In response to DNA damage, eukaryotic cells activate checkpoint pathways that arrest cell cycle progression and induce the expression of genes required for DNA repair. In budding yeast, the homothallic switching (HO) endonuclease creates a site-specific double-strand break at the mating type (MAT) locus. Continuous HO expression results in the phosphorylation of Rad53, which is dependent on products of the ataxia telangiectasia mutated-related MEC1 gene and other checkpoint genes, including DDC1, RAD9, and RAD24. Chromatin immunoprecipitation experiments revealed that the Ddc1 protein associates with a region near the MAT locus after HO expression. Ddc1 association required Rad24 but not Mec1 or Rad9. Mec1 also associated with a region near the cleavage site after HO expression, but this association is independent of Ddc1, Rad9, and Rad24. Thus, Mec1 and Ddc1 are recruited independently to sites of DNA damage, suggesting the existence of two separate mechanisms involved in recognition of DNA damage.

Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and to a wide variety of environmental stresses. MAPK cascades can be inactivated at the MAPK activation step by members of the MAPK phosphatase (MKP) family. However, the components that act in MKP-regulated pathways have not been well characterized in the context of whole organisms. Here we characterize the Caenorhabditis elegans vhp-1 gene, encoding an MKP that acts preferentially on the c-Jun N-terminal kinase (JNK) and p38 MAPKs. We found that animals defective in vhp-1 are arrested during larval development. This vhp-1 defect is suppressed by loss-of-function mutations in the kgb-1, mek-1, and mlk-1 genes encoding a JNK-like MAPK, an MKK7-type MAPKK, and an MLK-type MAPKKK, respectively. The genetic and biochemical data presented here demonstrate a critical role for VHP-1 in the KGB-1 pathway. Loss-of-function mutations in each component in the KGB-1 pathway result in hypersensitivity to heavy metals. These results suggest that VHP-1 plays a pivotal role in the integration and fine-tuning of the stress response regulated by the KGB-1 MAPK pathway.

In eukaryotes, the ATM and ATR family proteins play a critical role in the DNA damage and replication checkpoint controls. These proteins are characterized by a kinase domain related to the phosphatidylinositol 3-kinase, but they have the ability to phosphorylate proteins. In budding yeast, the ATR family protein Mec1/Esr1 is essential for checkpoint responses and cell growth. We have isolated the PIE1 gene in a two-hybrid screen for proteins that interact with Mec1, and we show that Pie1 interacts physically with Mec1 in vivo. Like MEC1, PIE1 is essential for cell growth, and deletion of the PIE1 gene causes defects in the DNA damage and replication block checkpoints similar to those observed in mec1⌬ mutants. Rad53 hyperphosphorylation following DNA damage and replication block is also decreased in pie1⌬ cells, as in mec1⌬ cells. Pie1 has a limited homology to fission yeast Rad26, which forms a complex with the ATR family protein Rad3. Mutation of the region in Pie1 homologous to Rad26 results in a phenotype similar to that of the pie1⌬ mutation. Mec1 protein kinase activity appears to be essential for checkpoint responses and cell growth. However, Mec1 kinase activity is unaffected by the pie1⌬ mutation, suggesting that Pie1 regulates some essential function other than Mec1 kinase activity. Thus, Pie1 is structurally and functionally related to Rad26 and interacts with Mec1 to control checkpoints and cell proliferation.When DNA replication is blocked and DNA damage occurs, checkpoints arrest the cell cycle, allowing DNA replication and repair to take place (13,19). Loss of checkpoint control results in cell death or genetic instability that can lead to cancer. Checkpoint pathways are an evolutionarily conserved feature of eukaryotic cells. This conservation is exemplified by the family of genes encoding high-molecular-weight protein kinases, including ATM (mammals), ATR (mammals), MEC1 (Saccharomyces cerevisiae), TEL1 (S. cerevisiae), rad3 ϩ (Schizosaccharomyces pombe), mei-41 (Drosophila melanogaster), and uvsB (Aspergillus nidulans) (6,9,18,22,23,30,37,40,48). Each of these genes falls into two family groups based on homology; ATM is related most closely to TEL1, while ATR is more related to MEC1, rad3 ϩ , mei-41, and uvsB (6, 40). This homology is not restricted to the kinase domain at the carboxyl terminus but extends over the length of the protein. The carboxyl-terminal kinase domain is structurally related to the catalytic domain of the phosphatidylinositol (PI) 3-kinases. Despite this similarity, none of these proteins has been shown to phosphorylate lipids. ATM, ATR, and Rad3 are all capable of phosphorylating protein substrates (5,8,28,29). However, it remains to be determined how the kinase activity of these proteins is controlled in checkpoint responses. Moreover, little is known about whether these proteins form a complex with other proteins, although Rad3 has been recently shown to form a complex with Rad26 (12). The only Rad26 homolog identified so far is A. nidulans UVSD (40), but it has not be...

RAD24 has been identified as a gene essential for the DNA damage checkpoint in budding yeast. Rad24 is structurally related to subunits of the replication factor C (RFC) complex, and forms an RFC-related complex with Rfc2, Rfc3, Rfc4, and Rfc5. The rad24⌬ mutation enhances the defect of rfc5-1 in the DNA replication block checkpoint, implicating RAD24 in this checkpoint. CHL12 (also called CTF18) encodes a protein that is structurally related to the Rad24 and RFC proteins. We show here that although neither chl12⌬ nor rad24⌬ single mutants are defective, chl12⌬ rad24⌬ double mutants become defective in the replication block checkpoint. We also show that Chl12 interacts physically with Rfc2, Rfc3, Rfc4, and Rfc5 and forms an RFC-related complex which is distinct from the RFC and RAD24 complexes. Our results suggest that Chl12 forms a novel RFC-related complex and functions redundantly with Rad24 in the DNA replication block checkpoint.Eukaryotic cells employ a set of surveillance mechanisms to coordinate the onset of one event and the completion of the preceding event during the cell cycle. The mechanisms that ensure the proper ordering of cell cycle events have been termed checkpoint controls in eukaryotes (11). When DNA is damaged or DNA replication is blocked, the activation of checkpoint pathways arrests the cell cycle and induces the transcription of genes that facilitate DNA repair and/or replication (5, 33).Checkpoint pathways are an evolutionarily conserved feature of eukaryotic cells. This feature is typified in the ATM and ATR family genes which encode phosphatidylinositol 3-kinaserelated proteins possessing protein kinase activity (33). In the budding yeast Saccharomyces cerevisiae, MEC1 encodes an ATR-related protein and plays a critical role in checkpoint controls (14,17,32). Mec1 physically interacts with Pie1 (also called Lcd1 or Ddc2), a protein that exhibits limited homology to the fission yeast Rad26 protein (17,19,32). Likewise, in fission yeast the ATR family protein Rad3 forms a complex with Rad26 (4). DNA damage responses have been well characterized in budding yeast and consist of the G 1 -, S-, and G 2 /M-phase damage checkpoints (14). Both Mec1 and Pie1 are essential for all three DNA damage checkpoints, as well as the DNA replication block checkpoint.In addition to MEC1 and PIE1, a number of genes that control the checkpoints in budding yeast have been identified. These include DDC1, MEC3, RAD9, RAD17, RAD24, and RAD53 (5,14,33). RAD53 encodes a protein kinase and functions downstream of MEC1 in the checkpoint pathway. Like Mec1, Rad53 plays an essential role in both the replication block and DNA damage checkpoints. Following DNA damage and replication block, the Rad53 protein is hyperphosphorylated and activated by a mechanism dependent on Mec1 (20, 26). Thus, Mec1 and Rad53 constitute a central checkpoint pathway in budding yeast. RAD9, RAD17, MEC3, DDC1, and RAD24 are also required for DNA damage checkpoints. Rad9 is hyperphosphorylated following DNA damage, and the phosphorylated Ra...

Genetic analysis has suggested that RAD17, RAD24, MEC3, and DDC1 play similar roles in the DNA damage checkpoint control in budding yeast. These genes are required for DNA damage-induced Rad53 phosphorylation and considered to function upstream of RAD53 in the DNA damage checkpoint pathway. Here we identify Mec3 as a protein that associates with Rad17 in a two-hybrid screen and demonstrate that Rad17 and Mec3 interact physically in vivo. The amino terminus of Rad17 is required for its interaction with Mec3, and the protein encoded by the rad17-1 allele, containing a missense mutation at the amino terminus, is defective for its interaction with Mec3 in vivo. Ddc1 interacts physically and cosediments with both Rad17 and Mec3, indicating that these three proteins form a complex. On the other hand, Rad24 is not found to associate with Rad17, Mec3, and Ddc1. DDC1 overexpression can partially suppress the phenotypes of the rad24⌬ mutation: sensitivity to DNA damage, defect in the DNA damage checkpoint and decrease in DNA damage-induced phosphorylation of Rad53. Taken together, our results suggest that Rad17, Mec3, and Ddc1 form a complex which functions downstream of Rad24 in the DNA damage checkpoint pathway.Eukaryotic cells employ a number of surveillance mechanisms to help ensure the orderly progression and completion of critical events such as chromosome replication and segregation during the cell division. The mechanisms that ensure the proper ordering of cell cycle events have been termed checkpoint controls (7). When DNA replication is delayed and DNA damage occurs, checkpoint controls activate cell cycle arrest, allowing DNA replication and repair (3,22).In the budding yeast Saccharomyces cerevisiae, checkpoint pathways induce cell cycle arrest in G 1 /S or G 2 /M and retard S-phase progression in response to DNA damage. Other checkpoints prevent cells with incompletely replicated DNA from exiting the S phase. Genetic studies have identified a number of genes that are involved in the DNA damage checkpoint and/or the replication block checkpoint (3,22). These include RAD9, RAD17, RAD24, MEC3, DDC1, POL2, RFC5, MEC1/ESR1, and RAD53/SPK1/MEC2/SAD1. Among these genes, RAD9, RAD17, RAD24, MEC3, and DDC1 are involved not only in the G 2 /M-phase but also in the G 1 -and S-phase DNA damage checkpoints (12,13,21,(28)(29)(30)(40)(41)(42). POL2 (17, 18) and RFC5 (33, 35) are required for the checkpoints responding to replication block and DNA damage in the S phase. POL2 encodes a large subunit of DNA polymerase ε, and RFC5 encodes a small subunit of replication factor C (RFC). We have recently demonstrated that Rad24 interacts physically and cosediments with Rfc5, suggesting that Rad24 is an associated component of the RFC complex (27). MEC1 and RAD53 are necessary for the checkpoints operating in response to both DNA damage and incomplete DNA replication (1, 42). RAD53 encodes a dual-specificity protein kinase (32), and Mec1 belongs to the phosphatidylinositol kinase family that includes the human ATM proteins (9, 25)....

RAD24 and RFC5 are required for DNA damage checkpoint control in the budding yeast Saccharomyces cerevisiae. Rad24 is structurally related to replication factor C (RFC) subunits and associates with RFC subunits Rfc2, Rfc3, Rfc4, and Rfc5. rad24⌬ mutants are defective in all the G 1 -, S-, and G 2 /M-phase DNA damage checkpoints, whereas the rfc5-1 mutant is impaired only in the S-phase DNA damage checkpoint. Both the RFC subunits and Rad24 contain a consensus sequence for nucleoside triphosphate (NTP) binding. To determine whether the NTP-binding motif is important for Rad24 function, we mutated the conserved lysine 115 residue in this motif. The rad24-K115E mutation, which changes lysine to glutamate, confers a complete loss-of-function phenotype, while the rad24-K115R mutation, which changes lysine to arginine, shows no apparent phenotype. Although neither rfc5-1 nor rad24-K115R single mutants are defective in the G 1 -and G 2 /M-phase DNA damage checkpoints, rfc5-1 rad24-K115R double mutants become defective in these checkpoints. Coimmunoprecipitation experiments revealed that Rad24 K115R fails to interact with the RFC proteins in rfc5-1 mutants. Together, these results indicate that RFC5, like RAD24, functions in all the G 1 -, S-and G 2 /M-phase DNA damage checkpoints and suggest that the interaction of Rad24 with the RFC proteins is essential for DNA damage checkpoint control.Eukaryotic cells employ a set of surveillance mechanisms to coordinate cell cycle events by permitting the onset of one event only after the completion of the preceding event. The mechanisms that ensure the proper ordering of cell cycle events have been termed checkpoint controls (10). DNA damage triggers the activation of checkpoint pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. Other checkpoints are activated when DNA replication is blocked. Failure to respond properly to DNA alterations may result in genomic instability, a mutagenic condition that predisposes organisms to cancer (5, 24).The cell cycle is transiently arrested at different stages depending on the phase at which DNA damage occurs. Three responses have been characterized in the budding yeast Saccharomyces cerevisiae, known as the G 1 -, S-and G 2 /M-phase DNA damage checkpoints (16). Genetic studies have identified genes that are involved in all three checkpoints. These include RAD9, RAD17, RAD24, MEC3, DDC1, MEC1(ESR1), and RAD53 (SPK1 or MEC2) (1,17,18,22,23,(30)(31)(32)(33)(43)(44)(45). Several lines of genetic evidence have suggested that RAD17, RAD24, MEC3, and DDC1 operate in the same checkpoint pathway, while RAD9 functions separately (17,18,20). Indeed, Ddc1, Mec3, and Rad17 physically interact with each other, suggesting that they function as a complex (13). RAD53 encodes a dual-specificity protein kinase (35), and Mec1 belongs to the ATM protein family (12,28). Rad53 is phosphorylated in response to DNA damage in a MEC1-dependent manner (26, 39). DNA damage-induced Rad53 phosphorylation is also dependent on R...

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