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Molecular and Cellular Biology, December 2005, p. 10652-10664, Vol. 25, No. 23
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.23.10652-10664.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Inactivation of Ku-Mediated End Joining Suppresses mec1{Delta} Lethality by Depleting the Ribonucleotide Reductase Inhibitor Sml1 through a Pathway Controlled by Tel1 Kinase and the Mre11 Complex

Yves Corda,1 Sang Eun Lee,2 Sylvine Guillot,1 André Walther,3 Julie Sollier,1 Ayelet Arbel-Eden,3 James E. Haber,3 and Vincent Géli1*

Laboratoire d'Ingénierie des Systèmes Macromoléculaires, IBSM, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille, Cedex 20, France,1 Rosenstiel Center, Brandeis University, 415 South Street, Waltham, Massachusetts 02454-9110,3 Department of Molecular Medicine/IBT, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, Texas 782452

Received 16 May 2005/ Returned for modification 9 June 2005/ Accepted 18 August 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 mec1{Delta}, but not rad53{Delta}, lethality. We show that suppression of mec1{Delta} 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, mec1{Delta} lethality is also suppressed by lif1{Delta}, which like yku70{Delta} and yku80{Delta}, prevents nonhomologous end joining. The viability of yku70{Delta} mec1{Delta} and yku80{Delta} mec1{Delta} 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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 mec1{Delta} and rad53{Delta} 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 yku70{Delta} 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 mec1{Delta} and/or rad53{Delta} lethality. We report that YKU70 or YKU80 deletion suppress mec1{Delta}, but not rad53{Delta}, 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 yku70{Delta} and yku80{Delta}. Our results bring new insights into the way cells respond to DNA lesions in yku70{Delta} and yku80{Delta} cells and unmask for the first time a connection between the NHEJ pathway and the checkpoint response.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains and plasmids. The genotypes of the yeast strains used in this study are listed in Table 1. The mec1{Delta}::KAN, rad24{Delta}::KAN, rad17{Delta}::KAN, and rif2{Delta}::KAN null mutations were introduced as described previously (16). The mec1{Delta}::TRP1, lcd1{Delta}::TRP1, ddc1{Delta}::TRP1, mrc1{Delta}::TRP1, chk1{Delta}::TRP1, mad2{Delta}::TRP1, rad9{Delta}::TRP1, mre11{Delta}::TRP1, tel1{Delta}::TRP1, dun1{Delta}::TRP1, rnr3{Delta}::TRP1, lif1{Delta}::TRP1, and yku80{Delta}::TRP1 null mutations were obtained after PCR amplification of a disruption cassette from plasmid pF6a-TRP1. The exo1{Delta}::URA3 mutation was introduced using SphI-linearized pDL684 plasmid (from David Lydall). The mec3{Delta}::TRP1 mutation was introduced as described previously (11). The rad53-K227A mutation was introduced using EcoRI-linearized pCH3 plasmid (39). est2{Delta}::NAT, mec1{Delta}::NAT, rad53{Delta}::NAT, sml1{Delta}::NAT, and tel1{Delta}::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 rad52{Delta}::TRP1 cassette.


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  		TABLE 1. S. cerevisiae strains used in this studya

 
Protein extracts and Western blot analysis. 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.

RNA extraction and RT-PCR. 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.

Analysis of the YFP-Sml1 level. For G1 phase, cells were grown to early log phase and arrested by the addition of {alpha}-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 (10 mM) 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.

Analysis of telomere length. 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.

Micromanipulation. Cells were micromanipulated with the MSM system from Singer Instruments (26).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Strains initially defective in yKu70 rescue mec1{Delta}, but not rad53{Delta}, lethality. 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 yku70{Delta} diploid yeast strains. As expected, we were not able to obtain mec1{Delta} spores after 3 days of growth at 30°C when the spores were obtained from a mec1{Delta}/MEC1 YKU70/YKU70 strain or from the heterozygous mec1{Delta}/MEC1 yku70{Delta}/YKU70 diploid. Surprisingly, tetrads derived from the mec1{Delta}/MEC1 yku70{Delta}/yku70{Delta} diploid gave more than two colonies (Fig. 1A). One hundred tetrads were dissected. A total of 160/200 mec1{Delta} yku70{Delta} spores germinated to form colonies, whereas 166/200 yku70{Delta} cells grew. Subsequently, several viable mec1{Delta} yku70{Delta} spores were backcrossed to unrelated wild-type strains. The resulting diploids were sporulated and dissected. We never obtained viable mec1{Delta} or mec1{Delta} yku70{Delta} colonies (not shown). These results were reproducibly obtained using multiple independent disruptions. We concluded that mec1{Delta} yku70{Delta} spores have not acquired an inheritable suppressor and that the viability of mec1{Delta} yku70{Delta} segregants is entirely dependent on the genotype of the parental diploid. mec1{Delta} is viable only in mec1{Delta} yku70{Delta} spores that were generated from diploids homozygous for yku70{Delta}. 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 yku70{Delta} and mec1{Delta} yku70{Delta} 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 mec1{Delta} yku70{Delta} and MEC1 yku70{Delta} 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 mec1{Delta}/MEC1 yku70{Delta}/yku70{Delta} gave four big colonies with a size of 100 to 150 cells each. If the mec1{Delta} yku70{Delta} cells are separated into individual cells 3 days after germination, each mec1{Delta} cell divides at the same rate as yku70{Delta}. In each case, the viable mec1{Delta} yku70{Delta} colonies were sensitive to hydroxyurea and UV irradiation. We checked whether the HU sensitivity is different in mec1{Delta} compared to mec1{Delta} yku70{Delta} cells. Since mec1{Delta} 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, mec1{Delta} and mec1{Delta} yku70{Delta} cells presented a similar sensitivity (Fig. 1B). Taken together, these results indicate that both types of cells, yku70{Delta} and mec1{Delta} yku70{Delta}, are fully viable but only if the parental diploid strain was homozygous yku70{Delta}/yku70{Delta}.



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  		FIG. 1. Deletion of YKU70 suppresses mec1{Delta} lethality. (A) mec1{Delta} lethality is suppressed in cells generated from diploids homozygous for yku70{Delta}. The diploid strains mec1{Delta}/MEC1 YKU70/YKU70, mec1{Delta}/MEC1 yku70{Delta}/YKU70, and mec1{Delta}/MEC1 yku70{Delta}/yku70{Delta} were sporulated and dissected. Tetrads were displayed vertically on a YPD plate and incubated at 30°C for 3 days. Four representative tetrads are shown for each dissection. The arrows show mec1{Delta} spore clones. (B) HU sensitivity of mec1{Delta} yku70{Delta} cells. Haploid wild-type (wt) cells were transformed with the 2µm URA3-marked plasmid expressing RNR1 (pRS426-RNR1). Subsequently, mec1{Delta} or yku70{Delta} and mec1{Delta} gene deletions were created. Tenfold serial dilutions of fresh stationary-phase cultures were plated on SD-Ura and on YPD plates containing 20 mM, 5 mM, or 2 mM HU and subsequently incubated for 3 days at 30°C. (C) Effects of mec1{Delta} on viability, silencing, and telomere length of the yku70{Delta} cells. (Left) Cells of the indicated genotypes were streaked multiple times on YPD plates. Cells from the first, second, and third restreaks are shown. (Middle) Telomeric position effect was assayed by 10-fold serial dilution of the culture cells on 5-fluoroorotic acid (5-FOA) plates. (Right) Telomere lengths of mec1{Delta} yku70{Delta} strains. (D) Mec1 is not required for the viability of yku70{Delta} cells. Haploid wild-type cells were transformed with the 2µm URA3-marked plasmid expressing MEC1 (pRS426-MEC1). Subsequently, yku70{Delta}, mec1{Delta}, or yku70{Delta} and mec1{Delta} gene deletions were created. Tenfold serial dilutions of fresh stationary-phase cultures were plated on SD-Ura and 5-FOA plates and incubated for 3 days at 30°C.

 
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. 1C, mec1{Delta} did not confer senescence to a yku70{Delta} strain and mec1{Delta} yku70{Delta} cells exhibit telomeric defects similar to those observed in yku70{Delta} cells. The suppression of mec1{Delta} by YKU70 or YKU80 gene deletion was confirmed by our ability to disrupt MEC1 in yku70{Delta} or yku80{Delta} haploid strains and by the discovery that yku70{Delta} mec1{Delta} cells were able to lose a pMEC1 expression plasmid, whereas mec1{Delta} cells were not (Fig. 1D). Deletion of YKU70 or YKU80 did not suppress rad53{Delta} lethality (not shown).

Genetic dependence of the suppression of mec1{Delta} lethality by yku70{Delta}. 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 mec1{Delta} lethality by yku70{Delta}. We constructed diploid strains homozygous for yku70{Delta} and heterozygous for mec1{Delta} 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 2 indicate that the suppression of mec1{Delta} lethality by yku70{Delta} 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 mec1{Delta} lethality by yku70{Delta}. This result indicates that RAD9 has an essential function in rescuing mec1{Delta} lethality in yku70{Delta} cells.


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  		TABLE 2. Genetic dependence of mec1{Delta} lethality suppression by yku70{Delta}a

 
EXO1 and MAD2 are required to activate checkpoint pathways in yku70{Delta} mutants at 37°C (30). We found that neither exo1{Delta} nor mad2{Delta} mutations affect the ability of yku70{Delta} mec1{Delta} mutants to form colonies (Table 2). Similar results were observed using haploid yku80{Delta} for analyzing the genetic dependence of suppression of mec1{Delta} 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 rad52{Delta} have been isolated (32). We asked if Rad52 was required in the suppression of mec1{Delta} by inactivation of YKU70 and observed that yku70{Delta} is able to suppress the lethality of mec1{Delta} cells even in the absence of RAD52.

The TEL1/MRX checkpoint pathway is required for yku70{Delta} suppression of mec1{Delta} lethality. 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 mec1{Delta} lethality. To test this, tetrads derived from the tel1{Delta}/TEL1 mec1{Delta}/MEC1 yku70{Delta}/yku70{Delta} and mre11{Delta}/MRE11 mec1{Delta}/MEC1 yku70{Delta}/yku70{Delta} diploid strains were analyzed. We observed the appearance of mec1{Delta} yku70{Delta}, tel1{Delta} yku70{Delta}, and mre11{Delta} yku70{Delta} but not of mec1{Delta} tel1{Delta} yku70{Delta} and mec1{Delta} mre11{Delta} yku70{Delta} spores. Thus, in the absence of TEL1 or MRE11, mec1{Delta} yku70{Delta} strains are not able to grow. These results suggest that TEL1 and MRE11 have an essential function in rescuing mec1{Delta} lethality in yku70{Delta} cells.

One interpretation of our data is that the absence of mec1{Delta} yku70{Delta} tel1{Delta} and mec1{Delta} yku70{Delta} mre11{Delta} 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 yku70{Delta} or a yku80{Delta} 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 tel1{Delta}/TEL1 mec1{Delta}/MEC1 yku70{Delta}/yku70{Delta} and analyzed the spores derived from this diploid. We never obtained mec1{Delta} tel1{Delta} yku70{Delta} and rif2{Delta} mec1{Delta} tel1{Delta} yku70{Delta} spores (not shown). We were also unable to obtain mec1{Delta} transformants in rif2{Delta} tel1{Delta} yku80{Delta} cells, although telomeres of rif2{Delta} tel1{Delta} yku80{Delta} cells were significantly longer than those of tel1{Delta} yku80{Delta} cells and slightly greater than in yku80{Delta} cells (Fig. 2A). These results predict that a DNA damage pathway controlled by Tel1 and the Mre11 complex is activated in yku70{Delta} and yku80{Delta} cells.



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  		FIG. 2. Tel1 and Mre11 are required for the mec1{Delta} lethality suppression by yku70{Delta}. (A) Telomere length analysis for rif2{Delta} in wild-type (wt), yku80{Delta}, and yku80{Delta} tel1{Delta} cells. Lane M, ladder DNA serving as size standard. (B) Mre11 is phosphorylated in yku70{Delta} and mec1{Delta} yku70{Delta} cells. The indicated strains contain a plasmid allowing the expression of either wild-type Mre11-ProtA (+) or the vector (–) (14). Protein extracts of the indicated strains were analyzed by Western blotting for protein phosphorylation. Arrows indicate the position of the basal and phosphorylated (*) Mre11-ProtA bands. MW, molecular mass.

 
Because Mre11 is phosphorylated after DNA damage in a Tel1-dependent manner (14), we investigated whether Mre11 is phosphorylated in yku70{Delta} and yku70{Delta} mec1{Delta} cells at 30°C by using a Mre11-ProtA fusion. Figure 2B shows that Mre11-ProtA migrates as a single band in extracts obtained from wild-type cells and also in mre11{Delta} cells complemented with pMre11-ProtA, whereas a slower-migrating form of Mre11, corresponding to the phosphorylated form of Mre11, was detected in yku70{Delta} mre11{Delta} and yku70{Delta} mec1{Delta} mre11{Delta} cell extracts. These results revealed a Mec1-independent DNA damage-induced phosphorylation of Mre11 in yku70{Delta} cells.

dun1{Delta} prevents the suppression of mec1{Delta} lethality by yku70{Delta} and yku80{Delta}. 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 mec1{Delta} (35, 43); this suppression appears to reflect Dun1's role in repressing Sml1 activity, as suppression of mec1{Delta} by sml1{Delta} did not require the activity of Dun1 (59). However, other studies have shown that a viable mec1 mutation and dun1{Delta} are synthetically lethal (13). We tested whether yku70{Delta} or yku80{Delta} suppression of mec1{Delta} 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 mec1{Delta} transformants in haploid yku70{Delta} and yku80{Delta} mutants but not in yku70{Delta} dun1{Delta}, yku80{Delta} dun1{Delta}, yku70{Delta} rad53-K227A, or yku80{Delta} rad53-K227A double mutants (not shown). Thus, RAD53 and DUN1 are required to rescue mec1{Delta} lethality in yku70{Delta} and yku80{Delta} cells. However, we observed by a gel autophosphorylation assay and by a Western blot mobility shift assay that yku70{Delta} and mec1{Delta} yku70{Delta} mutants do not show a high level of Rad53 phosphorylation.

Loss of essential components in nonhomologous end joining suppress mec1{Delta} lethality. 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 mec1{Delta} 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 yku80{Delta} haploid strain. Cells were then examined for the suppression of mec1{Delta} lethality. We found that yku80{Delta} cells expressing the yku80-PF mutant protein, which are affected in telomeric silencing (42) and telomere size (Fig. 3A), were not able to suppress mec1{Delta} lethality. However, cells carrying the empty vector allowed the suppression of mec1{Delta} lethality (not shown). The inability of the yku80-PF mutant to suppress mec1{Delta} lethality in yku80{Delta} cells suggested that desilencing of the telomere and its size control is not sufficient to suppress mec1{Delta} lethality. Consistent with this interpretation, none of the viable segregants from 100 tetrads derived from sporulation of the diploid strain mec1{Delta}/MEC1 sir3{Delta}/sir3{Delta} was mec1{Delta} sir3{Delta}. Moreover, yku70{Delta} mec1{Delta} sir3{Delta} cells are viable (data not shown). We concluded that the suppression of mec1{Delta} lethality in a yku{Delta} strain is not due to the loss of telomeric position effect.



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  		FIG. 3. Suppression of the end-joining function of yKu suppresses mec1{Delta} lethality. (A) Telomere size analysis of the telomeric defective/repair-proficient yku80-PF437,438AA mutant. Telomere size was examined in strains of the indicated genotypes carrying either a pRS413-YKU80 plasmid or a pRS413 plasmid expressing the mutant allele yku80-PF437,438AA (42). Lane M, ladder DNA serving as size standard. wt, wild type. (B) lif1{Delta} suppresses mec1{Delta} lethality. The mec1{Delta}/MEC1 lif1{Delta}/lif1{Delta} diploid strain was dissected. The presence of mec1{Delta} lif1{Delta} spores is indicated by arrows.

 
Given that yKu70/yKu80 and Lif1 proteins are involved in a common NHEJ pathway, it was conceivable that lif1{Delta} would be able to suppress mec1{Delta} lethality. To test this possibility, we attempted to generate mec1{Delta} in lif1{Delta} spores, starting from mec1{Delta}/MEC1 lif1{Delta}/lif1{Delta}. As shown in Fig. 3B, we were able to obtain mec1{Delta} lif1{Delta} viable spores. In agreement with this result, we were also able to obtain mec1{Delta} transformants in lif1{Delta} haploid cells, and lif1{Delta} mec1{Delta} 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 mec1{Delta} lethality in yku70{Delta} and yku80{Delta} cells is probably associated with the loss of repair function.

TEL1 mRNA expression is slightly affected by YKU80 deletion. Because TEL1 overexpression can suppress both cell lethality and hypersensitivity to DNA-damaging agents of the mec1{Delta} 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, yku80{Delta}, or tel1{Delta} cells, and TEL1 mRNA levels were examined by RT-PCR (Fig. 4). Our data indicate that TEL1 is not overexpressed in yku80{Delta} mutants. However, a reproducible 1.5 enrichment of TEL1 mRNA was detected in yku80{Delta} cells.



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  		FIG. 4. TEL1 mRNA level is slightly increased in yku80{Delta} cells. Total RNA was extracted from wild-type (wt), yku80{Delta}, and tel1{Delta} cells and processed for RT-PCR with the appropriate primers to measure TEL1 and ACT1 mRNA levels. The signals specific for the TEL1 transcripts were normalized to that of the actin signal. The TEL1 transcript level was arbitrarily set at 1 in the wild-type cells, and RNA levels in mutant cells were determined accordingly. Bar graphs represent an analysis of the results from three experiments.

 
Sml1 is depleted in G1 and G2 in yku80{Delta}. 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 yku80{Delta} mutants (Fig. 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. 5A) and Western blotting (Fig. 5B). The amount of YFP-Sml1 was clearly reduced, about 60%, in the yku80{Delta} mutant in G1 and in G2. YFP-Sml1 remained undetectable in S phase. When we reintroduced a wild-type YKU80 in the yku80{Delta} mutant, we restored the level of YFP-Sml1. Next, we tested whether the decrease of Sml1 levels in G1, S, and G2 phases in yku80{Delta} depended on Mec1. Since mec1{Delta} 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 yku80{Delta} mec1{Delta} strains compared with mec1{Delta} cells (Fig. 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 mec1{Delta} lethality by yku80{Delta}.



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  		FIG. 5. Regulation of Sml1 protein amount in yku80{Delta} cells. Sml1 levels are reduced in yku80{Delta} cells. A chromosomally encoded YFP-SML1 was introduced in the indicated strains. Exponentially growing cells of the indicated strains were blocked in G1 with alpha factor (left), in S phase with hydroxyurea (center), or in G2 with nocodazole (right), and the Sml1 level was analyzed either by epifluorescence microscopy (A) or by Western blot analysis (B) with anti-GFP antibodies (after confirmation of the efficiency of the G1, S, and G2 arrests by microscopic analyses). The arrowheads indicate the positions of YFP-Sml1. In each case, a band cross-reacting with anti-GFP antibodies is used as a loading control and the amount of YFP-Sml1 was normalized to that of the wild-type YFP-Sml1 level. Each value corresponds to the average of the results from three independent experiments. (C) Haploid cells of the indicated genotypes were transformed with a 2µm plasmid expressing RNR1 (pRS426-RNR1), and the Sml1 protein amount was analyzed by Western blot analysis. Here again, in each case, a band cross-reacting with anti-GFP antibodies is used as a loading control and the amount of YFP-Sml1 was normalized to the wild-type YFP-Sml1 level. MW, size standard.

 
To correlate YFP-Sml1 levels with suppression of mec1{Delta} lethality, we analyzed YFP-Sml1 levels in G1 and G2 phases, in mec3{Delta}, rad24{Delta}, rad9{Delta}, mre11{Delta}, tel1{Delta}, rad53-K227A, and dun1{Delta} mutants in the presence or absence of YKU80 (data not shown). Western blot analyses indicate that Sml1 depletion, at least in yku80{Delta} 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 lif1{Delta} cells and a wild-type level of Sml1 in the telomeric defective/repair-proficient yku80-PF437,438AA mutant which does not suppress mec1{Delta} lethality (not shown). To confirm the importance of Sml1 depletion in the suppression of mec1{Delta} lethality by YKU80 deletion, we deleted YKU80 and MEC1 genes in a GAL-SML1 haploid strain (57). We found that GAL-SML1 ku80{Delta} mec1{Delta} 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 mec1{Delta} yku70{Delta} combined with rad9{Delta}, tel1{Delta}, or mre11{Delta} deletion, in the absence of SML1. We were able to obtain sml1{Delta} rad9{Delta} mec1{Delta} yku70{Delta} viable spores that grew as well as rad9{Delta} yku70{Delta} spores (Fig. 6A). Genetic analysis also showed that a sml1{Delta} mutation rescues the lethality of mre11{Delta} mec1{Delta} yku70{Delta} and tel1{Delta} mec1{Delta} yku70{Delta} 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 mec1{Delta} lethality in yku70{Delta} and yku80{Delta} cells. This suggests that YKU70 and YKU80 deletions contribute to Sml1 degradation via both Tel1/MRX- and Mec1-dependent pathways.



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  		FIG. 6. SML1 deletion suppresses mec1{Delta} lethality in different genetic contexts. SML1 deletion rescues mec1{Delta} rad9{Delta} yku70{Delta} (A), mec1{Delta} mre11{Delta} yku70{Delta} (B), and mec1{Delta} tel1{Delta} yku70{Delta} (C) lethality. (A, B, C) Tetrads from diploids homozygous for yku70{Delta} and heterozygous for mec1{Delta} (left) or mec1{Delta} sml1{Delta} (right) and either rad9{Delta} (A), mre11{Delta} (B), or tel1{Delta} (C) were dissected and analyzed for the presence of auxotrophic markers. Four tetrads are shown for each and are displayed vertically. Open squares indicate the yku70{Delta} single mutant. Diamonds indicate sml1{Delta} yku70{Delta} mutants. Circles indicate mec1{Delta} yku70{Delta} mutants. Hexagons indicate sml1{Delta} mec1{Delta} yku70{Delta} mutants. Squares indicate rad9{Delta} yku70{Delta} (A), mre11{Delta} yku70{Delta} (B), or tel1{Delta} yku70{Delta} (C) mutants. Crosses indicate rad9{Delta} sml1{Delta} yku70{Delta} (A), mre11{Delta} sml1{Delta} yku70{Delta} (B), or tel1{Delta} sml1{Delta} yku70{Delta} (C) mutants. Triangles indicate mec1{Delta} rad9{Delta} yku70{Delta} (A), mec1{Delta} mre11{Delta} yku70{Delta} (B), or mec1{Delta} tel1{Delta} yku70{Delta} (C) mutants. Hearts indicate mec1{Delta} rad9{Delta} sml1{Delta} yku70{Delta} (A), mec1{Delta} mre11{Delta} sml1{Delta} yku70{Delta} (B), or mec1{Delta} tel1{Delta} sml1{Delta} yku70{Delta} (C) mutants.

 
Spontaneous Mre11 foci are observed in yku80{Delta} cells. 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 yku80{Delta} 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 yku80{Delta} cells (Fig. 7). When a plasmid that encoded the telomeric defective/repair-proficient yku80-PF437,438AA (yku80-PF) mutant (42) was introduced into the yku80{Delta} haploid strain, we observed a significant reduction of Mre11 foci compared to yku80{Delta} cells (Fig. 7). These in vivo observations are consistent with the presence of DNA damage associated with the loss of repair function in yku80{Delta} cells, providing an explanation for the Sml1 depletion.



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  		FIG. 7. Mre11 foci are detected in yku80{Delta} cells. Mre11 foci were analyzed in asynchronously growing cells in the absence (–) or presence (+) of MMS (0.05%). Cells were analyzed by YFP fluorescence and phase microscopy. The numbers indicate the percentages of cells that contained Mre11 foci. At least 500 cells were analyzed for each strain. Upper panels, YFP fluorescence; lower panels, phase contrast. wt, wild type.

 
Finally, because DSB induction appears to be the key event in suppressing mec1{Delta} lethality in yku80{Delta} cells, we analyzed whether a pMEC1 expression plasmid can be cured from mec1{Delta} 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 mec1{Delta} 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.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Suppression of mec1{Delta} lethality and implications for checkpoint proteins. 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 mec1{Delta} is viable in spores that were generated from diploids homozygous, but not heterozygous, for yku70{Delta}. One explanation for these results is that yku70{Delta} mec1{Delta} spores derived from heterozygous yku70{Delta}/YKU70 diploids were still phenotypically YKU70+ and failed to reach the point where they are phenotypically yku70{Delta}. These observations are strongly supported by the finding that one can also obtain mec1{Delta} deletions by direct transformation of yku70{Delta} and yku80{Delta} cells. In contrast, deletion of YKU70 or YKU80 does not suppress rad53{Delta} lethality. This result is analogous to the previous observation that the sml1{Delta} mutation, which elevates dNTP levels, strongly suppresses mec1{Delta} lethality but only partially restores the growth of rad53{Delta} (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 mec1{Delta} 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 mec1{Delta} made it clear that Mec1 is not truly essential (15, 22, 59). We established here that yku70{Delta} and yku80{Delta} cells only slightly elevate RNR3 (not shown) and TEL1 gene expression. It seems that yku{Delta} can alleviate dNTP limitations by nontranscriptional means.

Relationship between Sml1 levels and suppression of mec1{Delta} lethality in yku80{Delta} cells. SML1 deletion suppresses mec1{Delta} and rad53{Delta} lethality. Sml1 is normally removed during S phase and after DNA damage to provide sufficient dNTPs (57, 59). Deletion of yku80{Delta} 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 yku80{Delta} mutant than in wild-type cells can explain the suppression of mec1{Delta} lethality and suggests that unrepaired DNA damage or stress signals remain in yku80{Delta} and yku80{Delta} mec1{Delta} cells. The analysis of Mre11 foci in yku80{Delta} cells demonstrates the presence of DSBs. We found that Sml1 depletion in the yku80{Delta} mutants is dependent on Rad9, Rad53, and Dun1, but not on other checkpoint proteins, confirming the implication of these proteins in the suppression of mec1{Delta} 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 yku{Delta}-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 yku80{Delta} cells because a slight but reproducible Sml1 depletion is observed in yku80{Delta} MEC1 cells compared to yku80{Delta} mec1{Delta} 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 yku80{Delta} mutant, the Tel1/MRX and Mec1 pathways are responsible for the regulation of the Sml1 level. Consequently, in yku80{Delta} 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 mec1{Delta} YKU cells. Because Rad53 phosphorylation remains undetectable or very weak in yku70{Delta} and yku70{Delta} mec1{Delta} mutants, we suggest that Rad53 is activated in a different way, as was previously observed by Clerici et al. (10).

Implication of the NHEJ repair pathway in the suppression of mec1{Delta} lethality in yku70{Delta} cells. 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 yku{Delta} and yku{Delta} mec1{Delta} 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 yku70{Delta} and yku80{Delta} cells have been tested at 37°C. Previous studies have demonstrated that the cell cycle arrest exhibited by yku70{Delta} 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 yku80{Delta} cells, RAD24, RAD9, and RAD53 are associated with the checkpoint response (48). Our work suggests that, at 30°C, in yku70{Delta} and yk80{Delta} cells, the telomeric defects are not the major event responsible for the suppression of mec1{Delta} lethality. Consistent with such a suggestion, we show (i) that the suppression of mec1{Delta} lethality displayed by yku70{Delta} cells still occurred in an exo1{Delta} background that partially rescued the telomere defects of yku70{Delta} cells (5), (ii) that the lethality of yku80{Delta} tel1{Delta} mec1{Delta} and yku80{Delta} mre11{Delta} mec1{Delta} 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 mec1{Delta} lethality. These results point strongly to the involvement of an NHEJ deficiency in the suppression of mec1{Delta} lethality. This is consistent with the observation that lif1{Delta} mutants, which are defective in NHEJ but are not affected at their telomeres, allow the suppression of mec1{Delta} lethality. For all these reasons, we favor the hypothesis that the absence of viability observed for yku70{Delta} tel1{Delta} mec1{Delta}, yku80{Delta} tel1{Delta} mec1{Delta}, yku70{Delta} mre11{Delta} mec1{Delta}, and yku80{Delta} mre11{Delta} mec1{Delta} 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 sml1{Delta} partially rescues the lethality of yku80{Delta} tel1{Delta} mec1{Delta} and yku80{Delta} mre11{Delta} mec1{Delta} mutants despite their telomeric defects.

Mec1 is required for processing of potentially lethal lesions arising spontaneously during normal cellular life. The inability of mec1{Delta} cells to up-regulate dNTP synthesis is proposed to contribute to the mec1{Delta} 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 yku80{Delta} 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 lif1{Delta} 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 mec1{Delta} 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 sml1{Delta} cells exhibit an increased viability after DNA damage, these observations may suggest that the yku{Delta}-dependent increase of viability observed for checkpoint mutants after DNA damage is the consequence of the Sml1 depletion observed in yku{Delta} 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.


   ACKNOWLEDGMENTS
 
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.


   FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire d'Ingénierie des Systèmes Macromoléculaires, IBSM, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille, Cedex 20, France. Phone: 33 4 91 16 41 01. Fax: 33 4 91 71 21 24. E-mail: geli{at}ibsm.cnrs-mrs.fr. Back


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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