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RAD53 and MEC1 are essential Saccharomyces cerevisiae genes required for the DNA replication and DNA damage checkpoint responses. Their lethality can be suppressed by increasing the intracellular pool of deoxynucleotide triphosphates. We report that deletion of YKU70 or YKU80 suppresses mec1Δ, but not rad53Δ, lethality. We show that suppression of mec1Δ lethality is not due to Ku−-associated telomeric defects but rather results from the inability of Ku− cells to efficiently repair DNA double strand breaks by nonhomologous end joining. Consistent with these results, mec1Δ lethality is also suppressed by lif1Δ, which like yku70Δ and yku80Δ, prevents nonhomologous end joining. The viability of yku70Δ mec1Δ and yku80Δ mec1Δ cells depends on the ATM-related Tel1 kinase, the Mre11-Rad50-Xrs2 complex, and the DNA damage checkpoint protein Rad9. We further report that this Mec1-independent pathway converges with the Rad53/Dun1-regulated checkpoint kinase cascade and leads to the degradation of the ribonucleotide reductase inhibitor Sml1.
Cells maintain the integrity of their genetic information by activating complex pathways in response to DNA damage. The activation of these signal transduction pathways leads to a delay of cell cycle progression to ensure replication and/or segregation of damaged DNA molecules and to activate DNA repair pathways (61). In the budding yeast Saccharomyces cerevisiae, the central components of these checkpoint pathways are MEC1 and RAD53, the budding yeast homologs of the ATR-Rad3-related gene and CHK2 (CDS1), respectively (1, 50, 56). In addition to their involvement in checkpoint pathways, Mec1 and Rad53 are required to induce the transcription of repair genes and of the genes encoding ribonucleotide reductase (RNR) that catalyze the rate-limiting step in deoxynucleoside triphosphate (dNTP) synthesis (2).
In contrast to most other checkpoint genes, MEC1 and RAD53 are essential for cell viability. The lethality associated with the disruption of MEC1 and RAD53, but not their checkpoint defects, can be suppressed by increasing the intracellular concentration of dNTPs (15, 22, 56).
Sml1 inhibits Rnr1 through a direct interaction (59). Mec1 and Rad53 relieve the Sml1-Rnr1 interaction in S phase, allowing synthesis of sufficient amounts of dNTPs for DNA replication (59). Consistent with this idea, sml1 missense mutations that rescue mec1Δ and rad53Δ lethality abolish the Sml1-Rnr1 interaction (58). More recently, it has been shown that Dun1 phosphorylates and removes Sml1 during S phase (57, 60). From all these results, it has been proposed that the absence of Mec1 or Rad53 would lead to insufficient dNTP levels and subsequent cell death. Both MEC1 and RAD53 also regulate the activation of late-firing origins of DNA replication (44, 46). Firing of replication origins with insufficient nucleotides would effectively cause a condition of higher dNTP deprivation (15).
The Ku heterodimer is conserved in a wide range of eukaryotes and plays multiple roles in DNA metabolism in yeast. Ku is involved in double strand break repair by nonhomologous end joining (NHEJ) (4, 6, 33, 38). Inactivation of YKU70 or YKU80 also results in telomere shortening, loss of telomere clustering and silencing, deregulation of the normally cell cycle-dependent telomeric G overhang, earlier activation of replication origins close to telomeres, and synthetic lethality with mutations that impair telomere replication (3, 7, 12, 18, 25, 36, 40). yku70 and yku80 mutants are viable at 30°C but are unable to grow at 37°C, which reflects a defect in telomere maintenance rather than a more generalized DNA repair defect (18, 30, 31, 48). CHK1, MEC1, and RAD9 checkpoint genes contribute to the inhibition of cell division of yku70Δ mutants cultured at 37°C (30). Recently, it was suggested that Mec1, Rad9, and Rad53 inhibit degradation of double-stranded DNA in and near telomere repeats (23).
In this study, we initially asked whether the inactivation of YKU genes would affect mec1Δ and/or rad53Δ lethality. We report that YKU70 or YKU80 deletion suppress mec1Δ, but not rad53Δ, lethality. We showed that in the absence of Mec1, a deletion of YKU70 or YKU80 associated with a defective end-joining function induces a Tel1-Mre11-dependent response. Rad9, Rad53, and Dun1 are all required for the degradation of the RNR inhibitor Sml1 in yku70Δ and yku80Δ. Our results bring new insights into the way cells respond to DNA lesions in yku70Δ and yku80Δ cells and unmask for the first time a connection between the NHEJ pathway and the checkpoint response.
The genotypes of the yeast strains used in this study are listed in Table Table1.1. The mec1Δ::KAN, rad24Δ::KAN, rad17Δ::KAN, and rif2Δ::KAN null mutations were introduced as described previously (16). The mec1Δ::TRP1, lcd1Δ::TRP1, ddc1Δ::TRP1, mrc1Δ::TRP1, chk1Δ::TRP1, mad2Δ::TRP1, rad9Δ::TRP1, mre11Δ::TRP1, tel1Δ::TRP1, dun1Δ::TRP1, rnr3Δ::TRP1, lif1Δ::TRP1, and yku80Δ::TRP1 null mutations were obtained after PCR amplification of a disruption cassette from plasmid pF6a-TRP1. The exo1Δ::URA3 mutation was introduced using SphI-linearized pDL684 plasmid (from David Lydall). The mec3Δ::TRP1 mutation was introduced as described previously (11). The rad53-K227A mutation was introduced using EcoRI-linearized pCH3 plasmid (39). est2Δ::NAT, mec1Δ::NAT, rad53Δ::NAT, sml1Δ::NAT, and tel1Δ::NAT were obtained after PCR amplification of a disruption cassette containing the nourseothricin (nat) resistance gene (17). To disrupt TEL1 with URA3 or LEU2, we linearized plasmid pPG47 (URA3 or LEU2) with SacI and transformed the appropriate yeast strains (20). To construct strains carrying the RAD52 chromosomal deletion, we transformed the appropriate strains with the BamHI-linearized plasmid pSM21 (a gift from M. Fasullo, Loyola University, Chicago, IL), which carries a rad52Δ::TRP1 cassette.
Protein extracts for Western blot analysis were prepared from trichloroacetic acid-treated cells and resolved by electrophoresis on an appropriate sodium dodecyl sulfate-polyacrylamide gel (80:1 acrylamide-bisacrylamide). Immunoblots were performed with anti-protein A (Sigma) and anti-green fluorescent protein (GFP; Roche) monoclonal antibodies for Mre11-ProA detection and for yellow fluorescent protein (YFP)-Sml1 detection, respectively.
Total RNA was extracted from yeast cells using the SV total RNA isolation system kit (Promega). RNA (1 μg) was reverse transcribed using the Titan one-tube reverse transcriptase PCR (RT-PCR) kit (Roche) using specific primers for TEL1 (5′-CCACAGGATTGTCCCTGCC-3′and 5′-AGCTGCGACACCTTTTGTGTA-3′) and ACT1 (5′-CCAATTGCTCGAGAGATTTC-3′and 5′-CATGATACCTTGGTGTCTTG-3′). PCR cycling conditions for TEL1 and ACT1 were as follows: a denaturation step at 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 55°C for 30 s, and 68°C for 45 s, as well as a final extension of 68°C for 7 min. PCR products (30% of reaction mixture) were then separated on 2% agarose gels, and bands were visualized with ethidium bromide. The gels were scanned with a Molecular Dynamics PhosphorImager, and the signals were quantified with Kodak software. The TEL1 transcript levels were arbitrarily set at 1 in the wild-type cells, and TEL1 levels in mutant cells were normalized accordingly.
For G1 phase, cells were grown to early log phase and arrested by the addition of α-factor (5 μg/ml) for 60 to 90 min, at which point arrest was verified by the absence of budded cells. For S phase, cells were grown to early log phase and arrested by the addition of hydroxyurea (10mM) for 90 min. For G2 phase, cells were grown to early log phase and arrested by the addition of nocodazol (30 μg/ml) for 90 to 120 min. Cells were analyzed by fluorescence microscopy, and the YFP-Sml1 level was detected by Western blot analysis.
Genomic DNA was isolated from overnight cultures of the strains indicated, cut with XhoI, separated on a 0.8% agarose gel, and subjected to Southern blot analysis with poly(GT) telomeric probe, which was obtained by PCR using plasmid sp100 (a gift from E. Gilson, Ecole Normale Superieur, Lyon) as a template.
Cells were micromanipulated with the MSM system from Singer Instruments (26).
To test if the essential function of MEC1 could be suppressed by inactivation of YKU70, we disrupted one allele of MEC1 in either wild-type, heterozygous, or homozygous for yku70Δ diploid yeast strains. As expected, we were not able to obtain mec1Δ spores after 3 days of growth at 30°C when the spores were obtained from a mec1Δ/MEC1 YKU70/YKU70 strain or from the heterozygous mec1Δ/MEC1 yku70Δ/YKU70 diploid. Surprisingly, tetrads derived from the mec1Δ/MEC1 yku70Δ/yku70Δ diploid gave more than two colonies (Fig. (Fig.1A).1A). One hundred tetrads were dissected. A total of 160/200 mec1Δ yku70Δ spores germinated to form colonies, whereas 166/200 yku70Δ cells grew. Subsequently, several viable mec1Δ yku70Δ spores were backcrossed to unrelated wild-type strains. The resulting diploids were sporulated and dissected. We never obtained viable mec1Δ or mec1Δ yku70Δ colonies (not shown). These results were reproducibly obtained using multiple independent disruptions. We concluded that mec1Δ yku70Δ spores have not acquired an inheritable suppressor and that the viability of mec1Δ yku70Δ segregants is entirely dependent on the genotype of the parental diploid. mec1Δ is viable only in mec1Δ yku70Δ spores that were generated from diploids homozygous for yku70Δ. Similar results were obtained with the deletion of LCD1/DDC2 (not shown), the ATRIP partner of Mec1 that is involved in both its essential and checkpoint functions (37, 41, 53). The germinating yku70Δ and mec1Δ yku70Δ spores of several tetrads were examined microscopically after 4, 8, 18, and 72 h (not shown). All of them start to germinate after 8 h at 30°C. After 18 h, the spores grew into microcolonies consisting of at least 20 cells, but often more. We could not detect any difference in colony size between mec1Δ yku70Δ and MEC1 yku70Δ microcolonies after 18 h, indicating that these cells are growing at the same rate. As previously shown, after 3 days, most of the tetrads arising from mec1Δ/MEC1 yku70Δ/yku70Δ gave four big colonies with a size of 100 to 150 cells each. If the mec1Δ yku70Δ cells are separated into individual cells 3 days after germination, each mec1Δ cell divides at the same rate as yku70Δ. In each case, the viable mec1Δ yku70Δ colonies were sensitive to hydroxyurea and UV irradiation. We checked whether the HU sensitivity is different in mec1Δ compared to mec1Δ yku70Δ cells. Since mec1Δ strains are unable to grow in the presence of yKu70, their ability was maintained by the presence of a 2μm RNR1 plasmid (15). We found that even at a low dose of HU, mec1Δ and mec1Δ yku70Δ cells presented a similar sensitivity (Fig. (Fig.1B).1B). Taken together, these results indicate that both types of cells, yku70Δ and mec1Δ yku70Δ, are fully viable but only if the parental diploid strain was homozygous yku70Δ/yku70Δ.
An important role of yKu70 and yKu80 is maintenance of telomere integrity (40). Mec1 is associated with telomeres (47), and its absence causes a shortening of telomere length. We investigated whether the deletion of MEC1 (in the presence of SML1) aggravated the telomeric phenotypes associated with yku70 deletion. As shown in Fig. Fig.1C,1C, mec1Δ did not confer senescence to a yku70Δ strain and mec1Δ yku70Δ cells exhibit telomeric defects similar to those observed in yku70Δ cells. The suppression of mec1Δ by YKU70 or YKU80 gene deletion was confirmed by our ability to disrupt MEC1 in yku70Δ or yku80Δ haploid strains and by the discovery that yku70Δ mec1Δ cells were able to lose a pMEC1 expression plasmid, whereas mec1Δ cells were not (Fig. (Fig.1D).1D). Deletion of YKU70 or YKU80 did not suppress rad53Δ lethality (not shown).
Because the DNA damage pathway regulates dNTP levels by increasing RNR gene transcription (22) and RNR activity by phosphorylation-mediated removal of Sml1, an inhibitor of RNR (57), we next addressed the importance of known genetic pathways involved in the response to DNA damage in the suppression of the mec1Δ lethality by yku70Δ. We constructed diploid strains homozygous for yku70Δ and heterozygous for mec1Δ and for several DNA damage checkpoint genes. For each combination, the diploids were sporulated, 100 tetrads were dissected, and the genotypes of the viable spores were determined. The results shown in Table Table22 indicate that the suppression of mec1Δ lethality by yku70Δ does not depend on the PCNA-like proteins Rad17, Ddc1, and Mec3, the RFC-like protein Rad24, the DNA replication checkpoint Mrc1, or the downstream signal transduction kinase Chk1. In contrast, deletion of the DNA damage checkpoint gene RAD9 prevented suppression of mec1Δ lethality by yku70Δ. This result indicates that RAD9 has an essential function in rescuing mec1Δ lethality in yku70Δ cells.
EXO1 and MAD2 are required to activate checkpoint pathways in yku70Δ mutants at 37°C (30). We found that neither exo1Δ nor mad2Δ mutations affect the ability of yku70Δ mec1Δ mutants to form colonies (Table (Table2).2). Similar results were observed using haploid yku80Δ for analyzing the genetic dependence of suppression of mec1Δ lethality.
It has been suggested that the RAD52 recombinational repair pathway is required to repair double strand breaks (DSBs) caused by defective DNA replication in mec1 mutants. Indeed, several viable mec1 mutations that display synthetic lethality with rad52Δ have been isolated (32). We asked if Rad52 was required in the suppression of mec1Δ by inactivation of YKU70 and observed that yku70Δ is able to suppress the lethality of mec1Δ cells even in the absence of RAD52.
Rad9 is implicated with Tel1 and the Mre11-Rad50-Xrs2 (MRX) complex in a checkpoint pathway that recognizes unprocessed DSBs and parallels the Mec1 pathway (14, 51). We considered the Mre11 nuclease and the ATM-related Tel1 kinase, which are the first proteins detected at DSBs (27), as candidates that might function in suppressing mec1Δ lethality. To test this, tetrads derived from the tel1Δ/TEL1 mec1Δ/MEC1 yku70Δ/yku70Δ and mre11Δ/MRE11 mec1Δ/MEC1 yku70Δ/yku70Δ diploid strains were analyzed. We observed the appearance of mec1Δ yku70Δ, tel1Δ yku70Δ, and mre11Δ yku70Δ but not of mec1Δ tel1Δ yku70Δ and mec1Δ mre11Δ yku70Δ spores. Thus, in the absence of TEL1 or MRE11, mec1Δ yku70Δ strains are not able to grow. These results suggest that TEL1 and MRE11 have an essential function in rescuing mec1Δ lethality in yku70Δ cells.
One interpretation of our data is that the absence of mec1Δ yku70Δ tel1Δ and mec1Δ yku70Δ mre11Δ spores is the consequence of the synthetic lethality of the triple mutation due to telomere shortening and eventual cellular senescence. To address this possibility, we took advantage of the fact that deleting the RIF2 gene causes telomere elongation (even in a yku70Δ or a yku80Δ background) and bypasses the requirement for Mec1 and Tel1 kinases in telomere maintenance (9, 34). We disrupted one allele of RIF2 in the diploid yeast strain tel1Δ/TEL1 mec1Δ/MEC1 yku70Δ/yku70Δ and analyzed the spores derived from this diploid. We never obtained mec1Δ tel1Δ yku70Δ and rif2Δ mec1Δ tel1Δ yku70Δ spores (not shown). We were also unable to obtain mec1Δ transformants in rif2Δ tel1Δ yku80Δ cells, although telomeres of rif2Δ tel1Δ yku80Δ cells were significantly longer than those of tel1Δ yku80Δ cells and slightly greater than in yku80Δ cells (Fig. (Fig.2A).2A). These results predict that a DNA damage pathway controlled by Tel1 and the Mre11 complex is activated in yku70Δ and yku80Δ cells.
Because Mre11 is phosphorylated after DNA damage in a Tel1-dependent manner (14), we investigated whether Mre11 is phosphorylated in yku70Δ and yku70Δ mec1Δ cells at 30°C by using a Mre11-ProtA fusion. Figure Figure2B2B shows that Mre11-ProtA migrates as a single band in extracts obtained from wild-type cells and also in mre11Δ cells complemented with pMre11-ProtA, whereas a slower-migrating form of Mre11, corresponding to the phosphorylated form of Mre11, was detected in yku70Δ mre11Δ and yku70Δ mec1Δ mre11Δ cell extracts. These results revealed a Mec1-independent DNA damage-induced phosphorylation of Mre11 in yku70Δ cells.
Rad53 plays an essential role in both the DNA damage and replication block checkpoints (28). Its phosphorylation correlates with activation of the checkpoint pathways (43). Overexpression of the DUN1 gene can suppress the lethality of mec1Δ (35, 43); this suppression appears to reflect Dun1's role in repressing Sml1 activity, as suppression of mec1Δ by sml1Δ did not require the activity of Dun1 (59). However, other studies have shown that a viable mec1 mutation and dun1Δ are synthetically lethal (13). We tested whether yku70Δ or yku80Δ suppression of mec1Δ lethality is dependent on the product of DUN1 and RAD53 genes. Since RAD53 is essential, we used the viable checkpoint-deficient rad53 allele (rad53-K227A) that carries a substitution within the conserved kinase domain of Rad53 (39). We found that we were able to obtain mec1Δ transformants in haploid yku70Δ and yku80Δ mutants but not in yku70Δ dun1Δ, yku80Δ dun1Δ, yku70Δ rad53-K227A, or yku80Δ rad53-K227A double mutants (not shown). Thus, RAD53 and DUN1 are required to rescue mec1Δ lethality in yku70Δ and yku80Δ cells. However, we observed by a gel autophosphorylation assay and by a Western blot mobility shift assay that yku70Δ and mec1Δ yku70Δ mutants do not show a high level of Rad53 phosphorylation.
yKu70 and yKu80 are DNA end-binding proteins that play various roles at different kinds of DNA ends. At telomeres, yKu70 and yKu80 are part of the structure that protects the chromosome end, whereas at broken DNA ends, they promote DNA repair as part of the NHEJ pathway. To gain insight about which aspect of yKu function, when lost, leads to the suppression of mec1Δ lethality, we tested a separation-of-function mutant of YKU80. For these experiments, plasmids that carried the telomeric defective/repair-proficient yku80-PF437,438AA (yku80-PF) mutation (42) or the wild-type YKU80 gene were introduced individually into a yku80Δ haploid strain. Cells were then examined for the suppression of mec1Δ lethality. We found that yku80Δ cells expressing the yku80-PF mutant protein, which are affected in telomeric silencing (42) and telomere size (Fig. (Fig.3A),3A), were not able to suppress mec1Δ lethality. However, cells carrying the empty vector allowed the suppression of mec1Δ lethality (not shown). The inability of the yku80-PF mutant to suppress mec1Δ lethality in yku80Δ cells suggested that desilencing of the telomere and its size control is not sufficient to suppress mec1Δ lethality. Consistent with this interpretation, none of the viable segregants from 100 tetrads derived from sporulation of the diploid strain mec1Δ/MEC1 sir3Δ/sir3Δ was mec1Δ sir3Δ. Moreover, yku70Δ mec1Δ sir3Δ cells are viable (data not shown). We concluded that the suppression of mec1Δ lethality in a ykuΔ strain is not due to the loss of telomeric position effect.
Given that yKu70/yKu80 and Lif1 proteins are involved in a common NHEJ pathway, it was conceivable that lif1Δ would be able to suppress mec1Δ lethality. To test this possibility, we attempted to generate mec1Δ in lif1Δ spores, starting from mec1Δ/MEC1 lif1Δ/lif1Δ. As shown in Fig. Fig.3B,3B, we were able to obtain mec1Δ lif1Δ viable spores. In agreement with this result, we were also able to obtain mec1Δ transformants in lif1Δ haploid cells, and lif1Δ mec1Δ haploid cells are able to lose a pMEC1 expression plasmid (not shown). These results indicate that NHEJ defects in general rescue the lethal phenotype of Mec1-deficient yeast. We concluded that the suppression of mec1Δ lethality in yku70Δ and yku80Δ cells is probably associated with the loss of repair function.
Because TEL1 overexpression can suppress both cell lethality and hypersensitivity to DNA-damaging agents of the mec1Δ mutant, indicating that excess Tel1 can bypass the requirement for Mec1 (10, 43), it remains possible that YKU80 deletion increases TEL1 expression. Total RNA were prepared from wild-type, yku80Δ, or tel1Δ cells, and TEL1 mRNA levels were examined by RT-PCR (Fig. (Fig.4).4). Our data indicate that TEL1 is not overexpressed in yku80Δ mutants. However, a reproducible 1.5 enrichment of TEL1 mRNA was detected in yku80Δ cells.
Sml1 is phosphorylated and then degraded during S phase and after DNA damage to provide sufficient dNTPs to complete DNA replication (57, 60). We used a chromosomally integrated YFP-Sml1 fusion protein (kindly provided by R. Rothstein; unpublished results) to monitor the amount of Sml1 in yku80Δ mutants (Fig. (Fig.5).5). Cells were arrested either in G1, S, or G2. By analyzing the population of cells in the cultures, we confirmed the efficiency of the G1, S, and G2 arrests (not shown). Both epifluorescence and Western blot analysis were performed to determine the amount of YFP-Sml1. In agreement with previous results (60), YFP-Sml1 levels are reduced in S phase, whereas it is detected in G1 and G2, both by epifluorescence (Fig. (Fig.5A)5A) and Western blotting (Fig. (Fig.5B).5B). The amount of YFP-Sml1 was clearly reduced, about 60%, in the yku80Δ mutant in G1 and in G2. YFP-Sml1 remained undetectable in S phase. When we reintroduced a wild-type YKU80 in the yku80Δ mutant, we restored the level of YFP-Sml1. Next, we tested whether the decrease of Sml1 levels in G1, S, and G2 phases in yku80Δ depended on Mec1. Since mec1Δ strains are unable to grow in the presence of Sml1, their ability was maintained by the presence of a 2μm RNR1 plasmid (15). Sml1 levels are highly reduced at G1 and G2 phases in yku80Δ mec1Δ strains compared with mec1Δ cells (Fig. (Fig.5C).5C). We deduced from these results that the protein amount of Sml1 is influenced by yKu80 protein and that degradation of Sml1 can occur independently of Mec1. The lower level of Sml1 is a plausible explanation for the suppression of mec1Δ lethality by yku80Δ.
To correlate YFP-Sml1 levels with suppression of mec1Δ lethality, we analyzed YFP-Sml1 levels in G1 and G2 phases, in mec3Δ, rad24Δ, rad9Δ, mre11Δ, tel1Δ, rad53-K227A, and dun1Δ mutants in the presence or absence of YKU80 (data not shown). Western blot analyses indicate that Sml1 depletion, at least in yku80Δ mutant cells, depends on Rad9, on the kinase activity of Rad53, and also on Dun1 but not on Mec3 and Rad24. Deleting MRE11 and, to a lesser extent, TEL1 produces by itself a depletion of the Sml1 protein amount, a situation that occurs in the yku80 mutant; however, these mutations are likely to cause Sml1 degradation via the canonical Mec1-dependent pathway. On the other hand, we found Sml1 depletion in lif1Δ cells and a wild-type level of Sml1 in the telomeric defective/repair-proficient yku80-PF437,438AA mutant which does not suppress mec1Δ lethality (not shown). To confirm the importance of Sml1 depletion in the suppression of mec1Δ lethality by YKU80 deletion, we deleted YKU80 and MEC1 genes in a GAL-SML1 haploid strain (57). We found that GAL-SML1 ku80Δ mec1Δ strains grow normally in noninducible medium but not after galactose induction (not shown). Finally, we believed that it was important to check the viability of mec1Δ yku70Δ combined with rad9Δ, tel1Δ, or mre11Δ deletion, in the absence of SML1. We were able to obtain sml1Δ rad9Δ mec1Δ yku70Δ viable spores that grew as well as rad9Δ yku70Δ spores (Fig. (Fig.6A).6A). Genetic analysis also showed that a sml1Δ mutation rescues the lethality of mre11Δ mec1Δ yku70Δ and tel1Δ mec1Δ yku70Δ cells (Fig. 6B and C). However, these mutants both present a significant growth defect. On this base, we conclude that Rad9, Tel1, and Mre11 are required for Sml1 depletion and, consequently, for suppression of mec1Δ lethality in yku70Δ and yku80Δ cells. This suggests that YKU70 and YKU80 deletions contribute to Sml1 degradation via both Tel1/MRX- and Mec1-dependent pathways.
In response to DSBs, a number of DNA checkpoint and repair proteins in S. cerevisiae relocalize from a diffuse nuclear distribution to distinct subnuclear foci acting as centers of recombinational DNA repair. To investigate the presence of DNA damage in yku80Δ cells, we used a chromosomally integrated Mre11-YFP fusion protein (27). The Mre11-Rad50-Xrs2 complex proteins are the first proteins detected at DSBs. As reported previously (27), we found that spontaneous Mre11 foci form in a low percentage of wild-type cells (4.5%) even in the absence of exogenous DNA damage. In contrast, approximately 8-fold more Mre11 foci (35% of the cells) are detected in yku80Δ cells (Fig. (Fig.7).7). When a plasmid that encoded the telomeric defective/repair-proficient yku80-PF437,438AA (yku80-PF) mutant (42) was introduced into the yku80Δ haploid strain, we observed a significant reduction of Mre11 foci compared to yku80Δ cells (Fig. (Fig.7).7). These in vivo observations are consistent with the presence of DNA damage associated with the loss of repair function in yku80Δ cells, providing an explanation for the Sml1 depletion.
Finally, because DSB induction appears to be the key event in suppressing mec1Δ lethality in yku80Δ cells, we analyzed whether a pMEC1 expression plasmid can be cured from mec1Δ cells (in the presence of SML1) in a low concentration of methyl methanesulfonate (MMS) (0.01%, 0.005%, 0.001%, and 0.0005%). Our experiences indicate that MMS did not allow mec1Δ cells to lose the pMEC1 expression plasmid (not shown), suggesting that DSBs generated by the absence of yKu80 are not similar and/or proceeded differently than those induced by MMS.
RAD53 and MEC1 are essential S. cerevisiae genes required for the DNA damage response. Their lethality, but not their role in checkpoint responses, can be suppressed by increasing the intracellular pool of deoxynucleotides. This lethality can be explained both by observations that Mec1 and Rad53 are required to maintain sufficient dNTP levels (22) and by the findings that Mec1 and Rad53 stabilize stalled replication forks (whose occurrence would increase when dNTP levels are low) (29, 49). For example, certain alleles of mec1 (mec1-srf) accumulate short DNA replication intermediates that are suppressed by the inactivation of Sml1, which raises dNTP levels (32, 59). Moreover, alteration/reduction of Mec1 function leads to fork stalling, followed by chromosome breakage (8).
We found that mec1Δ is viable in spores that were generated from diploids homozygous, but not heterozygous, for yku70Δ. One explanation for these results is that yku70Δ mec1Δ spores derived from heterozygous yku70Δ/YKU70 diploids were still phenotypically YKU70+ and failed to reach the point where they are phenotypically yku70Δ. These observations are strongly supported by the finding that one can also obtain mec1Δ deletions by direct transformation of yku70Δ and yku80Δ cells. In contrast, deletion of YKU70 or YKU80 does not suppress rad53Δ lethality. This result is analogous to the previous observation that the sml1Δ mutation, which elevates dNTP levels, strongly suppresses mec1Δ lethality but only partially restores the growth of rad53Δ (58). It suggests that Rad53 has other, yet undefined functions that go beyond the regulation of dNTP levels (21, 45, 51).
We have found that yku70 and yku80 mutant suppression of mec1Δ lethality is dependent on the genes RAD9, TEL1, MRE11, RAD53, and DUN1. These genes can be imagined to be part of the Tel1-MRX signal transduction pathway (14, 51, 52) that has been characterized as responding to nonresected DSB ends.
The discovery that elevation of dNTP levels by overexpressing RNR or TEL1 genes would suppress mec1Δ made it clear that Mec1 is not truly essential (15, 22, 59). We established here that yku70Δ and yku80Δ cells only slightly elevate RNR3 (not shown) and TEL1 gene expression. It seems that ykuΔ can alleviate dNTP limitations by nontranscriptional means.
SML1 deletion suppresses mec1Δ and rad53Δ lethality. Sml1 is normally removed during S phase and after DNA damage to provide sufficient dNTPs (57, 59). Deletion of yku80Δ results in a decrease in Sml1 levels in both G1- and G2/M-arrested cells. The finding that the level of Sml1 protein is lower in a yku80Δ mutant than in wild-type cells can explain the suppression of mec1Δ lethality and suggests that unrepaired DNA damage or stress signals remain in yku80Δ and yku80Δ mec1Δ cells. The analysis of Mre11 foci in yku80Δ cells demonstrates the presence of DSBs. We found that Sml1 depletion in the yku80Δ mutants is dependent on Rad9, Rad53, and Dun1, but not on other checkpoint proteins, confirming the implication of these proteins in the suppression of mec1Δ lethality.
Based on recent observations from Lisby et al. (27) which indicate that Mre11 and Tel1 are the first proteins detected at DSBs, we propose a model to explain the functions of checkpoint proteins in responding to ykuΔ-induced DNA damage. According to this model, Mre11 is phosphorylated in a Tel1-dependent manner, leading to Sml1 phosphorylation and degradation by the subsequent activation of the Rad9, Rad53, and Dun1 pathway. We suggest that Mec1 itself might also participate in Sml1 degradation in yku80Δ cells because a slight but reproducible Sml1 depletion is observed in yku80Δ MEC1 cells compared to yku80Δ mec1Δ cells. In cells lacking both yKu and Mec1, the Rad9-, Rad53-, and Dun1-dependent checkpoint pathway is activated by Tel1 and the MRX complex independently of Rad24, Ddc1, Mec3, and Rad17, leading to Sml1 depletion. Thus, in the yku80Δ mutant, the Tel1/MRX and Mec1 pathways are responsible for the regulation of the Sml1 level. Consequently, in yku80Δ cells, the absence of Mec1 coupled with the absence of a member of the parallel pathway (Tel1/MRX) or of any one of the downstream checkpoints (Rad9, Rad53, or Dun1) is lethal for the cell, as it was in mec1Δ YKU cells. Because Rad53 phosphorylation remains undetectable or very weak in yku70Δ and yku70Δ mec1Δ mutants, we suggest that Rad53 is activated in a different way, as was previously observed by Clerici et al. (10).
One of the main problems that needs to be addressed is what type of DNA damage could be responsible for the depletion of Sml1 in ykuΔ and ykuΔ mec1Δ strains? It is well known that cells devoid of YKU70 or YKU80 exhibit a telomere length decline and present an excess of single-stranded G-rich DNA at their telomere. Such single-stranded overhangs are an important determinant for recognition as DNA damage (18, 19). The role of checkpoint genes in responding to telomeric defects in yku70Δ and yku80Δ cells have been tested at 37°C. Previous studies have demonstrated that the cell cycle arrest exhibited by yku70Δ cells at 37°C is dependent on the DNA damage checkpoint genes MEC1, CHK1, and RAD9 and on the spindle checkpoint gene MAD2 (30). Other studies have shown that, in yku80Δ cells, RAD24, RAD9, and RAD53 are associated with the checkpoint response (48). Our work suggests that, at 30°C, in yku70Δ and yk80Δ cells, the telomeric defects are not the major event responsible for the suppression of mec1Δ lethality. Consistent with such a suggestion, we show (i) that the suppression of mec1Δ lethality displayed by yku70Δ cells still occurred in an exo1Δ background that partially rescued the telomere defects of yku70Δ cells (5), (ii) that the lethality of yku80Δ tel1Δ mec1Δ and yku80Δ mre11Δ mec1Δ is not rescued by telomere lengthening induced by the deletion of the gene encoding the Rif2 protein, and (iii) that telomeric defective/repair-proficient yku80 mutants lost the capacity to suppress mec1Δ lethality. These results point strongly to the involvement of an NHEJ deficiency in the suppression of mec1Δ lethality. This is consistent with the observation that lif1Δ mutants, which are defective in NHEJ but are not affected at their telomeres, allow the suppression of mec1Δ lethality. For all these reasons, we favor the hypothesis that the absence of viability observed for yku70Δ tel1Δ mec1Δ, yku80Δ tel1Δ mec1Δ, yku70Δ mre11Δ mec1Δ, and yku80Δ mre11Δ mec1Δ cells is due to the deficiency of the Tel1/MRX checkpoint function of Tel1 and/or Mre11 rather than to the accumulation of telomeric defects. This is confirmed by the observation that sml1Δ partially rescues the lethality of yku80Δ tel1Δ mec1Δ and yku80Δ mre11Δ mec1Δ mutants despite their telomeric defects.
Mec1 is required for processing of potentially lethal lesions arising spontaneously during normal cellular life. The inability of mec1Δ cells to up-regulate dNTP synthesis is proposed to contribute to the mec1Δ lethality by inducing “replication stress,” that is, stalled forks that undergo irreversible collapse and/or are processed by recombination proteins into DSBs. In this study, we demonstrate that spontaneous DSBs arising in yku80Δ cells and, more generally, in NHEJ-deficient cells, activate the MRX-Tel1 pathway, leading to Sml1 depletion and subsequent elevation of the dNTP pool. We do not think that yKu proteins actively and directly regulate the Sml1 level in wild-type cells. The NHEJ-dependent depletion of Sml1 is a novel discovery. Since we also observed an Sml1 depletion in lif1Δ cells, we speculate that the failure of the NHEJ pathway is crucial for the depletion of Sml1, explaining why NHEJ mutants allow the suppression of mec1Δ lethality. This study unmasks for the first time a connection between the NHEJ pathway and the checkpoint response. The pathways regulating the checkpoint are conserved in organisms from yeasts to humans. The mechanisms that control the DNA damage checkpoints involve the activation of ATM (Tel1) and ATR (Mec1) kinases in mammalian cells. It was reported that Ku−/− cells have much stronger checkpoint responses than Ku+/+ cells (54, 55), suggesting that Ku proteins affect the checkpoint response in mammalian cells (54, 55). In agreement with this work, we also observed that the deletion of YKU increases the viability of some checkpoint mutants (Y. Corda and V. Géli, unpublished data). Because sml1Δ cells exhibit an increased viability after DNA damage, these observations may suggest that the ykuΔ-dependent increase of viability observed for checkpoint mutants after DNA damage is the consequence of the Sml1 depletion observed in ykuΔ cells. Interestingly, although no homology of Sml1 has yet been reported in mammalian cells, it has been shown that yeast Sml1 binds to the large subunit of mouse and human RNR and that the same Sml1 residues essential for the yeast RNR interaction and inhibition are also required for binding the human protein (58). These results suggest a conserved mechanism between yeasts and humans. If this is true, based on our data for yeast cells, interfering with KU gene expression could affect dNTP levels in human cells. Several lines of evidence suggest that the alteration of the dNTP pool is associated with spontaneous mutations and chromosome instability. Moreover, increased dNTP levels are often associated with resistance of tumor cells to drugs, and there is some evidence that down-regulation of the Ku system promotes progression of cancer from a mildly to highly aggressive malignant clinical behavior (24). In light of this, Ku and the various components of the NHEJ system may be appealing targets for cancer therapy.
We gratefully acknowledge R. Rothstein and M. Lisby for the YFP-Sml1 and Mre11-YFP strains for the pRS416-MEC1 construct and for helpful discussions and critical reading of the manuscript. We acknowledge S. P. Jackson for the yku80 mutants and for the Mre11-ProtA construct and M. Fasullo, M. Foiani, M. P. Longhese, D. Lydall, T. Petes, and S. H. Teo for plasmids pSM21, pCH3, pML54, pDL684, and pGP47, respectively. We thank Pierre Luciano and Pierre-Marie Dehe for helpful discussions and Martine Zalewski and Isabelle Varlet for technical assistance.
The work in the laboratory of V.G. was supported by the “Ligue Nationale Contre le Cancer” and the “Ministère de la Recherche.” Research in the J.E.H. lab has been supported by NIH grant GM61766, by DOE grant ER01-63229, and by the Sydney Kimmel Foundation for Cancer Research (to S.E.L.). A.W. was an NIH postdoctoral fellow.