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

Nature Structural & Molecular Biology volume 12, pages 183–190 (2005)Cite this article

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.

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Figure 1: Model for a ternary complex of the E. coli clamp loader, clamp and nucleic acid derived from the structure of the RFC–PCNA complex19.
Figure 2: Effects of γBCD mutants.
Figure 3: DNA-binding affinity for γBCD mutants.
Figure 4: Effects of δ′E mutants.
Figure 5: Specificity of DNA-stimulated clamp release.
Figure 6: DNA competition assays with hairpin DNAs measured by fluorescence anisotropy.
Figure 7

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Acknowledgements

We gratefully recognize the assistance of X. Cao for DNA mutagenesis, L. Leighton for the creation of illustrations, and M. Levitus, R. McNally, M. Lamers, J. Guenther, O. Rosenberg and H. Sondermann for helpful discussions. This work was supported by an American Cancer Society postdoctoral fellowship to E.R.G., and by grants from the US National Institutes of Health to J.K. (GM45547) and M.O.D. (GM38839).

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Authors and Affiliations

  1. Department of Molecular and Cell Biology, Department of Chemistry, Howard Hughes Medical Institute, University of California, Berkeley, 94720, California, USA

    Eric R Goedken, Steven L Kazmirski, Gregory D Bowman & John Kuriyan

  2. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Eric R Goedken, Steven L Kazmirski, Gregory D Bowman & John Kuriyan

  3. Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, 10021, New York, USA

    Mike O'Donnell

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Correspondence to John Kuriyan.

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Supplementary information

Supplementary Fig. 1

FRET controls. (PDF 511 kb)

Supplementary Fig. 2

δ raw FRET data. (PDF 547 kb)

Supplementary Fig. 3

δ ATPase data. (PDF 572 kb)

Supplementary Fig. 4

Alternate hairpin DNA competition. (PDF 162 kb)

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Goedken, E., Kazmirski, S., Bowman, G. et al. Mapping the interaction of DNA with the Escherichia coli DNA polymerase clamp loader complex. Nat Struct Mol Biol 12, 183–190 (2005). https://doi.org/10.1038/nsmb889

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