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The clamp-loading complex for processive DNA replication

Nature Structural & Molecular Biology volume 11, pages 632–636 (2004)Cite this article

Abstract

DNA polymerase requires two processing factors, sliding clamps and clamp loaders, to direct rapid and accurate duplication of genomic DNA. In eukaryotes, proliferating cell nuclear antigen (PCNA), the ring-shaped sliding clamp, encircles double-stranded DNA within its central hole and tethers the DNA polymerases onto DNA. Replication factor C (RFC) acts as the clamp loader, which correctly installs the sliding clamp onto DNA strands in an ATP-dependent manner. Here we report the three-dimensional structure of an archaeal clamp-loading complex (RFC–PCNA–DNA) determined by single-particle EM. The three-dimensional structure of the complex, reconstituted in vitro using a nonhydrolyzable ATP analog, reveals two components, a closed ring and a horseshoe-shaped element, which correspond to PCNA and RFC, respectively. The atomic structure of PCNA fits well into the closed ring, suggesting that this ternary complex represents a state just after the PCNA ring has closed to encircle the DNA duplex.

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Figure 1: Formation of the clamp-loading complex from Pyrococcus furiosus and electron micrograph.
Figure 2: Three-dimensional structure of the clamp-loading complex.
Figure 3: Streptavidin labeling of the clamp-loading complex.
Figure 4: Model of the PCNA-loading reaction by RFC.

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Acknowledgements

The authors are grateful to G. Bowman and J. Kuriyan for providing the atomic coordinates of the yeast RFC–PCNA complex before publication, and for comments on the manuscript. We thank K. Shimizu, H. Nishida and T. Shiraki for helpful discussions and M. Yuasa and S. Matsumiya for assistance with sample preparation. This work was partly supported by a research grant endorsed by the Japan New Energy and Industrial Technology Development Organization (NEDO). Y.I. was supported by the Human Frontier Science Program.

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Authors and Affiliations

  1. Department of Structural Biology, Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, Suita, 565-0874, Osaka, Japan

    Tomoko Miyata, Takuji Oyama, Kouta Mayanagi, Sonoko Ishino & Kosuke Morikawa

  2. Laboratory of Protein Chemistry and Engineering, Faculty of Agriculture, Kyushu University, Hakozaki, Higash-ku, Fukuoka-shi, Fukuoka, 812-8581, Japan

    Yoshizumi Ishino

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Correspondence to Kosuke Morikawa.

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Supplementary information

Supplementary Fig. 1

Biochemical analyses of the clamp-loading complex. (PDF 152 kb)

Supplementary Video. 1

A movie of EM three-dimensional reconstruction map of the clamp-loading complex. The first shot corresponds to the top view in Figure 2a. The particle rotates 360° around the horizontal axis via the side view (Fig. 2b) and the bottom view (Fig. 2c), and then turns along with the vertical axis. The final shot corresponds to Figure 2d, showing the central channel of the closed ring from the top view side. (MOV 1078 kb)

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Miyata, T., Oyama, T., Mayanagi, K. et al. The clamp-loading complex for processive DNA replication. Nat Struct Mol Biol 11, 632–636 (2004). https://doi.org/10.1038/nsmb788

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