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BIOCHEMISTRY
Open clamp structure in the clamp-loading complex visualized by electron microscopic image analysis

Tomoko Miyata ^* , Hirofumi Suzuki ^* , Takuji Oyama ^*, Kouta Mayanagi ^*, Yoshizumi Ishino , and Kosuke Morikawa ^*, 

^*Department of Structural Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan; and Laboratory of Protein Chemistry and Engineering, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka-shi, Fukuoka 812-8581, Japan

Communicated by John Kuriyan, University of California, Berkeley, CA, July 28, 2005 (received for review June 13, 2005)

Ring-shaped sliding clamps and clamp loader ATPases are essential factors for rapid and accurate DNA replication. The clamp ring is opened and resealed at the primer–template junctions by the ATP-fueled clamp loader function. The processivity of the DNA polymerase is conferred by its attachment to the clamp loaded onto the DNA. In eukarya and archaea, the replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) play crucial roles as the clamp loader and the clamp, respectively. Here, we report the electron microscopic structure of an archaeal RFC–PCNA–DNA complex at 12-Å resolution. This complex exhibits excellent fitting of each atomic structure of RFC, PCNA, and the primed DNA. The PCNA ring retains an open conformation by extensive interactions with RFC, with a distorted spring washer-like conformation. The complex appears to represent the intermediate, where the PCNA ring is kept open before ATP hydrolysis by RFC.

AAA⁺ ATPase | clamp loader | DNA replication | electron microscopy | single-particle analysis

Abbreviations: CTF, contrast transfer function; ds, double-stranded; PCNA, proliferating cell nuclear antigen; Pfu, Pyrococcus furiosus; priDNA, primed template DNA; RFC, replication factor C; RFCL, RFC large subunit; RFCS, RFC small subunit; ss, single-stranded.

T.M. and H.S. contributed equally to this work.

To whom correspondence should be addressed. E-mail: morikawa{at}beri.or.jp.

© 2005 by The National Academy of Sciences of the USA

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