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Structural Studies of the Mechanism of Clamp Loading by Clamp Loader Complexes

  • Author(s): Simonetta, Kyle Robert
  • Advisor(s): Kuriyan, John
  • et al.
Abstract

High-speed DNA replication is an intricate process that requires the coordinated efforts of many proteins at the replication fork. The replicative DNA polymerases require tethering to the DNA substrate in order to remain bound to the template and replicate the DNA processively. The polymerases are tethered to DNA by attachment to ring-shaped processivity factors, known as sliding clamps, which encircle DNA. Pentameric molecular machines comprised of AAA+ subunits, known as clamp loaders, are required to link the sliding clamps to DNA topologically. Clamp loaders, through the binding and hydrolysis of ATP, catalyze the opening of the ring-shaped sliding clamps and the placement of the clamps around DNA. Interaction with a sliding clamp requires that the subunits of a clamp loader be loaded with ATP. Once bound to the clamp, the clamp loader complex binds to a primer-template junction, in the process threading the DNA through the open interface of the clamp. Binding to the primer-template junction induces a conformational change in the clamp loader that acts as a switch, activating the ATPase activity of the AAA+ subunits and resulting in release of the clamp and DNA by the clamp loader. The structural mechanisms by which clamp loaders recognize primer-template junctions and hydrolyze ATP in response to DNA binding are not well understood. In this dissertation, I report the crystal structure of the E. coli clamp loader, γ complex, bound to primer-template DNA. The structure reveals that, when bound to DNA, the AAA+ domains of the clamp loader subunits adopt a highly symmetric spiral conformation that interacts with the helical DNA duplex, with the N-terminal domains of the subunits tracking the template strand of the primer-template junction. In this conformation, the ATP binding sites, which are formed at subunit-subunit interfaces within the AAA+ spiral, are all in the same ATPase activated conformation, suggesting a mechanism by which DNA binding promotes this conformation and thereby leads to ATP hydrolysis. An unexpected feature of this structure is that primer-template recognition is restricted primarily to the template strand, with virtually no contacts made with the primer strand. As a consequence of this mode of DNA binding in which contacts are restricted to the template strand, models for the recognition of RNA-DNA primer-template junctions, as well as the recognition of reverse polarity primer-templates, are proposed. A related structure which I also present, that of the E. coli clamp loader bound to DNA as well as a peptide derived from the N-terminal tail of the ψ protein, a clamp loader binding partner, suggests a mechanism whereby the binding of the ψ protein promotes the clamp and DNA binding activities of the clamp loader. Binding of this peptide promotes a conformational change within the collar domains of the clamp loader that is necessary for the clamp loader to adopt the highly symmetric spiral conformation of the AAA+ domains when bound to DNA.

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