Book of Abstracts: Albany 2007
June 19-23 2007
Structural view of the clamp-loading mechanism onto DNA
DNA replication is a highly coordinated process, which involves numerous proteins in nuclei. To promise the integrated system, proteins involved in this event constitute several kinds of molecular machinery. Archaeal systems generally exhibit attractive properties to study DNA metabolism; Their DNA binding proteins are very similar to those from eukarya in both functional and structural aspects, irrespective of their morphological difference, and hence they are good model systems for understanding eukaryotic DNA processing. We have been working on several DNA processing proteins from a hyperthermophilic archaeon, Pyrococcus furiosus (Pfu).
Replicative DNA polymerase requires two essential protein factors, a sliding clamp and a clamp loader, for rapid and accurate DNA duplication. In eukarya and archaea, a homo-trimeric proliferating cell nuclear antigen (PCNA) and a hetero-pentameric replication factor C (RFC) function as the clamp and the clamp loader, respectively. The ATP-dependent clamp-loading mechanism by RFC is particularly intriguing, because it requires opening and resealing of the PCNA ring. Both crystal structures of PCNA and the RFC small subunit were determined at the atomic resolution, indicating that their architectures are indeed similar to those from eukaryotic proteins. More recently, we determined the three-dimensional structure of an RFC?PCNA?DNA clamp-loading complex at 12A resolution by electron microscopy (EM)-single particle reconstruction. The X-ray structures of the RFC small subunit, PCNA and a DNA duplex could be well fitted into the image of the complex. Importantly, the structure of the ternary complex presents the first direct view of a washer-like open conformation of the PCNA ring in contact with RFC. In combination with the two X-ray structural data reported previously by the other group, our EM model implies an intriguing clamp loading mechanism.
References and Footnotes
- Atomic structure of the clamp loader small subunit from Pyrococcus furiosus.
T. Oyama, Y. Ishino, I. K. O. Cann, S. Ishino and K. Morikawa , Molec. Cell, 8, 455-463 (2001).
- Intermolecular ion pairs maintain the toroidal structure of Pirococcus furiosus PCNA, S. Matsumiya, S. Ishino, Y. Ishino and K. Morikawa, Protein Science 12, 823-831 (2003).
- Open clamp structure in the clamp-loading complex visualized by electron microscopic image analysis., T. Miyata, H. Suzuki, T. Ohyama, K. Mayanagi, Y. Ishino and K. Morikawa, Proc. Natl. Acad. Sci. USA 102, 13795-13800 (2005).
Open Laboratories of Advanced Bioscience and Biotechnology (OLABB),
Institute for Protein Research, Osaka University, Furuedai, Suita, Osaka 565-0874, Japan