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 loader͞DNA clamp pairs. When all eight subunits of the two checkpoint complexes are coexpressed in insect cells, a stable Rad17-RFC͞9-1-1 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. E ukaryotic cells exposed to genotoxic agents activate the DNA damage checkpoint signaling pathway, which arrests cellcycle progression and in so doing prevents cell death or mutations. Recent work has revealed that in mammalian cells, the ATM and ATR proteins, which belong to the phosphatidylinositide kinase-like kinase family, and the Rad17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complexes, which have structural similarities to the replication clamp loader and replication clamp RFC and proliferating cell nuclear antigen (PCNA), respectively, are involved in damage recognition, which activates the checkpoint response (reviewed in refs. 1-4). Studies with budding and fission yeasts have shown that the orthologs of these proteins perform similar functions. However, biochemical data on the specific roles of the phosphatidylinositide kinase-like kinase family members and the Rad17-RFC and 9-1-1 complexes are scarce, and hence the damage sensing step of the checkpoint response remains illdefined. We previously reported that ATR directly recognizes and is activated by damaged DNA (5). In this article, we investigate the interactions of Rad17-RFC and the 9-1-1 checkpoint complexes with DNA to gain some insight into their roles as damage sensors.Rad17-RFC is one of the three known RFC-like complexes in mammalian cells. In this form of RFC, the p140 subunit is replaced by the 75-kDa Rad17 protein, which has homology to all RFC subunits (6). Yeast genetic studies indicate that the orthologs of human Rad17 function exclusively in the DNA damage checkpoint response (7,8). The 9-1-1 checkpoint complex is a heterotrimer of Rad9, Rad1, and Hus1 proteins, which were predicted to have structural homology to PCNA (9-13). Previously, we showed that Rad17 associates with the four small RFC subunits to make an RFC-like complex, which by electron microscopy exhibits an RFC-like structure (14). Similarly, we found that Rad9, Rad1, and Hus1 f...

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The linkage of sister chromatids after DNA replication ensures the faithful inheritance of chromosomes by daughter cells. In budding yeast, the establishment of sister chromatid cohesion requires Ctf8, Dcc1, and Ctf18, a homologue of the p140 subunit of the replication factor C (RFC). In this report we demonstrate that in 293T cells, Flag-tagged Ctf18 forms a seven-subunit cohesion-RFC complex comprised of Ctf18, Dcc1, Ctf8, RFCp40, RFCp38, RFCp37, and RFCp36 (Ctf18 -RFC). We demonstrate that a stoichiometric heteroheptameric Ctf18 -RFC complex can be assembled by coexpressing the seven proteins in baculovirus-infected insect cells. In addition, the two other stable subcomplexes were formed, which include a pentameric complex comprised of Ctf18, RFCp40, RFCp38, RFCp37, and RFCp36 and a dimeric Dcc1-Ctf8. Both the five-and sevensubunit Ctf18 -RFC complexes bind to single-stranded and primed DNAs and possess weak ATPase activity that is stimulated by the addition of primed DNA and proliferating cell nuclear antigen (PCNA). These complexes catalyzed the ATP-dependent loading of PCNA onto primed and gapped DNA but not onto double-stranded nicked or single-stranded circular DNAs. Consistent with these observations, both Ctf18 -RFC complexes substituted for the replicative RFC in the PCNA-dependent DNA polymerase ␦-catalyzed DNA replication reaction. These results support a model in which sister chromatid cohesion is linked to DNA replication.F aithful inheritance of a complete set of the chromosome complement by daughter cells is essential for cell survival (1-4). In eukaryotes, newly synthesized sister chromatid DNAs are linked together physically by the cohesin complex (called cohesion) from the time they are replicated until their distribution between daughter cells in anaphase (1, 5-7). In budding yeast, the cohesin complex, composed of the four proteins Scc1͞Rad21, Scc3, SMC1, and SMC3, is loaded onto chromatin during the G 1 ͞S transition and leads to the association of sister chromatids after DNA replication. Mutations in any one of these subunits result in the precocious separation of sister chromatids before cohesion is severed in anaphase and ultimately leads to cell death. Recent studies have revealed important insights into the cohesin structure and have shown that the severance of cohesion is mediated by separase, a protease that cleaves Scc1 (1,5,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). The steps leading to cohesion between sister chromatids, however, remain unknown. Chromosome-loss assays have identified a number of genes required for cohesion of sister chromatids. Based on their putative roles, cohesion genes have been characterized as either deposition or establishment factors (18). The deposition factors such as Scc2 and Scc4, which interact and must function during S phase, are required for the loading of cohesin onto DNA but are not part of the cohesin complex (19). Establishment factors, which include Ctf7͞Eco1͞Eso1, Ctf4͞Pob1, Ctf18͞Chl12, Dcc1, and Ctf8, are essential for cohesion and have some ...

http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.10100245/-/DC1.