations suggest that initiation complex formation reactions catalyzed by ␶-containing DnaX complex might have different ATP binding and hydrolysis requirements than a ␤2 loading reaction that occurs in the absence of pol III

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Summary

Introduction

DNA polymerase III holoenzyme (pol III HE)2 exhibits features common to all other cellular replicases. These observations suggest that initiation complex formation reactions catalyzed by ␶-containing DnaX complex might have different ATP binding and hydrolysis requirements than a ␤2 loading reaction that occurs in the absence of pol III.

Results
Conclusion

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