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.

Although the Abelson (Abl) tyrosine kinase inhibitor imatinib mesylate has improved the treatment of breakpoint cluster region-Abl (Bcr-Abl)-positive leukemia, resistance is often reported in patients with advanced-stage disease. Although several Src inhibitors are more effective than imatinib and simultaneously inhibit Lyn, whose overexpression is associated with imatinib resistance, these inhibitors are less specific than imatinib.We have identified a specific dual Abl-Lyn inhibitor, NS-187 (elsewhere described as CNS-9), which is 25 to 55 times more potent than imatinib in vitro. NS-187 is also at least 10 times as effective as

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...

Rad9 is required for the activation of DNA damage checkpoint pathways in budding yeast. Rad9 is phosphorylated after DNA damage in a Mec1-and Tel1-dependent manner and subsequently interacts with Rad53. This Rad9-Rad53 interaction has been suggested to trigger the activation and phosphorylation of Rad53. Here we show that Mec1 controls the Rad9 accumulation at double-strand breaks (DSBs). Rad9 was phosphorylated after DSB induction and associated with DSBs. However, its phosphorylation and association with DSBs were significantly decreased in cells carrying a mec1⌬ or kinase-negative mec1 mutation. Mec1 phosphorylated the S/TQ motifs of Rad9 in vitro, the same motifs that are phosphorylated after DNA damage in vivo. In addition, multiple mutations in the Rad9 S/TQ motifs resulted in its defective association with DSBs. Phosphorylation of Rad9 was partially defective in cells carrying a weak mec1 allele (mec1-81), whereas its association with DSBs occurred efficiently in the mec1-81 mutants, as found in wild-type cells. However, the Rad9-Rad53 interaction after DSB induction was significantly decreased in mec1-81 mutants, as it was in mec1⌬ mutants. Deletion mutation in RAD53 did not affect the association of Rad9 with DSBs. Our results suggest that Mec1 promotes association of Rad9 with sites of DNA damage, thereby leading to full phosphorylation of Rad9 and its interaction with Rad53.