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- Title
Clamp and clamp loader structures of the human checkpoint protein complexes, Rad9-1-1 and Rad17-RFC.
- Authors
Shiomi, Yasushi; Shinozaki, Ayako; Nakada, Daisuke; Sugimoto, Katsunori; Usukura, Jiro; Obuse, Chikashi; Tsurimoto, Toshiki
- Abstract
Abstract Background: We have reported that protein imaging by transmission electron microscope observation based on low-angle platinum shadowing can reproduce characteristic ring structures of the replication clamp, proliferating cell nuclear antigen (PCNA), and the clamp loader protein, replication factor C (RFC). The checkpoint protein complexes, Rad9-Hus1-Rad1 (Rad9-1-1) and Rad17-RFCs2-5 (Rad17-RFC), have been predicted to function as novel clamp and clamp loader proteins, respectively, due to their amino acid sequence similarities with PCNA and RFC. Results: We reconstituted human Rad9-1-1 and Rad17-RFC complexes in insect cells using a baculovirus expression system and showed purified Rad9-1-1 to be composed of equimolar amounts of Rad9, Hus1 and Rad1 proteins, exhibiting a native molecular mass of 100 kDa, in line with a trimeric complex. When Rad17 was co-expressed with the four small subunits of RFC in insect cells, these proteins formed a complex of 240 kDa that displayed DNA binding, ATPase activity and binding to its predicted target protein, Rad9-1-1. Analyses of the molecular architecture of Rad9-1-1 and Rad17-RFC using transmission electron microscopy, in comparison with PCNA and RFC, revealed the Rad9-1-1 complex to have a characteristic ring structure indistinguishable from that of PCNA in shape and size. In addition, the Rad17–RFC complex was found to be oval in structure and 26 × 22 nm in size with a cleft, reminiscent of the structure of RFC. Conclusion: Our direct comparison of images from the two sets of clamp and clamp loader proteins indicated that Rad9-1-1 and Rad17-RFC are, respectively, structural orthologs of PCNA and RFC, with presumed functions as novel clamp and clamp-loader proteins in eukaryotes.
- Subjects
PROTEINS; ANTIGENS; AMINO acid sequence
- Publication
Genes to Cells, 2002, Vol 7, Issue 8, p861
- ISSN
1356-9597
- Publication type
Article
- DOI
10.1046/j.1365-2443.2002.00566.x