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Mapping the interaction of DNA with the Escherichia coli DNA polymerase clamp loader complex


Eric R Goedken, Steven L Kazmirski, Gregory D Bowman, Mike O'Donnell & John Kuriyan


Nat Struct Mol Biol (2005) 12:2, 183190. (local copy)

Abstract / Figures from the paper Movies /


Abstract:

Sliding clamps are loaded onto DNA by ATP-dependent clamp loader complexes. A recent crystal structure of a clamp loader-clamp complex suggested an unexpected mechanism for DNA recognition, in which the ATPase subunits of the loader spiral around primed DNA. We report the results of fluorescence-based assays that probe the mechanism of the Escherichia coli clamp loader and show that conserved residues clustered within the inner surface of the modeled clamp loader spiral are critical for DNA recognition, DNA-dependent ATPase activity and clamp release. Duplex DNA with a 5'-overhang single-stranded region (corresponding to correctly primed DNA) stimulates clamp release, as does blunt-ended duplex DNA, whereas duplex DNA with a 3' overhang and single-stranded DNA are ineffective. These results provide evidence for the recognition of DNA within an inner chamber formed by the spiral organization of the ATPase domains of the clamp loader.

Illustrations from the paper. Click on the small image to get a bigger one.













Accessory Illustrations





Movies

Crude model of E.coli clamp loader

and beta clamp with A-form RNA-DNA primer-template