Genetic inheritance requires exceptional genetic stability over many generations of cells and organisms. To ensure that cells pass accurate copies of their genomes on to the next generation, evolution has overlaid the core cell cycle machinery with a series of surveillance pathways, termed checkpoints, that provide the cells with the capacity to survive genotoxic insults. These protective mechanisms are signal transduction pathways specialized in detecting abnormal DNA structures and in coordinating cell cycle progression with DNA repair. Their activation leads to cell cycle progression delay and concomitant activation of DNA repair pathways, thus preventing replication or segregation of damaged DNA molecules. The keystone of the DNA damage checkpoint is a protein kinase family related to phosphoinositide 3-kinase, among which are Saccharomyces cerevisiae Mec1 (48, 61, 90) and Tel1 (26, 52), Schizosaccharomyces pombe Rad3 (5), Drosophila melanogaster Mei-41 (29), and mammalian ATR (5) and ATM (72). These protein kinases respond to various stresses by phosphorylating key proteins, thus regulating numerous processes, depending on the spectra of their substrates (for reviews, see references 1 and 75). In particular, S. cerevisiae Mec1 and S. pombe Rad3, more closely related to human ATR, are the prototype transducers of the DNA damage and replication stress signals; they respond to UV damage, double-strand breaks (DSBs), and stalled replication forks. Conversely, yeast Tel1, similar to human ATM, is likely involved only in the response to DSBs (for reviews, see references 57 and 75). Tel1 and Mec1 also contribute to telomere length maintenance. In fact, TEL1 deletion causes marked telomere shortening in yeast (26, 52), and mec1 tel1 cells show more-dramatic telomere shortening than each single mutant, similar to that seen in cells lacking active telomerase (67). The absence of Tel1 does not affect either telomerase catalytic activity or binding to telomeric DNA of the Cdc13 protein, which mediates telomerase action (9, 83). However, when telomerase is artificially targeted to telomeres, their lengthening is at least as effective in mec1 tel1 as in wild-type cells, suggesting that Mec1 and Tel1 may act by recruiting to telomeres either telomerase or telomerase-activating factors (83). One characteristic of ATR-related proteins is their need for an accessory protein. Mec1 physically interacts with the checkpoint protein Ddc2 (also called Lcd1 or Pie1) (60, 69, 89), functionally related to Rad26 and ATRIP, which bind S. pombe Rad3 and human ATR, respectively (12, 20). Although the Mec1-Ddc2 complex plays a key role in the DNA damage checkpoint, full Mec1-dependent activation of downstream targets requires other factors, such as the Ddc1/Rad17/Mec3 and Rad24/Rfc2 to -5 complexes (for a review, see reference 42), presumed to be structure-specific DNA damage sensors based on their similarities to the proliferating cell nuclear antigen (PCNA) and its accessory factor replication factor C (RFC), respectively (7, 25, 46, 79). Once DNA perturbations are sensed, checkpoint signals are propagated through the protein kinases Chk1 and Rad53, which also undergo phosphorylation in response to DNA damage in a Mec1-dependent manner (70, 71). While Rad53 is required for proper response to DNA damage in all the cell cycle phases, Chk1 contributes only to the activation of the G2/M checkpoint in a Rad53-independent way (71). The DNA damage-sensing functions are then linked with the downstream effectors through DNA damage-specific or S-phase-specific mediators. In particular, while Rad9 is required to activate Rad53 in response to DNA damage by acting as a scaffold protein upon which Rad53 may autophosphorylate and self-activate (24, 73), Mrc1 seems to act as the Rad9 counterpart in activating Rad53 in response to DNA replication blocks (2, 58). Whereas human ATM plays a key role in responding to DSBs in human cells, S. cerevisiae Tel1 has only a secondary role in the DNA damage response. In fact, Tel1, which is primarily involved in telomere metabolism, seems to respond to unprocessed DSBs by controlling a checkpoint that becomes apparent only in the absence of Mec1, and converges with the canonical Mec1 pathway on the effector kinase Rad53 (84). A trimeric complex, known as MRN (Mre11-Rad50-Nbs1) in mammals and MRX (Mre11-Rad50-Xrs2) in S. cerevisiae, is a substrate for the ATM/Tel1 kinase. In fact, mammalian Mre11 and Nbs1 are phosphorylated in response to DNA damage, and their phosphorylation depends on ATM (21, 22, 39, 91, 93). This response is evolutionarily conserved, as DNA damage also stimulates Tel1-dependent phosphorylation of the S. cerevisiae Mre11 and Xrs2 proteins (15, 27). In both yeast and humans, this complex plays multiple roles in chromosome metabolism and DNA repair. It has been implicated in telomere maintenance, regulation of DNA replication, nonhomologous end joining, and meiotic DSB formation or resection (for reviews, see references 16, 62, and 86). Moreover, it may control DSB processing during mitotic homologous recombination, thus amplifying the checkpoint signal and stimulating the Mec1/Tel1 kinases (84). Finally, the MRX complex is involved in the S. cerevisiae checkpoint response as part of the DNA damage-sensing apparatus, since mre11Δ, rad50Δ, and xrs2Δ yeast cells are defective in S-phase checkpoint activation in response to DSB-inducing agents and hydroxyurea (HU) (15, 27). Some processes involving the MRX complex also require the SAE2/COM1 gene, whose loss-of-function alleles were originally identified together with the rad50s and mre11s separation-of-function alleles in a search for mutants whose meiotic products died if DSBs were made but could survive when meiotic recombination and reductional chromosome segregation were prevented by mutations inactivating the SPO11 gene (49, 65). The topoisomerase II-like Spo11 protein is necessary for the creation of meiotic DSBs, which are subjected to rapid resection of their 5′ strand termini, yielding molecules with 3′ single-stranded tails, presumably used to form strand exchange products during recombination (62). While the MRX complex participates in both the formation and the processing of DSBs at meiotic recombination hot spots, the Sae2 protein seems to participate only in DSB processing. In fact, in contrast to mrx-null alleles, which are unable to initiate meiotic DSBs, both sae2Δ and the rad50s and mre11s separation-of-function alleles were shown to allow SPO11-mediated DSB formation, but not single-strand endonucleolytic removal of Spo11 from the DNA ends, thus uncoupling cleavage of DNA strands from subsequent exonucleolytic resection (34). Since the MRX complex has nuclease activity, the lack of Sae2 might directly modify its in vivo nuclease function, and the rad50s allele might affect its ability to interact with Sae2, as previously suggested (66). Although Sae2, as well as the MRX complex, is dispensable for some mitotic homologous recombination processes requiring Rad52 and Rad51 (6, 32, 47, 82), several observations indicate that Sae2 may also have mitotic functions. (i) sae2Δ cells have been shown to be hypersensitive to the alkylating agent methyl methanesulfonate (MMS), and the fidelity of mitotic DSB repair in these cells has been shown to be affected (49, 66). (ii) Sae2, along with MRX, is also required for repair of mitotic hairpin-capped DSBs induced by inverted Alu sequences, indicating that Sae2 may participate in making protected DNA ends accessible to resection in mitotic cells also (40). (iii) A lack of Sae2 enhances Tel1-mediated Rad53 phosphorylation after DNA damage, and this enhancement requires the MRX complex, suggesting that sae2Δ cells may accumulate DNA lesions specifically sensed by the Tel1/MRX-dependent checkpoint (84). (iv) High levels of Sae2 not only cause telomere lengthening in a Tel1-dependent manner, but also accelerate the rebalancing of telomere length when sudden telomere elongation is induced by TEL1 overexpression, suggesting a role for Sae2 in unprotected telomeric end processing (87). We now show that the functions of Sae2 in DNA repair and recombination require the checkpoint pathway that responds to DNA damage during the mitotic cell cycle. Sae2, whose lack delays the recovery from checkpoint-mediated cell cycle arrest, undergoes Mec1- and Tel1-dependent phosphorylation both periodically during the unperturbed cell cycle and in response to DNA damage independently of cell cycle progression. Site-directed mutagenesis of the favored ATM/ATR phosphorylation sites indicates that they are essential not only for DNA damage-induced Sae2 phosphorylation but also for Sae2 functions in vivo.