Clamp and clamp loader structures of the human checkpoint protein complexes, Rad9-1-1 and Rad17-RFC.
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 x 22 nm in size with a cleft, reminiscent of the structure of RFC.


CONCLUSIONS
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.
Shiomi
Yasushi
Y
Graduate School of Biological Science, Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-0101 Japan.
Shinozaki
Ayako
A
Nakada
Daisuke
D
Sugimoto
Katsunori
K
Usukura
Jiro
J
Obuse
Chikashi
C
Tsurimoto
Toshiki
T
D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000648:ultrastructure; D017931:DNA Primers; D004268:DNA-Binding Proteins; D006801:Humans; D008854:Microscopy, Electron; D011487:Protein Conformation
D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C069930:rad9 protein