The ATM-related Tel1 protein of Saccharomyces cerevisiae controls a checkpoint response following phleomycin treatment

MEC1 and TEL1 encode ATR- and ATM-related proteins in the budding yeast Saccharomyces cerevisiae, respectively. Phleomycin is an agent that catalyzes double-strand breaks in DNA. We show here that both Mec1 and Tel1 regulate the checkpoint response following phleomycin treatment. MEC1 is required for Rad53 phosphorylation and cell-cycle progression delay following phleomycin treatment in G1, S or G2/M phases. The tel1Δ mutation confers a defect in the checkpoint responses to phleomycin treatment in S phase. In addition, the tel1Δ mutation enhances the mec1 defect in activation of the phleomycin-induced checkpoint pathway in S phase. In contrast, the tel1Δ mutation confers only a minor defect in the checkpoint responses in G1 phase and no apparent defect in G2/M phase. Methyl methanesulfonate (MMS) treatment also activates checkpoints, inducing Rad53 phosphorylation in S phase. MMS-induced Rad53 phosphorylation is not detected in mec1Δ mutants during S phase, but occurs in tel1Δ mutants similar to wild-type cells. Finally, Xrs2 is phosphorylated after phleomycin treatment in a TEL1-dependent manner during S phase, whereas no significant Xrs2 phosphorylation is detected after MMS treatment. Together, our results support a model in which Tel1 contributes to checkpoint control in response to phleomycin-induced DNA damage in S phase.     

Sae2 Function at DNA Double-Strand Breaks Is Bypassed by Dampening Tel1 or Rad53 Activity

The MRX complex together with Sae2 initiates resection of DNA double-strand breaks (DSBs) to generate single-stranded DNA (ssDNA) that triggers homologous recombination. The absence of Sae2 not only impairs DSB resection, but also causes prolonged MRX binding at the DSBs that leads to persistent Tel1- and Rad53-dependent DNA damage checkpoint activation and cell cycle arrest. Whether this enhanced checkpoint signaling contributes to the DNA damage sensitivity and/or the resection defect of sae2Δ cells is not known. By performing a genetic screen, we identify rad53 and tel1 mutant alleles that suppress both the DNA damage hypersensitivity and the resection defect of sae2Δ cells through an Sgs1-Dna2-dependent mechanism. These suppression events do not involve escaping the checkpoint-mediated cell cycle arrest. Rather, defective Rad53 or Tel1 signaling bypasses Sae2 function at DSBs by decreasing the amount of Rad9 bound at DSBs. As a consequence, reduced Rad9 association to DNA ends relieves inhibition of Sgs1-Dna2 activity, which can then compensate for the lack of Sae2 in DSB resection and DNA damage resistance. We propose that persistent Tel1 and Rad53 checkpoint signaling in cells lacking Sae2 increases the association of Rad9 at DSBs, which in turn inhibits DSB resection by limiting the activity of the Sgs1-Dna2 resection machinery.     

A Ddc2-Rad53 fusion protein can bypass the requirements for RAD9 and MRC1 in Rad53 activation

Activation of Rad53p by DNA damage plays an essential role in DNA damage checkpoint pathways. Rad53p activation requires coupling of Rad53p to Mec1p through a “mediator” protein, Rad9p or Mrc1p. We sought to determine whether the mediator requirement could be circumvented by making fusion proteins between the Mec1 binding partner Ddc2p and Rad53p. Ddc2-Rad53p interacted with Mec1p and other Ddc2-Rad53p molecules under basal conditions and displayed an increased oligomerization upon DNA damage. Ddc2-Rad53p was activated in a Mec1p- and Tel1p-dependent manner upon DNA damage. Expression of Ddc2-Rad53p in Δrad9 or Δrad9Δmrc1 cells increased viability on plates containing the alkylating agent methyl methane sulfonate. Ddc2-Rad53p was activated at least partially by DNA damage in Δrad9Δmrc1 cells. In addition, expression of Ddc2-Rad53p in Δrad24Δrad17Δmec3 cells increased cell survival. These results reveal minimal requirements for function of a core checkpoint signaling system.     

Rad53 phosphorylation site clusters are important for Rad53 regulation and signaling

Budding yeast Rad53 is an essential protein kinase that is phosphorylated and activated in a MEC1- and TEL1-dependent manner in response to DNA damage. We studied the role of Rad53 phosphorylation through mutation of consensus phosphorylation sites for upstream kinases Mec1 and Tel1. Alanine substitution of the Rad53 amino-terminal TQ cluster region reduced viability and impaired checkpoint functions. These substitution mutations spared the basal interaction with Asf1 and the DNA damage-induced interactions with Rad9. However, they caused a decrease in DNA damage-induced Rad53 kinase activity and an impaired interaction with the protein kinase Dun1. The Dun1 FHA (Forkhead-associated) domain recognized the amino-terminal TQ cluster of Rad53 after DNA damage or replication blockade. Thus, the phosphorylation of Rad53 by upstream kinases is important not only for Rad53 activation but also for creation of an interface between Rad53 and Dun1.     

Nej1 interacts with Sae2 at DNA double-stranded breaks to inhibit DNA resection

The two major pathways of DNA double-strand break repair, nonhomologous end-joining and homologous recombination, are highly conserved from yeast to mammals. The regulation of 5′-DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends, and more recently, it was shown to participate in DNA end-bridging and in the inhibition of 5′-resection mediated by the nuclease/helicase complex Dna2–Sgs1. Here, we show that Nej1 interacts with Sae2 to impact DSB repair in three ways. First, we show that Nej1 inhibits interaction of Sae2 with the Mre11–Rad50–Xrs2 complex and Sae2 localization to DSBs. Second, we found that Nej1 inhibits Sae2-dependent recruitment of Dna2 independently of Sgs1. Third, we determined that NEJ1 and SAE2 showed an epistatic relationship for end-bridging, an event that restrains broken DNA ends and reduces the frequency of genomic deletions from developing at the break site. Finally, we demonstrate that deletion of NEJ1 suppressed the synthetic lethality of sae2Δ sgs1Δ mutants, and that triple mutant viability was dependent on Dna2 nuclease activity. Taken together, these findings provide mechanistic insight to how Nej1 functionality inhibits the initiation of DNA resection, a role that is distinct from its involvement in end-joining repair at DSBs.     

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.     

Mek1 stabilizes Hop1-Thr318 phosphorylation to promote interhomolog recombination and checkpoint responses during yeast meiosis

Red1, Hop1 and Mek1 are three yeast meiosis-specific chromosomal proteins that uphold the interhomolog (IH) bias of meiotic recombination. Mek1 is also an effector protein kinase in a checkpoint that responds to aberrant DNA and/or axis structure. The activation of Mek1 requires Red1-dependent Hop1-Thr(T)318 phosphorylation, which is mediated by Mec1 and Tel1, the yeast homologs of the mammalian DNA damage sensor kinases ATR and ATM. As the ectopic expression of Mek1-glutathione S-transferase (GST) was shown to promote IH recombination in the absence of Mec1/Tel1-dependent checkpoint function, it was proposed that Mek1 might play dual roles during meiosis by directly phosphorylating targets that are involved in the recombination checkpoint. Here, we report that Mek1 has a positive feedback activity in the stabilization of Mec1/Tel1-mediated Hop1-T318 phosphorylation against the dephosphorylation mediated by protein phosphatase 4. Our results also reveal that GST-Mek1 or Mek1-GST further increases Hop1-T318 phosphorylation. This positive feedback function of Mek1 is independent of Mek1’s kinase activity, but dependent on Mek1’s forkhead-associated (FHA) domain and its arginine 51 residue. Arginine 51 directly mediates the interaction of Mek1-FHA and phosphorylated Hop1-T318. We suggest that the Hop1–Mek1 interaction is similar to the Rad53-Dun1 signaling pathway, which is mediated through the interaction of phosphorylated Rad53 and Dun1-FHA.     

Amino acid changes in Xrs2p,Dun1p,and Rfa2p that remove the preferred targets of the ATM family of protein kinases do not affect DNA repair or telomere length in Saccharomyces cerevisiae

In eukaryotes, mutations in a number of genes that affect DNA damage checkpoints or DNA replication also affect telomere length [Curr. Opin. Cell Biol. 13 (2001) 281]. Saccharomyces cerevisae strains with mutations in the TEL1 gene (encoding an ATM-like protein kinase) have very short telomeres, as do strains with mutations in XRS2, RAD50, or MRE11 (encoding members of a trimeric complex). Xrs2p and Mre11p are phosphorylated in a Tel1p-dependent manner in response to DNA damage [Genes Dev. 15 (2001) 2238; Mol. Cell 7 (2001) 1255]. We found that Xrs2p, but not Mre11p or Rad50p, is efficiently phosphorylated in vitro by immunopreciptated Tel1p. Strains with mutations eliminating all SQ and TQ motifs in Xrs2p (preferred targets of the ATM kinase family) had wild-type length telomeres and wild-type sensitivity to DNA damaging agents. We also showed that Rfa2p (a subunit of RPA) and the Dun1p checkpoint kinase, which are required for DNA damage repair and which are phosphorylated in response to DNA damage in vivo, are in vitro substrates of the Tel1p and Mec1p kinases. In addition, Dun1p substrates with no SQ or TQ motifs are phosphorylated by Mec1p in vitro very inefficiently, but retain most of their ability to be phosphorylated by Tel1p. We demonstrated that null alleles of DUN1 and certain mutant alleles of RFA2 result in short telomeres. As observed with Xrs2p, however, strains with mutations of DUN1 or RFA2 that eliminate SQ motifs have no effect on telomere length or DNA damage sensitivity.     

Requirement of the FATC domain of protein kinase Tel1 for localization to DNA ends and target protein recognition

Two large phosphatidylinositol 3-kinase–related protein kinases (PIKKs), ATM and ATR, play a central role in the DNA damage response pathway. PIKKs contain a highly conserved extreme C-terminus called the FRAP-ATM-TRRAP-C-terminal (FATC) domain. In budding yeast, ATM and ATR correspond to Tel1 and Mec1, respectively. In this study, we characterized functions of the FATC domain of Tel1 by introducing substitution or truncation mutations. One substitution mutation, termed tel1-21, and a truncation mutation, called tel1-ΔC, did not significantly affect the expression level. The tel1-21 mutation impaired the cellular response to DNA damage and conferred moderate telomere maintenance defect. In contrast, the tel1-ΔC mutation behaved like a null mutation, conferring defects in both DNA damage response and telomere maintenance. Tel1-21 protein localized to DNA ends as effectively as wild-type Tel1 protein, whereas Tel1-ΔC protein failed. Introduction of a hyperactive TEL1-hy mutation suppressed the tel1-21 mutation but not the tel1-ΔC mutation. In vitro analyses revealed that both Tel1-21 and Tel1-ΔC proteins undergo efficient autophosphorylation but exhibit decreased kinase activities toward the exogenous substrate protein, Rad53. Our results show that the FATC domain of Tel1 mediates localization to DNA ends and contributes to phosphorylation of target proteins.     

Hyperactivation of the yeast DNA damage checkpoint by TEL1 and DDC2 overexpression.

The evolutionarily conserved yeast Mec1 and Tel1 protein kinases, as well as the Mec1-interacting protein Ddc2, are involved in the DNA damage checkpoint response. We show that regulation of Tel1 and Ddc2-Mec1 activities is important to modulate both activation and termination of checkpoint-mediated cell cycle arrest. In fact, overproduction of either Tel1 or Ddc2 causes a prolonged cell cycle arrest and cell death in response to DNA damage, impairing the ability of cells to recover from checkpoint activation. This cell cycle arrest is independent of Mec1 in UV-irradiated Tel1-overproducing cells, while it is strictly Mec1 dependent in similarly treated DDC2-overexpressing cells. The Rad53 checkpoint kinase is instead required in both cases for cell cycle arrest, which correlates with its enhanced and persistent phosphorylation, suggesting that unscheduled Rad53 phosphorylation might prevent cells from re-entering the cell cycle after checkpoint activation. In addition, Tel1 overproduction results in transient nuclear division arrest and concomitant Rad53 phosphorylation in the absence of exogenous DNA damage independently of Mec1 and Ddc1.     

Protein kinase C controls activation of the DNA integrity checkpoint

The protein kinase C (PKC) superfamily plays key regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whose main function is cell wall integrity maintenance. In this work, we connect the Pkc1 protein to the maintenance of genome integrity in response to genotoxic stresses. Pkc1 and its kinase activity are necessary for the phosphorylation of checkpoint kinase Rad53, histone H2A and Xrs2 protein after deoxyribonucleic acid (DNA) damage, indicating that Pkc1 is required for activation of checkpoint kinases Mec1 and Tel1. Furthermore, Pkc1 electrophoretic mobility is delayed after inducing DNA damage, which reflects that Pkc1 is post-translationally modified. This modification is a phosphorylation event mediated by Tel1. The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint. Finally, downregulation of PKCδ activity in HeLa cells caused a defective activation of checkpoint kinase Chk2 when DNA damage was induced. Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans.     

Molecular Basis of the Essential S Phase Function of the Rad53 Checkpoint Kinase

The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.     

A Tel1/MRX-dependent checkpoint inhibits the metaphase-to-anaphase transition after UV irradiation in the absence of Mec1

In Saccharomyces cerevisiae, Mec1/ATR plays a primary role in sensing and transducing checkpoint signals in response to different types of DNA lesions, while the role of the Tel1/ATM kinase in DNA damage checkpoints is not as well defined. We found that UV irradiation in G(1) in the absence of Mec1 activates a Tel1/MRX-dependent checkpoint, which specifically inhibits the metaphase-to-anaphase transition. Activation of this checkpoint leads to phosphorylation of the downstream checkpoint kinases Rad53 and Chk1, which are required for Tel1-dependent cell cycle arrest, and their adaptor Rad9. The spindle assembly checkpoint protein Mad2 also partially contributes to the G(2)/M arrest of UV-irradiated mec1Delta cells independently of Rad53 phosphorylation and activation. The inability of UV-irradiated mec1Delta cells to undergo anaphase can be relieved by eliminating the anaphase inhibitor Pds1, whose phosphorylation and stabilization in these cells depend on Tel1, suggesting that Pds1 persistence may be responsible for the inability to undergo anaphase. Moreover, while UV irradiation can trigger Mec1-dependent Rad53 phosphorylation and activation in G(1)- and G(2)-arrested cells, Tel1-dependent checkpoint activation requires entry into S phase independently of the cell cycle phase at which cells are UV irradiated, and it is decreased when single-stranded DNA signaling is affected by the rfa1-t11 allele. This indicates that UV-damaged DNA molecules need to undergo structural changes in order to activate the Tel1-dependent checkpoint. Active Clb-cyclin-dependent kinase 1 (CDK1) complexes also participate in triggering this checkpoint and are required to maintain both Mec1- and Tel1-dependent Rad53 phosphorylation, suggesting that they may provide critical phosphorylation events in the DNA damage checkpoint cascade.     

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.     

Mechanism of Dun1 activation by Rad53 phosphorylation in Saccharomyces cerevisiae

Despite extensive studies, the molecular mechanism of DNA damage checkpoint activation remains incompletely understood. To better dissect this mechanism, we developed an activity-based assay for Dun1, a downstream DNA damage check-point kinase in yeast, using its physiological substrate Sml1. Using this assay, we confirmed the genetic basis of Dun1 activation. Rad53 was found to be directly responsible for Dun1 activation. We reconstituted the activation of Dun1 by Rad53 and found that phosphorylation of Thr-380 in the activation loop of Dun1 by Rad53 is responsible for Dun1 activation. Interestingly, phosphorylation of the evolutionarily conserved Thr-354 in the activation loop of Rad53 is also important for the regulation of Rad53 activity. Thus, this conserved mode of activation loop phosphorylation appears to be a general mechanism for the activation of Chk2 family kinases.     

The Saccharomyces cerevisiae Sae2 protein negatively regulates DNA damage checkpoint signalling

Double-strand breaks (DSBs) elicit a DNA damage response, resulting in checkpoint-mediated cell-cycle delay and DNA repair. The Saccharomyces cerevisiae Sae2 protein is known to act together with the MRX complex in meiotic DSB processing, as well as in DNA damage response during the mitotic cell cycle. Here, we report that cells lacking Sae2 fail to turn off both Mec1- and Tel1-dependent checkpoints activated by a single irreparable DSB, and delay Mre11 foci disassembly at DNA breaks, indicating that Sae2 may negatively regulate checkpoint signalling by modulating MRX association at damaged DNA. Consistently, high levels of Sae2 prevent checkpoint activation and impair MRX foci formation in response to unrepaired DSBs. Mec1- and Tel1-dependent Sae2 phosphorylation is necessary for these Sae2 functions, suggesting that the two kinases, once activated, may regulate checkpoint switch off through Sae2-mediated inhibition of MRX signalling.     

Cdc5 blocks in vivo rad53 activity,but not in situ activity (ISA)

DNA damage promotes the activation of a signal transduction cascade referred to as the DNA damage checkpoint. This pathway initiates with the Mec1/ATR kinase, which then phosphorylates the Rad53/Chk2 kinase. Mec1 phosphorylation of Rad53 is then thought to promote Rad53 autophosphorylation, ultimately leading to a fully active Rad53 molecule that can go on to phosphorylate substrates important for DNA damage resistance. In the absence of DNA repair, this checkpoint is eventually downregulated in a Cdc5-dependent process referred to as checkpoint adaptation. Recently, we showed that overexpression of Cdc5 leads to checkpoint inactivation and loss of the strong electrophoretic shift associated with Rad53 inactivation. Interestingly, this same overexpression did not strongly inhibit Rad53 autophosphorylation activity as measured by the in situ assay (ISA). The ISA involves incubating the re-natured Rad53 protein with γ ³²P labeled ATP after electrophoresis and western blotting. Using a newly identified Rad53 target, we show that despite strong ISA activity, Rad53 does not maintain phosphorylation of this substrate. We hypothesize that, during adaptation, Rad53 may be in a unique state in which it maintains some Mec1 phosphorylation but does not have the auto-phosphorylations required for full activity towards exogenous substrates.Key words: DNA damage, checkpoint, adaptation, CDC5, RAD53, ISA     

Mec1/ATR regulates the generation of single‐stranded DNA that attenuates Tel1/ATM signaling at DNA ends

Tel1/ATM and Mec1/ATR checkpoint kinases are activated by DNA double‐strand breaks (DSBs). Mec1/ATR recruitment to DSBs requires the formation of RPA‐coated single‐stranded DNA (ssDNA), which arises from 5′–3′ nucleolytic degradation (resection) of DNA ends. Here, we show that Saccharomyces cerevisiae Mec1 regulates resection of the DSB ends. The lack of Mec1 accelerates resection and reduces the loading to DSBs of the checkpoint protein Rad9, which is known to inhibit ssDNA generation. Extensive resection is instead inhibited by the Mec1‐ad mutant variant that increases the recruitment near the DSB of Rad9, which in turn blocks DSB resection by both Rad53‐dependent and Rad53‐independent mechanisms. The mec1‐ad resection defect leads to prolonged persistence at DSBs of the MRX complex that causes unscheduled Tel1 activation, which in turn impairs checkpoint switch off. Thus, Mec1 regulates the generation of ssDNA at DSBs, and this control is important to coordinate Mec1 and Tel1 signaling activities at these breaks.