Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro

The human DNA damage sensors, Rad17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complex, are thought to be involved in the early steps of the DNA damage checkpoint response. Rad17-RFC and the 9-1-1 complex have been shown to be structurally similar to the replication factors, RFC clamp loader and proliferating cell nuclear antigen polymerase clamp, respectively. Here, we demonstrate functional similarities between the replication and checkpoint clamp loader/DNA clamp pairs. When all eight subunits of the two checkpoint complexes are coexpressed in insect cells, a stable Rad17-RFC/9-1-1 checkpoint supercomplex forms in vivo and is readily purified. The two individually purified checkpoint complexes also form a supercomplex in vitro, which depends on ATP and is mediated by interactions between Rad17 and Rad9. Rad17-RFC binds to nicked circular, gapped, and primed DNA and recruits the 9-1-1 complex in an ATP-dependent manner. Electron microscopic analyses of the reaction products indicate that the 9-1-1 ring is clamped around the DNA.

[1]  T. Weinert,et al.  Toward maintaining the genome: DNA damage and replication checkpoints. , 2002, Annual review of genetics.

[2]  S. Elledge,et al.  Checking on the fork: the DNA-replication stress-response pathway. , 2002, Trends in cell biology.

[3]  K. Sugimoto,et al.  Clamp and clamp loader structures of the human checkpoint protein complexes, Rad9‐1‐1 and Rad17‐RFC , 2002, Genes to cells : devoted to molecular & cellular mechanisms.

[4]  S. Jackson,et al.  Interfaces Between the Detection, Signaling, and Repair of DNA Damage , 2002, Science.

[5]  J. Griffith,et al.  Preferential binding of ATR protein to UV-damaged DNA , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  J. Griffith,et al.  Structures of the Human Rad17-Replication Factor C and Checkpoint Rad 9-1-1 Complexes Visualized by Glycerol Spray/Low Voltage Microscopy* , 2002, The Journal of Biological Chemistry.

[7]  S. Elledge,et al.  Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin. , 2002, Genes & development.

[8]  R. St.Onge,et al.  DNA Damage-dependent and -independent Phosphorylation of the hRad9 Checkpoint Protein* , 2001, The Journal of Biological Chemistry.

[9]  D. Toczyski,et al.  Two checkpoint complexes are independently recruited to sites of DNA damage in vivo. , 2001, Genes & development.

[10]  Kunihiro Matsumoto,et al.  Recruitment of Mec1 and Ddc1 Checkpoint Proteins to Double-Strand Breaks Through Distinct Mechanisms , 2001, Science.

[11]  J. Hurwitz,et al.  Purification and characterization of human DNA damage checkpoint Rad complexes , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[12]  R. Abraham.  Cell cycle checkpoint signaling through the ATM and ATR kinases. , 2001, Genes & development.

[13]  L. Karnitz,et al.  Reconstitution and Molecular Analysis of the hRad9-hHus1-hRad1 (9-1-1) DNA Damage Responsive Checkpoint Complex* , 2001, The Journal of Biological Chemistry.

[14]  S. Gygi,et al.  Identification of RFC(Ctf18p, Ctf8p, Dcc1p): an alternative RFC complex required for sister chromatid cohesion in S. cerevisiae. , 2001, Molecular cell.

[15]  F. Spencer,et al.  Saccharomyces cerevisiae CTF18 and CTF4 Are Required for Sister Chromatid Cohesion , 2001, Molecular and Cellular Biology.

[16]  Ceslovas Venclovas,et al.  Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamp-loading complexes , 2000, Nucleic Acids Res..

[17]  T. Caspari,et al.  Characterization of Schizosaccharomyces pombeHus1: a PCNA-Related Protein That Associates with Rad1 and Rad9 , 2000, Molecular and Cellular Biology.

[18]  Č. Venclovas,et al.  A Sliding Clamp Model for the Rad1 Family of Cell Cycle Checkpoint Proteins , 1999, Cell.

[19]  Z. Kelman,et al.  Studies on the interactions between human replication factor C and human proliferating cell nuclear antigen. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  L. Karnitz,et al.  Human Homologs of Schizosaccharomyces pombe Rad1, Hus1, and Rad9 Form a DNA Damage-responsive Protein Complex* , 1999, The Journal of Biological Chemistry.

[21]  B. Stillman,et al.  The DNA replication fork in eukaryotic cells. , 1998, Annual review of biochemistry.

[22]  D. Lydall,et al.  G2 / M checkpoint genes of Saccharomyces cerevisiae : further evidence for roles in DNA replication and / or repair , 1997, Molecular and General Genetics MGG.

[23]  P. Burgers,et al.  Overproduction and Affinity Purification of Saccharomyces cerevisiae Replication Factor C* , 1997, The Journal of Biological Chemistry.

[24]  Z. Kelman,et al.  Escherichia coli expression vectors containing a protein kinase recognition motif, His6-tag and hemagglutinin epitope. , 1995, Gene.

[25]  A. Carr,et al.  Fission yeast rad17: a homologue of budding yeast RAD24 that shares regions of sequence similarity with DNA polymerase accessory proteins. , 1995, The EMBO journal.

[26]  J. Griffith,et al.  Electron microscope visualization of chromatin and other DNA-protein complexes. , 1978, Annual review of biophysics and bioengineering.