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Clamp and clamp loader structures of the human checkpoint protein complexes, Rad9‐1‐1 and Rad17‐RFC
Author(s) -
Shiomi Yasushi,
Shinozaki Ayako,
Nakada Daisuke,
Sugimoto Katsunori,
Usukura Jiro,
Obuse Chikashi,
Tsurimoto Toshiki
Publication year - 2002
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1046/j.1365-2443.2002.00566.x
Subject(s) - biology , clamp , replication factor c , proliferating cell nuclear antigen , dna replication , processivity , microbiology and biotechnology , biophysics , dna polymerase delta , dna , biochemistry , eukaryotic dna replication , computer science , rna , reverse transcriptase , clamping , gene , computer vision
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.