The checkpoint protein Ddc2, functionally related to S. pombe Rad26, interacts with Mec1 and is regulated by Mec1-dependent phosphorylation in budding yeast

Eukaryotic cells ensure genetic integrity after DNA damage or inhibition of DNA replication through a complex network of surveillance mechanisms known as checkpoints, which provide the cells with the capacity to survive genotoxic insults (Weinert and Hartwell 1988). These protective mechanisms are signal-transduction pathways specialized in detecting abnormal DNA structures. Their activation leads to delay of cell cycle progression, preventing replication or segregation of damaged DNA molecules. Checkpoint pathways are conserved from yeast to human cells, and failure to respond properly to DNA damage allows the cells to replicate and segregate damaged DNA molecules, resulting in increased mutagenesis and genetic instability, which may lead to cancer in multicellular organisms (for review, see Hartwell and Kastan 1994). Studies in different organisms, including the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, have allowed the partial dissection of the checkpoint pathways. In S. cerevisiae, the activation of the checkpoint-mediated response to DNA damage leads to a delay of the G1–S transition (Siede et al. 1993), slows down progression through S phase (Paulovich and Hartwell 1995), and delays nuclear division (Weinert and Hartwell 1988; Weinert et al. 1994), when DNA is damaged in G1, during DNA synthesis, or in G2, respectively. This response requires the proteins encoded by the RAD9 gene and by the genes of the RAD24 epistasis group, including RAD17, RAD24, MEC3, and DDC1 (Weinert et al. 1994; Longhese et al. 1996; Longhese et al. 1997; Paulovich et al. 1997a; de la Torre-Ruiz et al. 1998). This subfamily of checkpoint proteins is thought to act at an early step of the pathway by recognizing changes in DNA structure and initiating the signal-transduction cascade (for review, see Longhese et al. 1998; Weinert 1998; Lowndes and Murguia 2000). The finding that Ddc1, Rad17, and Mec3 interact physically with each other provides the evidence that these putative sensor proteins, which were inferred from genetic studies to operate in the same pathway, do indeed interact biochemically (Paciotti et al. 1998; Kondo et al. 1999). Whereas Rad24 has been shown to have homology with and to interact with subunits of replication factor C (RFC) (Griffiths et al. 1995; Lydall and Weinert 1997; Green et al. 1999), both Rad17 and Ddc1 have been reported to be structurally related to PCNA (proliferating cell nuclear antigen) (Thelen et al. 1999). It has been suggested that the Rad24–RFC complex might have a DNA structure-specific activity allowing the loading of PCNA-like checkpoint proteins on particular DNA structures (Thelen et al. 1999; Caspari et al. 2000). Central to the checkpoint-mediated responses to DNA damage and to incomplete DNA replication are highly conserved phosphatidilinositol-related protein kinases (PIKs), including Mec1 in S. cerevisiae, the gene product of rad3+ in S. pombe, and human ATM (for review, see Carr 1997). Since Mec1 is required for Rad9 and Ddc1 phosphorylation, it has been proposed that Mec1 might participate with Ddc1, Rad9, and, possibly, with Rad17, Rad24, and Mec3 at an early step of the DNA damage recognition process (Emili 1998; Paciotti et al. 1998; Sun et al. 1998; Vialard et al. 1998). Moreover, the finding that cell cycle progression in the presence of irreparable DNA damage is controlled by Mec1, but does not require Rad9 and the Rad24 group of proteins, suggests that Mec1 plays a primary role in the S-phase damage-sensing pathway (Neecke et al. 1999). Altogether these data suggest that Mec1 acts in the DNA structure recognition step, and its activity may be modulated by the association with regulatory subunits (for review, see Longhese et al. 1998; Weinert 1998). Recent support for this hypothesis has come from studies of the fission yeast Mec1 homolog Rad3. The Rad3 protein was found to be associated with Rad26, whose DNA damage-induced phosphorylation depends on Rad3, but not on the other known checkpoint proteins, suggesting that the Rad3–Rad26 complex may function at early steps in the DNA damage recognition process (Edwards et al. 1999). Once DNA perturbations are sensed, checkpoint signals are propagated through the protein kinase Rad53, which becomes phosphorylated and activated in response to genotoxic agents and whose phosphorylation depends on the above-listed checkpoint proteins (Sanchez et al. 1996; Sun et al. 1996). After DNA damage, Rad53 binds specifically to hyperphosphorylated Rad9, raising the possibility that Rad53 kinase activity might be influenced by association with other checkpoint proteins (Emili 1998; Sun et al. 1998; Vialard et al. 1998). In addition to their involvement in the checkpoint responses, Rad53 and Mec1 are essential for cell viability. Their essential function can be bypassed by increasing expression of genes encoding ribonucleotide reductase (Desany et al. 1998) or by deleting the SML1 gene (Zhao et al. 1998), which negatively affects dNTP pools, possibly through post-translational regulation of ribonucleotide reductase activity. Although many factors involved in the DNA damage checkpoint pathways have been identified, our knowledge of the molecular details of these mechanisms is still limited. Here, we describe a novel DNA integrity checkpoint protein, which we named Ddc2 (DNA damage checkpoint). We show that Ddc2 is required for all known DNA damage checkpoints and for preventing spindle elongation when DNA synthesis is inhibited. The Ddc2 protein physically interacts with Mec1 and is phosphorylated both during an unperturbed cell cycle and in response to DNA damage. In addition, Mec1 is absolutely required both in vitro and in vivo for Ddc2 phosphorylation. Moreover, Ddc2 is required for phosphorylation of the Ddc1, Pds1, and Rad53 checkpoint proteins. Conversely, Ddc2 phosphorylation does not require any of the other known DNA damage checkpoint proteins, suggesting that the Ddc2–Mec1 complex may respond to DNA insults independently of the other checkpoint factors.