DNA damage activates cell cycle checkpoint signaling pathways that coordinate cell cycle arrest and DNA repair. Three of the proteins involved in checkpoint signaling, Rad1, Hus1, and Rad9, have been shown to interact by immunoprecipitation and yeast two-hybrid studies. However, it is not known how these proteins interact and assemble into a complex. In the present study we demonstrated that in human cells all the hRad9 and hHus1 and approximately one-half of the cellular pool of hRad1 interacted as a stable, biochemically discrete complex, with an apparent molecular mass of 160 kDa. This complex was reconstituted by co-expression of all three recombinant proteins in a heterologous system, and the reconstituted complex exhibited identical chromatographic behavior as the endogenous complex. Interaction studies using differentially tagged proteins demonstrated that the proteins did not self-multimerize. Rather, each protein had a binding site for the other two partners, with the N terminus of hRad9 interacting with hRad1, the N terminus of hRad1 interacting with hHus1, and the N terminus of hHus1 interacting with the C terminus of hRad9's predicted PCNA-like region. Collectively, these analyses suggest a model of how these three proteins assemble to form a functional checkpoint complex, which we dubbed the 9-1-1 complex.DNA damage provokes multiple cellular responses, including the activation of DNA damage checkpoint signaling pathways, which arrest cell cycle progression in G 1 , S, and G 2 /M and possibly coordinate repair of the damage. Loss of the DNA damage checkpoint response leads to increased sensitivity to DNA-damaging agents, demonstrating that these pathways are critical for efficient recovery from DNA damage (reviewed in Refs. 1-6). The components of the checkpoint signaling pathways were initially identified by genetic analyses in the yeasts, Schizosaccharomyces pombe and Saccharomyces cerevisiae, and recent studies demonstrated that the checkpoint genes are conserved between yeasts and humans (7-23). Collectively, these studies have given rise to a conserved model in which DNA damage activates members of the ATM (ataxia telangiectasia-mutated) family of phosphatidylinositol 3-kinase-related kinases. These protein kinases are then required to activate the downstream protein kinases Chk1 and Chk2 (20,(22)(23)(24)(25), which regulate cell cycle arrest and possibly other DNA damage-induced responses.Despite the recent progress in the dissection of the downstream protein kinase-mediated portions of the checkpoint signaling pathways, yeast genetic studies demonstrated that additional checkpoint genes also regulate the DNA damage response (reviewed in Refs. 2-6). For example, in S. pombe spRad1, spHus1, spRad9, and spRad17 (using S. pombe nomenclature) are all essential for DNA damage-induced cell cycle arrest and activation of the spChk1 protein kinase after DNA damage (4 -6). Thus, these results suggest that Rad1, Hus1, Rad9, and Rad17 function upstream of Chk1 activation in the DNA damage-signaling pathw...

DNA damage activates cell cycle checkpoints that prevent progression through the cell cycle. In yeast, the DNA damage checkpoint response is regulated by a series of genes that have mammalian homologs, including rad1, rad9, hus1, and rad17. On the basis of sequence homology, yeast and human Rad1, Rad9, and Hus1 protein homologs are predicted to structurally resemble the sliding clamp PCNA. Likewise, Rad17 homologs have extensive homology with replication factor C (RFC) subunits (p36, p37, p38, p40, and p140), which form a clamp loader for PCNA. These observations predict that Rad1, Hus1, and Rad9 might interact with Rad17 as a clampclamp loader pair during the DNA damage response. In this report, we demonstrate that endogenous human Rad17 (hRad17) interacts with the PCNA-related checkpoint proteins hRad1, hRad9, and hHus1. Mutational analysis of hRad1 and hRad17 demonstrates that this interaction has properties similar to the interaction between RFC and PCNA, a well characterized clampclamp loader pair. Moreover, we show that DNA damage affects the association of hRad17 with the clamp-like checkpoint proteins. Collectively, these data provide the first experimental evidence that hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9 in manner similar to the interaction between RFC and PCNA.In response to DNA damage, eukaryotic cells block cell cycle progression in a process commonly known as the DNA damageinduced checkpoint response. Studies in genetically tractable yeast model systems have identified a large number of genes, dubbed checkpoint genes, that are essential for DNA damageinducible checkpoint activation (reviewed in Refs. 1-5). Epistasis and biochemical analyses in yeasts and humans have provisionally ordered the checkpoint proteins into a signaling pathway in which DNA damage relays activating signals through the phosphatidylinositol 3-kinase-related kinases, which include spRad3, scMec1, ATR, and ATM. The phosphatidylinositol 3-kinase-related kinases regulate activation of the serine-threonine protein kinases Chk1 and Chk2 (6 -10), which phosphorylate the cell-cycle phosphatase Cdc25 (7,9,11,12). Phosphorylation of Cdc25 inhibits its activity (13,14) and its accumulation in the nucleus (15, 16), thereby preventing activation of the CyclinB-Cdc2 complex and blocking the G 2 /M transition after DNA damage.Studies in Schizosaccharomyces pombe and Saccharomyces cerevisiae demonstrated that the checkpoint proteins spRad1, spHus1, spRad9, and spRad17 (using S. pombe nomenclature) or their homologs are essential for DNA damage-activated checkpoint responses (reviewed in Refs. 1 and 3-5). Furthermore, these studies suggest that all four proteins act early in the DNA damage-induced signaling pathway. Sequence analyses provide a few clues regarding potential functions of these proteins. Yeast, human, and fly Rad1 exhibit sequence homology with Ustilago maydis Rec1 (17-23), a checkpoint protein and a 3Ј-5Јexonuclease (24), suggesting that Rad1 may also be a nuclease. However, a highly conserved D...

Rad17, Rad1, Hus1, and Rad9 are key participants in checkpoint signaling pathways that block cell cycle progression in response to genotoxins. Biochemical and molecular modeling data predict that Rad9, Hus1, and Rad1 form a heterotrimeric complex, dubbed 9-1-1, which is loaded onto chromatin by a complex of Rad17 and the four small replication factor C (RFC) subunits (Rad17-RFC) in response to DNA damage. It is unclear what checkpoint proteins or checkpoint signaling events regulate the association of the 9-1-1 complex with DNA. Here we show that genotoxin-induced chromatin binding of 9-1-1 does not require the Rad9-inducible phosphorylation site (Ser-272). Although we found that Rad9 undergoes an additional phosphatidylinositol 3-kinase-related kinase (PIKK)-dependent posttranslational modification, we also show that genotoxin-triggered 9-1-1 chromatin binding does not depend on the catalytic activity of the PIKKs ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), or DNA-PK. Additionally, 9-1-1 chromatin binding does not require DNA replication, suggesting that the complex can be loaded onto DNA in response to DNA structures other than stalled DNA replication forks. Collectively, these studies demonstrate that 9-1-1 chromatin binding is a proximal event in the checkpoint signaling cascade.DNA damage and replication stress activate checkpoint signaling pathways that block cell cycle progression, activate programmed cell death, and influence DNA repair. Biochemical and genetic studies in yeasts and mammals have identified many of the key components and arranged them into checkpoint signaling cascades (reviewed Refs. 1 and 2). The phosphatidylinositol-3-kinase-related kinases (PIKK) 1 ATM and ATR are central components of the evolutionarily conserved checkpoint signaling pathways (3). In response to genotoxins, ATM and ATR phosphorylate and activate the protein kinases Chk1 and Chk2 (4). Activated ATM, ATR, Chk1, and Chk2 then phosphorylate additional checkpoint proteins, including Rad9, BRCA1, p53, Cdc25A, Cdc25C, Nbs1, and other proteins that mediate checkpoint activation and DNA repair.In addition to the PIKKs, the checkpoint proteins, Rad9, Hus1, Rad1, and Rad17 (using Schizosaccharomyces pombe nomenclature) are key elements of checkpoint signaling pathways that are conserved from the yeasts to humans. Disruption of these genes in S. pombe blocks genotoxin-induced Chk1 activation (5). Correspondingly, Hus1 and Rad17 are also required for genotoxin-induced Chk1 activation in mammals (6, 7). Such studies suggest that these proteins function early in the checkpoint signaling pathway. Recent biochemical studies further support this notion. Rad9, Hus1, and Rad1 form a stable heterotrimeric complex (the 9-1-1 complex) (8, 9) that, based on biochemical, biophysical, and molecular modeling studies, is predicted to resemble PCNA (8, 10 -14). PCNA subunits assemble into a toroidal clamp complex that is loaded around DNA by the clamp loader, replication factor C (p140-RFC), a protein compl...