Polymerase Chaperoning and Multiple ATPase Sites Enable the E. coli DNA Polymerase III Holoenzyme to Rapidly Form Initiation Complexes.

Abstract:

Cellular replicases include three subassemblies: a DNA polymerase, a sliding clamp processivity factor, and a clamp loader complex. The Escherichia coli clamp loader is the DnaX complex (DnaX(3)δδ'χψ), where DnaX occurs either as τ or as the shorter γ that arises by translational frameshifting. Complexes composed of either form of DnaX are fully active clamp loaders, but τ confers important replicase functions including chaperoning the polymerase to the newly loaded clamp to form an initiation complex for processive replication. The kinetics of initiation complex formation were explored for DnaX complexes reconstituted with varying τ and γ stoichiometries, revealing that τ-mediated polymerase chaperoning accelerates initiation complex formation by 100-fold. Analyzing DnaX complexes containing one or more K51E variant DnaX subunits demonstrated that only one active ATP binding site is required to form initiation complexes, but the two additional sites increase the rate by ca 1000-fold. For τ-containing complexes, the ATP analogue ATPγS was found to support initiation complex formation at 1/1000th the rate with ATP. In contrast to previous models that proposed ATPγS drives hydrolysis-independent initiation complex formation by τ-containing complexes, the rate and stoichiometry of ATPγS hydrolysis coincide with those for initiation complex formation. These results show that although one ATPase site is sufficient for initiation complex formation, the combination of polymerase chaperoning and the binding and hydrolysis of three ATPs dramatically accelerates initiation complex formation to a rate constant (25-50 s(-1)) compatible with double-stranded DNA replication.

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