Clamp and clamp loader structures of the human checkpoint protein complexes, Rad9-1-1 and Rad17-RFC

Yasushi Shiomi, Ayako Shinozaki, Daisuke Nakada, Katsunori Sugimoto, Jiro Usukura, Chikashi Obuse, Toshiki Tsurimoto

Research output: Contribution to journalArticle

77 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)861-868
Number of pages8
JournalGenes to Cells
Volume7
Issue number8
DOIs
Publication statusPublished - Aug 1 2002

Fingerprint

Replication Protein C
Proliferating Cell Nuclear Antigen
Proteins
rad9 protein
Insect Proteins
Baculoviridae
Platinum
Eukaryota
Transmission Electron Microscopy
Insects
Adenosine Triphosphatases
Amino Acid Sequence

All Science Journal Classification (ASJC) codes

  • Genetics
  • Cell Biology

Cite this

Clamp and clamp loader structures of the human checkpoint protein complexes, Rad9-1-1 and Rad17-RFC. / Shiomi, Yasushi; Shinozaki, Ayako; Nakada, Daisuke; Sugimoto, Katsunori; Usukura, Jiro; Obuse, Chikashi; Tsurimoto, Toshiki.

In: Genes to Cells, Vol. 7, No. 8, 01.08.2002, p. 861-868.

Research output: Contribution to journalArticle

Shiomi, Yasushi ; Shinozaki, Ayako ; Nakada, Daisuke ; Sugimoto, Katsunori ; Usukura, Jiro ; Obuse, Chikashi ; Tsurimoto, Toshiki. / Clamp and clamp loader structures of the human checkpoint protein complexes, Rad9-1-1 and Rad17-RFC. In: Genes to Cells. 2002 ; Vol. 7, No. 8. pp. 861-868.
@article{41724c3d9bb74a17b330856fb153a160,
title = "Clamp and clamp loader structures of the human checkpoint protein complexes, Rad9-1-1 and Rad17-RFC",
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.",
author = "Yasushi Shiomi and Ayako Shinozaki and Daisuke Nakada and Katsunori Sugimoto and Jiro Usukura and Chikashi Obuse and Toshiki Tsurimoto",
year = "2002",
month = "8",
day = "1",
doi = "10.1046/j.1365-2443.2002.00566.x",
language = "English",
volume = "7",
pages = "861--868",
journal = "Genes to Cells",
issn = "1356-9597",
publisher = "Wiley-Blackwell",
number = "8",

}

TY - JOUR

T1 - Clamp and clamp loader structures of the human checkpoint protein complexes, Rad9-1-1 and Rad17-RFC

AU - Shiomi, Yasushi

AU - Shinozaki, Ayako

AU - Nakada, Daisuke

AU - Sugimoto, Katsunori

AU - Usukura, Jiro

AU - Obuse, Chikashi

AU - Tsurimoto, Toshiki

PY - 2002/8/1

Y1 - 2002/8/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0036668474&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036668474&partnerID=8YFLogxK

U2 - 10.1046/j.1365-2443.2002.00566.x

DO - 10.1046/j.1365-2443.2002.00566.x

M3 - Article

C2 - 12167163

AN - SCOPUS:0036668474

VL - 7

SP - 861

EP - 868

JO - Genes to Cells

JF - Genes to Cells

SN - 1356-9597

IS - 8

ER -