Three Distinct Modes of Mec1/ATR and Tel1/ATM Activation Illustrate Differential Checkpoint Targeting during Budding Yeast Early Meiosis

Recombination and synapsis of homologous chromosomes are hallmarks of meiosis in many organisms. Meiotic recombination is initiated by Spo11-induced DNA double-strand breaks (DSBs), whereas chromosome synapsis is mediated by a tripartite structure named the synaptonemal complex (SC). Previously, we proposed that budding yeast SC is assembled via noncovalent interactions between the axial SC protein Red1, SUMO chains or conjugates, and the central SC protein Zip1. Incomplete synapsis and unrepaired DNA are monitored by Mec1/Tel1-dependent checkpoint responses that prevent exit from the pachytene stage. Here, our results distinguished three distinct modes of Mec1/Tec1 activation during early meiosis that led to phosphorylation of three targets, histone H2A at S129 (γH2A), Hop1, and Zip1, which are involved, respectively, in DNA replication, the interhomolog recombination and chromosome synapsis checkpoint, and destabilization of homology-independent centromere pairing. γH2A phosphorylation is Red1 independent and occurs prior to Spo11-induced DSBs. DSB- and Red1-dependent Hop1 phosphorylation is activated via interaction of the Red1-SUMO chain/conjugate ensemble with the Ddc1-Rad17-Mec3 (9-1-1) checkpoint complex and the Mre11-Rad50-Xrs2 complex. During SC assembly, Zip1 outcompetes 9-1-1 from the Red1-SUMO chain ensemble to attenuate Hop1 phosphorylation. In contrast, chromosome synapsis cannot attenuate DSB-dependent and Red1-independent Zip1 phosphorylation. These results reveal how DNA replication, DSB repair, and chromosome synapsis are differentially monitored by the meiotic checkpoint network.     

Pch2 Prevents Mec1/Tel1-Mediated Hop1 Phosphorylation Occurring Independently of Red1 in Budding Yeast Meiosis

A prominent feature of meiosis in most sexually reproducing organisms is interhomolog recombination whereby a significant fraction of the programmed meiotic double-strand breaks are repaired using intact homologous non-sister chromatids rather than sister chromatids. Budding yeast DNA damage checkpoint kinases Mec1 and Tel1 act together with the axial element protein Red1 to promote interhomolog recombination by phosphorylating another axial element protein Hop1. Mec1 and Tel1 also phosphorylate γH2A and the synaptonemal complex protein Zip1 independently of Red1 to facilitate premeiotic DNA replication and to destabilize homology-independent centromere pairing, respectively. It has been unclear why Hop1 phosphorylation is Red1-dependent. Here, we report that the pachytene checkpoint protein 2 (Pch2) specifically prevents Red1-independent Hop1 phosphorylation. Our findings reveal a new function for Pch2 in linking two axial element proteins Red1 and Hop1 thus coordinating their effects in meiotic recombination and the checkpoint network.     

The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization

The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form.     

Cdc25 Inhibited In Vivo and In Vitro by Checkpoint Kinases Cds1 and Chk1

In the fission yeast Schizosaccharomyces pombe, the protein kinase Cds1 is activated by the S-M replication checkpoint that prevents mitosis when DNA is incompletely replicated. Cds1 is proposed to regulate Wee1 and Mik1, two tyrosine kinases that inhibit the mitotic kinase Cdc2. Here, we present evidence from in vivo and in vitro studies, which indicates that Cds1 also inhibits Cdc25, the phosphatase that activates Cdc2. In an in vivo assay that measures the rate at which Cdc25 catalyzes mitosis, Cds1 contributed to a mitotic delay imposed by the S-M replication checkpoint. Cds1 also inhibited Cdc25-dependent activation of Cdc2 in vitro. Chk1, a protein kinase that is required for the G2-M damage checkpoint that prevents mitosis while DNA is being repaired, also inhibited Cdc25 in the in vitro assay. In vitro, Cds1 and Chk1 phosphorylated Cdc25 predominantly on serine-99. The Cdc25 alanine-99 mutation partially impaired the S-M replication and G2-M damage checkpoints in vivo. Thus, Cds1 and Chk1 seem to act in different checkpoint responses to regulate Cdc25 by similar mechanisms.     

Mec1 and Tel1: an arresting dance of resection

At sites of damage DNA is resected to enable the optimal form of repair, directed by homology. But how does the cell regulate resection while coordinating with the cell cycle? In this issue of The EMBO Journal, Clerici et al define missing links between DNA resection and cell cycle arrest during repair, showing that Mec1 can inhibit resection to subsequently activate arrest by Tel1.See also: M Clerici et al (December 2013)     

Mad3/BubR1 Phosphorylation during Spindle Checkpoint Activation Depends on both Polo and Aurora Kinases in Budding Yeast

During mitosis the spindle assembly checkpoint (SAC) delays the onset of anaphase and mitotic exit until all chromosomes are bipolarly attached to spindle fibers. Both lack of attachment due to spindle/kinetochore defects and lack of tension across kinetochores generate the “wait anaphase” signal transmitted by the SAC, which involves the evolutionarily conserved Mad1, Mad2, Mad3/BubR1, Bub1, Bub3 and Mps1 proteins, and inhibits the activity of the ubiquitin ligase Cdc20/APC, that promotes both sister chromatid dissociation in anaphase and mitotic exit. In particular, Mad3/BubR1 is directly implicated, together with Mad2, in Cdc20 inactivation in both human and yeast cells, suggesting that its activity is likely finely regulated. We show that budding yeast Mad3, like its human orthologue BubR1, is a phosphoprotein that is hyperphosphorylated during mitosis and when SAC activation is triggered by microtubule depolymerizing agents, kinetochore defects or lack of kinetochore tension. In vivo Mad3 phosphorylation depends on the Polo kinase Cdc5 and, to a minor extent, the Aurora B kinase Ipl1. Accordingly, replacing with alanines five serine residues belonging to Polo kinase-dependent putative phosphorylation sites dramatically reduces Mad3 phosphorylation, suggesting that Mad3 is likely an in vivo target of Cdc5.     

Separable Crossover-Promoting and Crossover-Constraining Aspects of Zip1 Activity during Budding Yeast Meiosis

Accurate chromosome segregation during meiosis relies on the presence of crossover events distributed among all chromosomes. MutSγ and MutLγ homologs (Msh4/5 and Mlh1/3) facilitate the formation of a prominent group of meiotic crossovers that mature within the context of an elaborate chromosomal structure called the synaptonemal complex (SC). SC proteins are required for intermediate steps in the formation of MutSγ-MutLγ crossovers, but whether the assembled SC structure per se is required for MutSγ-MutLγ-dependent crossover recombination events is unknown. Here we describe an interspecies complementation experiment that reveals that the mature SC is dispensable for the formation of Mlh3-dependent crossovers in budding yeast. Zip1 forms a major structural component of the budding yeast SC, and is also required for MutSγ and MutLγ-dependent crossover formation. Kluyveromyces lactis ZIP1 expressed in place of Saccharomyces cerevisiae ZIP1 in S. cerevisiae cells fails to support SC assembly (synapsis) but promotes wild-type crossover levels in those nuclei that progress to form spores. While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery. Moreover, K. lactis Zip1-mediated crossovers rely on S. cerevisiae synapsis initiation proteins Zip3, Zip4, Spo16, as well as the Mlh3 protein, as do the crossovers mediated by S. cerevisiae Zip1. Surprisingly, however, K. lactis Zip1-mediated crossovers are largely Msh4/Msh5 (MutSγ)-independent. This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast. Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates.     

In Vivo Role for Actin-regulating Kinases in Endocytosis and Yeast Epsin Phosphorylation

The yeast actin-regulating kinases Ark1p and Prk1p are signaling proteins localized to cortical actin patches, which may be sites of endocytosis. Interactions between the endocytic proteins Pan1p and End3p may be regulated by Prk1p-dependent threonine phosphorylation of Pan1p within the consensus sequence [L/I]xxQxTG. We identified two Prk1p phosphorylation sites within the Pan1p-binding protein Ent1p, a yeast epsin homologue, and demonstrate Prk1p-dependent phosphorylation of both threonines. Converting both threonines to either glutamate or alanine mimics constitutively phosphorylated or dephosphorylated Ent1p, respectively. Synthetic growth defects were observed in a pan1-20 ENT1(EE) double mutant, suggesting that Ent1p phosphorylation negatively regulates the formation/activity of a Pan1p-Ent1p complex. Interestingly, pan1-20 ent2 Delta but not pan1-20 ent1 Delta double mutants had improved growth and endocytosis over the pan1-20 mutant. We found that actin-regulating Ser/Thr kinase (ARK) mutants exhibit endocytic defects and that overexpressing either wild-type or alanine-substituted Ent1p partially suppressed phenotypes associated with loss of ARK kinases, including growth, endocytosis, and actin localization defects. Consistent with synthetic growth defects of pan1-20 ENT1(EE) cells, overexpressing glutamate-substituted Ent1p was deleterious to ARK mutants. Surprisingly, overexpressing the related Ent2p protein could not suppress ARK kinase mutant phenotypes. These results suggest that Ent1p and Ent2p are not completely redundant and may perform opposing functions in endocytosis. These data support the model that, as for clathrin-dependent recycling of synaptic vesicles, yeast endocytic protein phosphorylation inhibits endocytic functions.     

Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway

Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends.     

The Spindle Checkpoint of Budding Yeast Depends on a Tight Complex between the Mad1 and Mad2 Proteins

The spindle checkpoint arrests the cell cycle at metaphase in the presence of defects in the mitotic spindle or in the attachment of chromosomes to the spindle. When spindle assembly is disrupted, the budding yeast mad and bub mutants fail to arrest and rapidly lose viability. We have cloned the MAD2 gene, which encodes a protein of 196 amino acids that remains at a constant level during the cell cycle. Gel filtration and co-immunoprecipitation analyses reveal that Mad2p tightly associates with another spindle checkpoint component, Mad1p. This association is independent of cell cycle stage and the presence or absence of other known checkpoint proteins. In addition, Mad2p binds to all of the different phosphorylated isoforms of Mad1p that can be resolved on SDS-PAGE. Deletion and mutational analysis of both proteins indicate that association of Mad2p with Mad1p is critical for checkpoint function and for hyperphosphorylation of Mad1p.     

CDK-Dependent Nuclear Localization of B-Cyclin Clb1 Promotes FEAR Activation during Meiosis I in Budding Yeast

Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1''s phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.     

chk-1 is an Essential Gene and is Required for an S-M Checkpoint During Early Embryogenesis

The chk1 gene was first discovered in screens for radiation sensitive mutants in S. pombe [1]. Genetic analysis revealed that chk1 is involved in a DNA damage G2-M checkpoint. Chk1 becomes activated in response to DNA damage and prevents entry into mitosis by inhibiting the cell cycle machinery. This checkpoint decreases the risk of defective DNA being inherited by daughter cells, therefore reducing the risk of genetic instability. In higher eukaryotes, chk1 homologues have similar checkpoint functions. For example, an avian B-lymphoma cell line that is defective for Chk1 fails to arrest in G2-M after DNA damage. Nonetheless, these Chk1 defective cells are viable indicating that Chk1 is not essential for normal somatic cells to divide [2]. In spite of this, mouse and Drosophila homozygous Chk1 mutants die during embryogenesis suggesting that this is an essential gene for embryonic cell cycles [3, 4]. What particular role does Chk1 have in directing embryonic cell divisions? Here we used the model organism, C. elegans, to address the role of chk-1 during development. As expected, disruption of chk-1 by RNAi eliminated the DNA damage checkpoint response in C. elegans. In addition, we revealed that chk-1 was predominantly expressed during embryogenesis and in the postembryonic germline. Indeed, we found that chk-1 had an essential role in embryo and germline development. More specifically, disruption of chk-1 expression resulted in embryo lethality, which was attributed to a defect in an intrinsic S-M hence causing premature entry into M-phase.     

Role of a Complex Containing Rad17, Mec3, and Ddc1 in the Yeast DNA Damage Checkpoint Pathway

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 rad24Δ 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.     

Mre11 Nuclease Activity and Ctp1 Regulate Chk1 Activation by Rad3ATR and Tel1ATM Checkpoint Kinases at Double-Strand Breaks

Rad3, the Schizosaccharomyces pombe ortholog of human ATR and Saccharomyces cerevisiae Mec1, activates the checkpoint kinase Chk1 in response to DNA double-strand breaks (DSBs). Rad3^ATR/Mec1 associates with replication protein A (RPA), which binds single-stranded DNA overhangs formed by DSB resection. In humans and both yeasts, DSBs are initially detected and processed by the Mre11-Rad50-Nbs1^Xrs2 (MRN) nucleolytic protein complex in association with the Tel1^ATM checkpoint kinase and the Ctp1^CtIP/Sae2 DNA-end processing factor; however, in budding yeast, neither Mre11 nuclease activity or Sae2 are required for Mec1 signaling at irreparable DSBs. Here, we investigate the relationship between DNA end processing and the DSB checkpoint response in fission yeast, and we report that Mre11 nuclease activity and Ctp1 are critical for efficient Rad3-to-Chk1 signaling. Moreover, deleting Ctp1 reveals a Tel1-to-Chk1 signaling pathway that bypasses Rad3. This pathway requires Mre11 nuclease activity, the Rad9-Hus1-Rad1 (9-1-1) checkpoint clamp complex, and Crb2 checkpoint mediator. Ctp1 negatively regulates this pathway by controlling MRN residency at DSBs. A Tel1-to-Chk1 checkpoint pathway acting at unresected DSBs provides a mechanism for coupling Chk1 activation to the initial detection of DSBs and suggests that ATM may activate Chk1 by both direct and indirect mechanisms in mammalian cells.DNA double-strand breaks (DSBs), formed by clastogens or from endogenous damage, trigger multiple cellular responses that are critical for maintaining genome integrity. Of particular importance is the cell cycle checkpoint that restrains the onset of mitosis while DSB repair is under way. Chk1 is the critical effector of this checkpoint in the fission yeast Schizosaccharomyces pombe and mammalian cells, whereas the budding yeast Saccharomyces cerevisiae uses both Chk1 and Rad53 (orthologous to human Chk2 and fission yeast Cds1) to delay anaphase entry and mitotic exit. These kinases are regulated by ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) checkpoint kinases (5). Curiously, the regulatory connections between ATM/ATR and Chk1/Chk2 orthologs are not strictly conserved between species (Fig. ​(Fig.1A).1A). In mammals, ATM activates Chk2 while ATR activates Chk1. In S. cerevisiae and S. pombe, ATR orthologs (Mec1 and Rad3, respectively) activate Chk2 orthologs and Chk1, while Tel1 (ATM ortholog) is primarily involved in telomere maintenance (14, 38, 40).Open in a separate windowFIG. 1.Deletion of Ctp1 restores the DNA damage checkpoint in rad3Δ cells. (A) Regulatory connections between ATM/ATR and Chk1/Chk2 orthologs in mammals, S. cerevisiae, and S. pombe. ATM phosphorylates Chk2 and ATR phosphorylates Chk1. CtIP mediates an ATM-to-ATR switch through DNA end resection in mammals (44, 53). ATM promotes Chk1 activation by stimulating CtIP-dependent resection through an unknown mechanism. In S. cerevisiae, Mec1 phosphorylates both Rad53 and Chk1. Deleting Sae2 uncovers a Tel1-to-Rad53 signaling pathway and enhances Rad53 activation (47). In S. pombe, Cds1 and Chk1 activation is dependent on Rad3. (B) Chk1 phosphorylation peaks in wild-type (wt) (top panel) and ctp1Δ cells (bottom panel) 30 min after exposure to 90 Gy of IR in log-phase cultures. Chk1 phosphorylation in ctp1Δ cells prior to IR exposure likely arises from an inability to repair spontaneous DNA damage (23). Immunoblots were probed for the HA epitope-tagged Chk1 or Cdc2 as a loading control. (C) Chk1 phosphorylation is reduced at least 2-fold in ctp1Δ cells relative to the wild type. Quantification of blots from panel B expressed as a ratio of phospho-Chk1 (upper band) versus nonphospho-Chk1 (lower band) was performed. The phospho-Chk1 signal in untreated ctp1Δ cells was subtracted from the IR-treated samples to more accurately measure the IR-induced phosphorylation. (D) The ctp1Δ mutation restores Chk1 phosphorylation in rad3Δ cells. Cells were harvested immediately after mock or 90-Gy IR treatment and blotted for HA epitope tag. Ponceau staining shows equal loading. (E) Quantitation of Chk1 phosphorylation. Error bars represent the standard errors from three independent experiments. (F) The checkpoint arrest is restored in ctp1Δ rad3Δ cells. Cells synchronized in G₂ by elutriation were mock treated or exposed to 100 Gy of IR. Cell cycle progression was tracked by microscopic observation.The functions of ATM and ATR orthologs are intimately tied to the detection and nucleolytic processing of DSBs. ATM^Tel1 localizes at DSBs by interacting with Mre11-Rad50-Nbs1^Xrs2 (MRN) protein complex, which directly binds DNA ends (12, 20, 24, 50, 52). The MRN complex is essential for ATM^Tel1 function in all species. The Mre11 subunit of MRN complex has DNase activities that are critical for radioresistance in S. pombe and mice but not in budding yeast (3, 19, 22, 50). In fission yeast, MRN complex also recruits Ctp1 DNA end-processing factor to DSBs (25, 49). Ctp1 is structurally and functionally related to CtIP in mammals and Sae2 in budding yeast, the latter of which has nuclease activity in vitro (21, 23, 43). Ctp1 and CtIP are essential for survival of ionizing radiation and other clastogens (23, 43, 54), whereas sae2Δ mutants are not radiosensitive except at very high doses of ionizing radiation (IR), although both Ctp1 and Sae2 are required for repair of meiotic DSBs formed by a Spo11/Rec12-dependent mechanism (17, 23, 36). Genetic and biochemical studies indicate that Sae2/Ctp1/CtIP collaborate with MRN complex to initiate the 5′-to-3′ resection of DSBs (7, 23, 28, 43, 53, 55), which leads to the generation of 3′ single-strand overhangs (SSOs) that are critical for DSB repair by homologous recombination (HR). Replication protein A (RPA) binding to SSOs is essential for HR repair of DSBs, but it is also important for recruiting ATR^Rad3/Mec1, which interacts with RPA through its regulatory subunit ATRIP (Rad26 in fission yeast, Ddc2 in budding yeast) (5, 56). Subsequent phosphorylation of Chk1 by ATR also requires the Rad9-Hus1-Rad1 (9-1-1) checkpoint clamp, which is loaded at the single-strand/double-strand DNA junctions (26, 48, 57), the ATR activating protein TopBP1 (Cut5 in fission yeast), and a checkpoint mediator protein such as Crb2 in fission yeast (34, 41, 48).In this mechanism of DNA damage checkpoint signaling, DNA end resection is critical for ATR (Rad3/Mec1) activation, and therefore resection defective mutants should be unable to mount a fully active checkpoint response (44). However, Rad53 activation is not diminished in budding yeast sae2Δ mutants that suffer an irreparable DSB by expressing HO endonuclease. In fact, there is a defect in turning off the checkpoint signal (6). A similar effect is observed in S. cerevisiae strains expressing the mre11-H125N nuclease-defective form of Mre11. Moreover, overexpression of SAE2 strongly inhibits Rad53 activation (6). The reasons for these phenotypes are unknown, since neither Sae2 nor Mre11 nuclease activity are required for DSB resection or radioresistance. However, deleting Sae2 delays resection while at the same time enhancing a cryptic Tel1-to-Rad53 checkpoint pathway (6, 47). These effects correlate with delayed disassembly of Mre11 foci at DSBs in sae2Δ cells, suggesting that Sae2 may negatively regulate checkpoint signaling by modulating Mre11 association at damaged DNA (1, 6, 24). Enhancement of a Tel1-to-Rad53 checkpoint pathway by eliminating Sae2 suggests that the signaling pathways between ATM/ATR and Chk1/Chk2 checkpoint kinases are not hard wired but are adaptable to changes in DNA end processing (47). However, as yet there is no evidence that ATM^Tel1 can activate Chk1 in any organism.Since SAE2 deletion or overexpression has unexpected effects on Rad53 activation in budding yeast, we decided to explore the relationship between Ctp1 and Chk1 activation in fission yeast. Here, we show that Chk1 activation is substantially diminished in ctp1Δ cells exposed to ionizing radiation. These data are consistent with studies showing that CtIP is required for efficient Chk1 activation in mammalian cells treated with camptothecin (CPT), a topoisomerase I poison that causes replication fork collapse (43, 53). We also investigate the role of Mre11 nuclease activity and find that while ablating Mre11 nuclease activity enhances Rad53 activation in budding yeast, the equivalent Mre11 mutation in fission yeast severely impairs Chk1 activation by ionizing radiation. Furthermore, we find that deleting Ctp1 reveals a previously unknown Tel1-to-Chk1 signaling pathway in S. pombe, a finding analogous to the enhancement of a Tel1-to-Rad53 checkpoint pathway by eliminating Sae2 in S. cerevisiae (47). This Tel1-to-Chk1 pathway also requires Mre11 nuclease activity. These data establish that Tel1^ATM can activate Chk1 independently of Rad3^ATR, which has implications for studies linking ATM to Chk1 activation in mammalian cells (16, 31). Characterization of this pathway allows us to propose a more detailed model of how Chk1 is activated in response to DSBs.     

Mec1 function in the DNA damage response does not require its interaction with Tel2

The essential, conserved Tel2 protein plays a role in the response to DNA damage and replication stress in a wide range of eukaryotes. Tel2 interacts physically with multiple members of the PI3-kinase related protein kinase (PIKK) family in mammalian cells and fission yeast. In mammalian cells, loss of Tel2 leads to destabilization of PIKKs. Our previous work in the yeast Saccharomyces cerevisiae showed that Tel2 interacts with the PIKK Tel1 (yeast ATM kinase), and that this interaction is abrogated by the only known non-lethal TEL2 mutation in S. cerevisiae, tel2-1. We showed that this mutation specifically disrupts the function of Tel1 and not the function of the closely related protein Mec1 (yeast ATR kinase) in DNA damage responses. Here we show that Tel2 and Mec1 interact in S. cerevisiae, and that surprisingly, this physical interaction is also disrupted by the tel2-1 mutation. Although the tel2-1 mutation leads to moderately lower Mec1 levels, the ability of Mec1 to localize to a site of DNA damage and to function in DNA damage signaling remains intact. These results suggest that the model of Tel2 as solely a global regulator of PIKK stability is insufficient. Rather, Tel2 can specifically and differentially regulate the function of individual PIKKs.     

The telomerase-recruitment domain of the telomere binding protein Cdc13 is regulated by Mec1p/Tel1p-dependent phosphorylation

The DNA damage-responsive protein kinases ATM and ATR phosphorylate SQ/TQ motifs that lie in clusters in most of their in vivo targets. Budding yeast Cdc13p contains two clusters of SQ/TQ motifs, suggesting that it might be a target of Mec1p/Tel1p (yeast ATR/ATM). Here we demonstrated that the telomerase recruitment domain of Cdc13p is phosphorylated by Mec1p and Tel1p. Gel analysis showed that Cdc13p contains a Mec1/Tel1-dependent post-translational modification. Using an immunoprecipitate (IP)-kinase assay, we showed that Mec1p phosphorylates Cdc13p on serine 225, 249, 255 and 306, and Tel1p phosphorylates Cdc13p on serine 225, 249 and 255 in vitro. Phenotypic analysis in vivo revealed that the mutations in the Cdc13p SQ motifs phosphorylated by Mec1p and Tel1p caused multiple telomere and growth defects. In addition, normal telomere length and growth could be restored by expressing a Cdc13–Est1p hybrid protein. These results demonstrate the telomerase recruitment domain of Cdc13p as an important new telomere-specific target of Mec1p/Tel1p.