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Structural analysis of a eukaryotic sliding DNA clamp–clamp loader complex


Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. Here we report the crystal structure of the five-protein clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding clamp (proliferating cell nuclear antigen, PCNA). Tight interfacial coordination of the ATP analogue ATP-γS by RFC results in a spiral arrangement of the ATPase domains of the clamp loader above the PCNA ring. Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA double helix. This model, in which the clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer–template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding clamp on DNA.

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Figure 1: Overview of the RFC:PCNA complex.
Figure 2: The AAA + domains of the RFC assembly form a right-handed spiral.
Figure 3: Nucleotide is tightly bound between proximal and distal faces of the RFC-A and RFC-B AAA + modules.
Figure 4: A model for primed DNA interacting with the RFC:PCNA complex.
Figure 5: Conserved residues in domain I of clamp loader subunits at the proposed DNA-interacting surface.
Figure 6: Inactive and active clamp loader complexes.


  1. 1

    Huang, C. C., Hearst, J. E. & Alberts, B. M. Two types of replication proteins increase the rate at which T4 DNA polymerase traverses the helical regions in a single-stranded DNA template. J. Biol. Chem. 256, 4087–4094 (1981)

    CAS  PubMed  Google Scholar 

  2. 2

    Prelich, G., Kostura, M., Marshak, D. R., Mathews, M. B. & Stillman, B. The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro. Nature 326, 471–475 (1987)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Stukenberg, P. T., Studwell-Vaughan, P. S. & O'Donnell, M. Mechanism of the sliding β-clamp of DNA polymerase III holoenzyme. J. Biol. Chem. 266, 11328–11334 (1991)

    CAS  PubMed  Google Scholar 

  4. 4

    Kong, X. P., Onrust, R., O'Donnell, M. & Kuriyan, J. Three-dimensional structure of the β subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell 69, 425–437 (1992)

    CAS  Article  Google Scholar 

  5. 5

    Krishna, T. S., Kong, X. P., Gary, S., Burgers, P. M. & Kuriyan, J. Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell 79, 1233–1243 (1994)

    CAS  Article  Google Scholar 

  6. 6

    Warbrick, E. The puzzle of PCNA's many partners. Bioessays 22, 997–1006 (2000)

    CAS  Article  Google Scholar 

  7. 7

    Tsurimoto, T. & Stillman, B. Purification of a cellular replication factor, RF-C, that is required for coordinated synthesis of leading and lagging strands during simian virus 40 DNA replication in vitro. Mol. Cell. Biol. 9, 609–619 (1989)

    CAS  Article  Google Scholar 

  8. 8

    Tsurimoto, T. & Stillman, B. Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4. Proc. Natl Acad. Sci. USA 87, 1023–1027 (1990)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Lee, S. H., Kwong, A. D., Pan, Z. Q. & Hurwitz, J. Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase δ. J. Biol. Chem. 266, 594–602 (1991)

    CAS  PubMed  Google Scholar 

  10. 10

    Gomes, X. V. & Burgers, P. M. ATP utilization by yeast replication factor C. I. ATP-mediated interaction with DNA and with proliferating cell nuclear antigen. J. Biol. Chem. 276, 34768–34775 (2001)

    CAS  Article  Google Scholar 

  11. 11

    Jeruzalmi, D., O'Donnell, M. & Kuriyan, J. Crystal structure of the processivity clamp loader γ complex of E. coli DNA polymerase III. Cell 106, 429–441 (2001)

    CAS  Article  Google Scholar 

  12. 12

    Oyama, T., Ishino, Y., Cann, I. K., Ishino, S. & Morikawa, K. Atomic structure of the clamp loader small subunit from Pyrococcus furiosus. Mol. Cell 8, 455–463 (2001)

    CAS  Article  Google Scholar 

  13. 13

    Neuwald, A. F., Aravind, L., Spouge, J. L. & Koonin, E. V. AAA + : A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 9, 27–43 (1999)

    CAS  PubMed  Google Scholar 

  14. 14

    Guenther, B., Onrust, R., Sali, A., O'Donnell, M. & Kuriyan, J. Crystal structure of the δ′ subunit of the clamp-loader complex of E. coli DNA polymerase III. Cell 91, 335–345 (1997)

    CAS  Article  Google Scholar 

  15. 15

    Yu, R. C., Hanson, P. I., Jahn, R. & Brunger, A. T. Structure of the ATP-dependent oligomerization domain of N-ethylmaleimide sensitive factor complexed with ATP. Nature Struct. Biol. 5, 803–811 (1998)

    CAS  Article  Google Scholar 

  16. 16

    Lenzen, C. U., Steinmann, D., Whiteheart, S. W. & Weis, W. I. Crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein. Cell 94, 525–536 (1998)

    CAS  Article  Google Scholar 

  17. 17

    Goedken, E. R. et al. Fluorescence measurements on the E.coli DNA polymerase clamp loader: implications for conformational changes during ATP and clamp binding. J. Mol. Biol. 336, 1047–1059 (2004)

    CAS  Article  Google Scholar 

  18. 18

    Ason, B. et al. Mechanism of loading the Escherichia coli DNA polymerase III β sliding clamp on DNA. Bona fide primer/templates preferentially trigger the γ complex to hydrolyze ATP and load the clamp. J. Biol. Chem. 278, 10033–10040 (2003)

    CAS  Article  Google Scholar 

  19. 19

    Heras, B. et al. Dehydration converts DsbG crystal diffraction from low to high resolution. Structure (Camb.) 11, 139–145 (2003)

    CAS  Article  Google Scholar 

  20. 20

    Shamoo, Y. & Steitz, T. A. Building a replisome from interacting pieces: sliding clamp complexed to a peptide from DNA polymerase and a polymerase editing complex. Cell 99, 155–166 (1999)

    CAS  Article  Google Scholar 

  21. 21

    Gulbis, J. M., Kelman, Z., Hurwitz, J., O'Donnell, M. & Kuriyan, J. Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA. Cell 87, 297–306 (1996)

    CAS  Article  Google Scholar 

  22. 22

    Lee, S. et al. The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state. Cell 115, 229–240 (2003)

    CAS  Article  Google Scholar 

  23. 23

    Niwa, H., Tsuchiya, D., Makyio, H., Yoshida, M. & Morikawa, K. Hexameric ring structure of the ATPase domain of the membrane-integrated metalloprotease FtsH from Thermus thermophilus HB8. Structure (Camb.) 10, 1415–1423 (2002)

    CAS  Article  Google Scholar 

  24. 24

    Menz, R. I., Walker, J. E. & Leslie, A. G. Structure of bovine mitochondrial F1-ATPase with nucleotide bound to all three catalytic sites: implications for the mechanism of rotary catalysis. Cell 106, 331–341 (2001)

    CAS  Article  Google Scholar 

  25. 25

    Yang, W., Gao, Y. Q., Cui, Q., Ma, J. & Karplus, M. The missing link between thermodynamics and structure in F1-ATPase. Proc. Natl Acad. Sci. USA 100, 874–879 (2003)

    ADS  CAS  Article  Google Scholar 

  26. 26

    Waga, S. & Stillman, B. The DNA replication fork in eukaryotic cells. Annu. Rev. Biochem. 67, 721–751 (1998)

    CAS  Article  Google Scholar 

  27. 27

    Saenger, W. Principles of Nucleic Acid Structure (Springer, New York, 1984)

    Google Scholar 

  28. 28

    Xiong, Y. & Sundaralingam, M. Crystal structure of a DNA RNA hybrid duplex with a polypurine RNA r(gaagaagag) and a complementary polypyrimidine DNA d(CTCTTCTTC). Nucleic Acids Res. 28, 2171–2176 (2000)

    CAS  Article  Google Scholar 

  29. 29

    Jones, S., van Heyningen, P., Berman, H. M. & Thornton, J. M. Protein–DNA interactions: A structural analysis. J. Mol. Biol. 287, 877–896 (1999)

    CAS  Article  Google Scholar 

  30. 30

    Jeruzalmi, D. et al. Mechanism of processivity clamp opening by the δ subunit wrench of the clamp loader complex of E. coli DNA polymerase III. Cell 106, 417–428 (2001)

    CAS  Article  Google Scholar 

  31. 31

    Story, R. M., Weber, I. T. & Steitz, T. A. The structure of the E. coli recA protein monomer and polymer. Nature 355, 318–325 (1992)

    ADS  CAS  Article  Google Scholar 

  32. 32

    Hortnagel, K. et al. Saturation mutagenesis of the E. coli RecA loop L2 homologous DNA pairing region reveals residues essential for recombination and recombinational repair. J. Mol. Biol. 286, 1097–1106 (1999)

    CAS  Article  Google Scholar 

  33. 33

    Mirshad, J. K. & Kowalczykowski, S. C. Biochemical characterization of a mutant RecA protein altered in DNA-binding loop 1. Biochemistry 42, 5945–5954 (2003)

    CAS  Article  Google Scholar 

  34. 34

    Notarnicola, S. M., Park, K., Griffith, J. D. & Richardson, C. C. A domain of the gene 4 helicase/primase of bacteriophage T7 required for the formation of an active hexamer. J. Biol. Chem. 270, 20215–20224 (1995)

    CAS  Article  Google Scholar 

  35. 35

    Washington, M. T., Rosenberg, A. H., Griffin, K., Studier, F. W. & Patel, S. S. Biochemical analysis of mutant T7 primase/helicase proteins defective in DNA binding, nucleotide hydrolysis, and the coupling of hydrolysis with DNA unwinding. J. Biol. Chem. 271, 26825–26834 (1996)

    CAS  Article  Google Scholar 

  36. 36

    Skordalakes, E. & Berger, J. M. Structure of the Rho transcription terminator: mechanism of mRNA recognition and helicase loading. Cell 114, 135–146 (2003)

    CAS  Article  Google Scholar 

  37. 37

    Sawaya, M. R., Guo, S., Tabor, S., Richardson, C. C. & Ellenberger, T. Crystal structure of the helicase domain from the replicative helicase-primase of bacteriophage T7. Cell 99, 167–177 (1999)

    CAS  Article  Google Scholar 

  38. 38

    Singleton, M. R., Sawaya, M. R., Ellenberger, T. & Wigley, D. B. Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell 101, 589–600 (2000)

    CAS  Article  Google Scholar 

  39. 39

    Tsurimoto, T. & Stillman, B. Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer–template junction by eukaryotic DNA polymerases and their accessory proteins. J. Biol. Chem. 266, 1950–1960 (1991)

    CAS  PubMed  Google Scholar 

  40. 40

    Huang, H., Chopra, R., Verdine, G. L. & Harrison, S. C. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science 282, 1669–1675 (1998)

    ADS  CAS  Article  Google Scholar 

  41. 41

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    CAS  Article  Google Scholar 

  42. 42

    Gonzalez, A. Optimizing data collection for structure determination. Acta Crystallogr. D 59, 1935–1942 (2003)

    Article  Google Scholar 

  43. 43

    Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999)

    CAS  Article  Google Scholar 

  44. 44

    La Fortelle, E. D. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement in the MIR and MAD methods. Methods Enzymol. 276, 476–494 (1997)

    Google Scholar 

  45. 45

    Collaborative Computational Project Number 4, The CCP4 suite programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  46. 46

    Kleywegt, G. J. & Jones, T. A. Efficient rebuilding of protein structures. Acta Crystallogr. D 50, 829–832 (1996)

    Article  Google Scholar 

  47. 47

    Brünger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

  48. 48

    Lu, G. TOP: A new method for protein structure comparisons and similarity searches. J. Appl. Crystallogr. 33, 176–183 (2000)

    CAS  Article  Google Scholar 

  49. 49

    Miyata, T. et al. Direct view of the clamp-loading complex for processive DNA replication. Nature Struct. Mol. Biol. (in the press)

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We thank J. Berger, E. Goedken, M. Hingorani, D. Jeruzalmi, A. Johnson, S. Kazmirski, I. Nodelman, M. Podobnik, N. Yao and M. Young for scientific discussions, S. Chung and J. Finkelstein for technical assistance, K. Morikawa for sharing data prior to publication, L. Leighton for help with figure preparation, D. King for mass spectroscopy analysis, and members of the Kuriyan laboratory for support and helpful discussions. We appreciate the assistance of C. Ralston, G. McDermott and the scientific staff of the Advanced Light Source (LBNL, Berkeley) in data collection. This work was supported by grants from the National Institutes of Health (J.K. and M.O.D.) and a Ruth L. Kirschstein National Research Service Award through the National Institute of General Medical Sciences (G.D.B.).

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Correspondence to John Kuriyan.

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Supplementary information

Supplementary Figure 1

Removal of the RFC “arginine finger” residues severely compromises the RFC ATPase. (JPG 18 kb)

Supplementary Figure 2

Experimental MAD-phased electron density maps surrounding bound nucleotides reveal the triphosphate character for RFC-A, B, C and D. (JPG 70 kb)

Supplementary Figure 3

The A:B interface of the RFC complex is more closely packed than those of NSF and HslU. (JPG 77 kb)

Supplementary Figure Legends (DOC 20 kb)

Supplementary Table (DOC 26 kb)

Supplementary Methods (DOC 23 kb)

Supplementary References

References for supplementary methods and figures. (DOC 20 kb)

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Bowman, G., O'Donnell, M. & Kuriyan, J. Structural analysis of a eukaryotic sliding DNA clamp–clamp loader complex. Nature 429, 724–730 (2004).

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