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RESEARCH COMMUNICATION
1 Department of Biochemistry, University of California, San Francisco, California 94143, USA; 2 Department of Biopharmaceutical Sciences, University of California, San Francisco, California 94143, USA; 3 Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA; 4 California Institute for Quantitative Biomedical Research, University of California, San Francisco, California 94143, USA
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Abstract |
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[Keywords: DNA damage signaling; checkpoint; Clk-2; PI3 kinase-like kinase; 
-superhelical protein; telomere]]
Received December 21, 2007; revised version accepted February 11, 2008.
The phoshoinositide 3-kinase-like kinases (PIKKs) ATM and ATR control the response to DNA damage (Abraham 2001
). These proteins share structural homology and perform partially redundant roles. ATM (Tel1 in Saccharomyces cerevisiae) responds mainly to double-strand breaks (DSBs), whereas ATR (S. cerevisiae Mec1) is activated in response to ssDNA (Abraham 2001). ATR/Mec1 constitutively associates with ATRIP (Ddc2 in S. cerevisiae); the ATR–ATRIP complex binds to regions of exposed ssDNA via an interaction with the single-stranded binding protein RPA (Rouse and Jackson 2002; Zou and Elledge 2003). In contrast, ATM/Tel1 localizes to DSBs via an interaction with the Mre11–Rad50–Nbs1 complex (Mre11–Rad50–Xrs2 in S. cerevisiae, called MRX) (Nakada et al. 2003a; Falck et al. 2005; You et al. 2005).
Tel2 is an essential gene conserved throughout eukaryotes. Evidence from various organisms has implicated Tel2 in the response to DNA damage (Ahmed et al. 2001; Collis et al. 2007; Shikata et al. 2007; Takai et al. 2007). In Caenorhabditis elegans, two mutant alleles of TEL2, rad-5(mn159) and clk-2(qm37), each cause sensitivity to DNA damaging agents and defects in the DNA damage checkpoint and the S-phase replication checkpoint (Hartman and Herman 1982; Gartner et al. 2000; Ahmed et al. 2001). The radiation sensitivity of clk-2 mutants is enhanced by mutations in mrt-2 or hus-1, components of the 9–1–1 complex (Ahmed et al. 2001), suggesting that Clk-2 acts independently of this complex. Furthermore, wild-type Clk-2 is not required for recruitment of ATL-1, the C. elegans ortholog of ATR/Mec1, to stalled replication forks (Garcia-Muse and Boulton 2005). A study of human cells found a physical association between Tel2 and ATR, ATRIP, and Chk1, although ATR activation and recruitment to sites of damage were not significantly affected by Tel2 depletion (Collis et al. 2007). The Schizosaccharomyces pombe Tel2 ortholog is required for the response to replication stress (Shikata et al. 2007). Repression of tel2+ expression abrogated phosphorylation of Mrc1 and Cds1 (S. cerevisiae Rad53) after treatment with hydroxyurea (HU), indicating that Tel2 functions upstream of Mrc1 and Cds1 in the response to replication stress. However, the exact function of Tel2 has remained unknown.
Here, we report that S. cerevisiae Tel2 functions at a specific step in the ATM/Tel1 pathway in the response to DNA damage. Analyses of damage sensitivity, cell cycle progression after DNA damage, and phosphorylation of key proteins of the DNA damage signaling network together showed that Tel2 is an upstream component of the TEL1 signaling pathway. We demonstrate that Tel1 and Tel2 interact, and that the tel2-1 mutation completely disrupts the Tel1–Tel2 interaction and interferes with localization of Tel1 to an induced DSB in vivo. While loss of the Tel1–Tel2 interaction modestly decreases the total amount of Tel1 protein in cells, we demonstrate that the loss of Tel1 function caused by the tel2-1 mutation is not a result of lower protein levels of either Tel2 or Tel1. Computational analysis showed structural similarity of Tel2 to Ddc2 (ATRIP in vertebrates), a binding partner of Mec1 required for recruitment of Mec1 to sites of DNA damage. We show that like Ddc2, Tel2 interacts with an -superhelical region in a portion of Tel1 N-terminal to the kinase domain. These findings reveal that the interaction of -superhelical modules is general strategy used by the PIKKs to interact with their partner proteins.
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). In plate growth assays, the tel2-1 mutation alone did not confer damage sensitivity (Fig. 1A; Supplemental Fig. S1), but when combined with a deletion of MEC1, cells grew more slowly on normal medium and were more sensitive to DNA-damaging agents than either single mutant (Fig. 1A). This is reminiscent of the previously reported damage sensitivity of tel1
strains, which, similarly, is uncovered in a mec1 background (Fig. 1A; Morrow et al. 1995). In contrast, tel1 tel2-1 cells showed no damage sensitivity. Notably, the phenotypes of the double mutants mec1 tel2-1 and mec1 tel1, and the triple mutant mec1 tel1 tel2-1, were all indistinguishable from each other (Fig. 1A; Supplemental Fig. S1B). These data therefore provided evidence that TEL2 acts in the TEL1 pathway of DNA damage signaling.
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). The tel2-1 mutation alone caused a delay in Rad53 phosphorylation after treatment with DNA-damaging agents (Fig. 1B). This delay occurred when damage was inflicted in either G1 or S phase of the cell cycle, but not in G2/M (Supplemental Fig. S2A); there was a corresponding failure of tel2-1 cells to halt the cell cycle properly after DNA damage was inflicted in G1 or S, but not G2/M (Supplemental Fig. S2B–D). To abolish Rad53 phosphorylation, both MEC1 and TEL1 must be deleted. Strikingly, double mutant tel2-1 mec1 cells completely failed to phosphorylate Rad53 after DNA damage (Fig. 1B). In contrast, in tel1 tel2-1 cells, the phosphorylation of Rad53 after phleomycin treatment occurred to a similar extent, and at approximately the same rate, as in each of the single mutants. Hence, we conclude that tel2-1 disrupts the Tel1 pathway, rather than the Mec1 pathway, of DNA damage signaling.
We next examined the stage in the Tel1 DNA damage response signaling pathway at which the tel2-1 mutation exerted its effect. Two proteins, Mrc1 and Rad9, act in parallel pathways downstream from Mec1 and Tel1 to activate Rad53 (Fig. 1C; Alcasabas et al. 2001; Tanaka and Russell 2001). In tel2-1 cells, following DNA damage the phosphorylation of these two proteins was significantly delayed (Fig. 1D), demonstrating that Tel2 acts upstream of Rad9 and Mrc1.
Xrs2 is temporally one of the earliest proteins to localize to sites of DNA damage and is required for the TEL1-dependent response to DSBs (Wu et al. 2000; Zhao et al. 2000; Usui et al. 2001). Xrs2 is phosphorylated in a strictly TEL1-dependent manner after DNA damage and is a substrate for Tel1 in vitro (D’Amours and Jackson 2001; Usui et al. 2001; Mallory et al. 2003; Nakada et al. 2003b). We found that tel2-1 significantly delayed phosphorylation of Xrs2 after DNA damage (Fig. 1D). Taken together, all these results indicate that Tel2 acts as an upstream component of the TEL1 signaling pathway.
Mec1 has been shown to associate constitutively with Ddc2, which is required for the localization of Mec1 to sites of DNA damage (Rouse and Jackson 2002). TEL2 and DDC2 encode proteins of similar size and share 24% identity at the primary sequence level (Supplemental Fig. S3). To test for structural similarity between these proteins, we used sequence–structure threading. This computational method uses both primary sequence and predicted secondary structure to compare a given protein against a database of known protein folds (see Materials and Methods). Strikingly, for both Tel2 and Ddc2, the highest-scored protein fold prediction identified the same protein: importin-β (Supplemental Fig. S4A). These predictions were made with high confidence (P-value < 0.0001 for Ddc2 and P-value < 0.001 for Tel2). Importin-β belongs to the ARM repeat superfamily of protein folds, as defined by the SCOP classification of protein structures (Murzin et al. 1995). This includes the HEAT repeat, Armadillo repeat, and other protein families, all of which adopt -superhelical three-dimensional structures. -Superhelices are found in proteins of diverse function, but many share the property of interaction with other -superhelices.
Tel1 and Mec1 have been predicted previously to contain -superhelices in regions N-terminal to their kinase domains (Perry and Kleckner 2003). Our independent analysis of Tel1 and Mec1 corroborated these predictions (Supplemental Fig. S4B). Specifically, the N-terminal part of each protein was predicted to consist of two ARM repeat -superhelices separated by another -superhelix composed of tetratricopeptide (TP) repeats; the C-terminal segment was predicted to contain a domain structurally similar to the PIK catalytic domain (Supplemental Fig. S4B). Based on the predictions of helical regions in the structures of Mec1 and Tel1, and because Ddc2 is known to interact with the N-terminal -superhelical region of Mec1 (Wakayama et al. 2001), we predicted that Tel2 and Tel1 would also interact.
Indeed, epitope-tagged Tel2 and Tel1 expressed from their native promoters could be coimmunoprecipitated from whole-cell yeast extracts (Fig. 2A,B), and this interaction was mediated by the ARM/TP repeat region of Tel1 (Supplemental Figs. S5, S6). The interaction did not require the Tel1 kinase domain itself. The interaction detected by coimmunoprecipitation was unchanged by DNA damage (Fig. 2A,B). In contrast, Tel2-1 protein failed to coimmunoprecipitate with Tel1 (Fig. 2C), indicating that the tel2-1 point mutation disrupts the interaction of Tel2 with Tel1. The expression levels of Tel2 and the mutant Tel2-1 were identical (Figs. 2C [input lanes], 3B). TEL2 is essential, yet cells expressing tel2-1 as their only copy of the TEL2 gene are alive and grow apparently normally (Fig. 1A), indicating that the Tel2-1 protein is not globally misfolded or degraded.
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; Falck et al. 2005; You et al. 2005). However, this Tel1–Xrs2 interaction remained intact in tel2-1 cells (Fig. 2D). Furthermore, the interaction between Tel1 and Tel2 did not require XRS2 (Fig. 2E). Finally, no interaction between Tel2 and Xrs2 was detected by coimmunoprecipitation (data not shown). We conclude that the Tel1–Tel2 interaction does not involve Xrs2.
We found that the tel2-1 mutation abrogated recruitment of Tel1 to a DSB. First, we found that tel2-1 did not alter nuclear localization of Tel1 (Fig. 3C), so the effects of the tel2-1 mutation cannot be explained by a failure of Tel1 to reach its DNA damage targets in the nucleus. We then performed chromatin immunoprecipitation (ChIP) using strains in which a single DSB could be induced by growth in galactose (Fig. 4A). Flag-Tel1 was specifically enriched at the break, as reported previously (Nakada et al. 2003a; Lisby et al. 2004). In tel2-1 cells, enrichment of Flag-Tel1 at the break dropped from 
30-fold to 7.5-fold over the background signal from an untagged control strain (Fig. 4A). A recent study of Tel2 in mammalian cells reported that lack of Tel2 caused destabilization of ATM protein (Takai et al. 2007). The tel2-1 mutation, which we found disrupts the Tel1–Tel2 interaction, also led to somewhat lower Tel1 levels; however, in yeast, the drop in protein levels was less dramatic than reported in the mammalian situation, with Flag-Tel1 still being maintained at 60%–80% of its normal level (Fig. 3A). Loss of protein stability is commonly seen when a protein cannot interact with one of its usual partners; for example, we also observed lower Tel1 protein levels when XRS2 was deleted (Fig. 2E). We ruled out the possibility that reduced Tel1 protein levels might have accounted for the reduced association with the DSB. We expressed a second copy of Flag-Tel1 in tel2-1 cells from a plasmid, thereby boosting the Flag-Tel1 expression level to above the level found in a TEL2 strain (Fig. 4B). In these tel2-1 cells, Flag-Tel1 localization to a DSB was still defective (Fig. 4B) in spite of its overexpression. Hence, the tel2-1 mutation impairs Tel1 recruitment to a site of damage, and this impairment is not a result of lowered protein level of the PIKK Tel1.
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; Lisby et al. 2004; Falck et al. 2005; You et al. 2005). We found that Xrs2 localization to a DSB remained normal in tel2-1 cells (Fig. 4C). Hence, in tel2-1 cells, Tel1 remains competent to interact with Xrs2 (Fig. 2D) and Xrs2 binds normally to a DSB, yet Tel1 localization to a DSB is impaired.
We were unable to detect an interaction of Tel2 itself with a DNA break in vivo by ChIP or immunofluorescence in strains with an induced HO cut, or by immunofluorescence on meiotic chromosome spreads (data not shown). Therefore we propose that the Tel1–Tel2 complex substantially or completely dissociates prior to (or upon) binding of Tel1 to a DSB. This is consistent with experiments on purified human ATM, in which localization to dsDNA in vitro required only Mre11–Rad50–Nbs1 (Lee and Paull 2005). Our results now suggest the possibility that the lack of added Tel2 in those experiments may help explain why that study did not recapitulate certain aspects of ATM activation in vivo.
Our data show that the Tel1–Tel2 interaction regulates Tel1 localization to DSBs and its ability to activate its downstream targets. In an independent report on findings made during the same time as the research described in the present paper (Takai et al. 2007), it was suggested that the entire effect of mammalian Tel2 on the ATM pathway is due to destabilization of ATM protein. This decreased stability upon experimentally induced loss or mutation of Tel2 may reflect ATM’s or Tel1’s loss of normal interaction with its binding partner. While we find that loss of the yeast Tel1–Tel2 interaction leads to moderately lower Tel1 protein levels, our work clearly demonstrates that a lower Tel1 protein level cannot account for the effects of Tel2 on the Tel1 signaling pathway; rather, our results show that Tel2 is necessary for localization of Tel1 to a DSB even with full levels of Tel1 present.
The defects in tel2-1 cells were largely specific for the Tel1 pathway. Studies in S. pombe and mammalian cells have demonstrated a physical interaction between Tel2 and other PIKKs, a family that includes Tel1/ATM and Mec1/ATR (Collis et al. 2007; Hayashi et al. 2007; Takai et al. 2007). Our data do not rule out an interaction between Tel2 and Mec1 (or other PIKKs) in budding yeast. Indeed, we note that Rad53 phosphorylation after DNA damage was slightly delayed in tel1 tel2-1 cells compared with each single mutant, consistent with Tel2 having additional functions that do not lie upstream of Tel1. Such additional roles are also suggested by the fact that TEL2 is essential for viability, while TEL1 is not. However, the available data indicate that TEL2 is not essential due to an interaction with the essential MEC1 gene, because we found that sml1, which rescues the lethality of mec1, could not rescue the lethality of tel2 (data not shown). Therefore Tel2 is likely to have an essential function or functions unrelated to Mec1/Tel1-dependent DNA damage signaling. Studies in other organisms suggest these other functions may include a role in DNA replication or survival of replication stress (Garcia-Muse and Boulton 2005; Collis et al. 2007; Shikata et al. 2007).
Our work reveals that both Tel2 and Ddc2 are structurally similar to importin-β, adopting a common -superhelical fold. Like Ddc2, Tel2 interacts with the ARM repeat-containing N-terminal region of Tel1. Therefore, this mode of protein–protein interaction via interactions between -superhelical regions may represent a general strategy by which various classes of proteins (including the PIKKs) can interact with their binding partners.
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Materials and methods |
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Bacterial and yeast plasmids were obtained and constructed as described in the Supplemental Material.
Yeast strains
All strains were constructed in the S288C strain background and were isogenic with BY4736 (EHB13029) (Brachmann et al. 1998), as described in the Supplemental Material except as noted in Supplemental Table 1. The tel2-1 mutation was introduced into the S288C strain background by crossing AJL1291D (a gift from A. Lustig) to BY4736, then backcrossing to BY4736 at least five times. Rad53 was HA-tagged in wild-type and tel2-1 strains by integration of plasmid pEHB3212 (see the Supplemental Material). The MEC1, SML1, TEL1, and XRS2 ORFs were deleted by PCR-mediated gene disruption (Longtine et al. 1998). Tel2, Mrc1, Tel1, Rad9, and Xrs2 were tagged as described in the Supplemental Material. Haploid strains with an inducible DSB were made as described in the Supplemental Material.
Damage sensitivity
For analysis of damage sensitivity on plates, logarithmically growing cells were counted on a hemocytometer, and fivefold serial dilutions were prepared from a starting concentration of 4 x 106 cells per milliliter. After spotting on plates, some of the plates were irradiated at 254 nm in a Stratalinker (Stratagene) as indicated. Plates were photographed after incubation for 2 d at 30°C.
Cell cycle experiments
All yeast cultures were grown at 30°C with shaking at 225 rpm. The G1, S-phase, and G2/M checkpoints and responses were tested using -factor- or nocodazole-arrest protocols as described in the Supplemental Material. For flow cytometry, fixed cells were stained with SYBR Green I as described (Fortuna et al. 2001). Data were collected on a FACScalibur (BD Biosciences).
Computational methods
The alignment of Tel2 and Ddc2 (Supplemental Fig. S3) was obtained using the T-Coffee server (Notredame et al. 2000).
To structurally characterize Tel2, Ddc2, Tel1, and Mec1 (Supplemental Fig. S4), we determined their domain architectures and a three-dimensional fold of each domain using the PSIPRED protein structure prediction server (McGuffin et al. 2000). Specifically, the folds were assigned by threading the whole sequence through a library of all known folds, relying on similarities in sequence, predicted secondary structure, and statistical potentials for residue accessibilities and residue–residue distances as implemented in GenTHREADER (McGuffin and Jones 2003). For each predicted fold, a P-value was also calculated that estimates the probability of a false prediction, and a confidence level was assigned that relates to the P-value within a particular range. The current implementation of GenTHREADER requires that the target sequence is shorter than 1000 residues. When the target was longer, it was divided into segments of 1000 residues that overlapped by 300 residues, each of which was independently processed by PSIPRED. Next, the top-scoring nonoverlapping folds that maximized the coverage of the entire sequence were selected. The fold families were assigned using the structural classification of protein domains, SCOP (Murzin et al. 1995). All folds were predicted at the highest confidence level (CERTAIN, P-value < 0.0001), except for Tel2 and the first domain of Tel1, which were predicted at the second-highest level (HIGH, P-value < 0.001). For both Tel2 and Ddc2, the highest-scored protein fold prediction identified the same protein, importin-β (PDB ID: 2BKU, chain B). The two closest structures for the first ARM repeats of Tel1 and Mec1 were importin-β structures solved independently (PDB IDs: 2BKU, chain B and 1QGR, respectively). The closest known structures to the TP repeat domain (PDB ID: 1W3B) as well as to the second ARM repeat together with PI3K catalytic domain (PDB ID for both: 1E8X
[PDB]
) were the same for Tel1 and Mec1.
Western blotting
For detection of Rad53-HA and Flag-Tel1, protein was prepared by TCA precipitation. For detection of Mrc1-Myc, Xrs2-Myc, HA-Rad9, Tel2-Myc, and Tel2-HA, protein was prepared by bead-beating in urea buffer. Detailed protein preparation, electrophoresis, and blotting conditions are described in the Supplemental Material.
Coimmunoprecipitation
Whole-cell extracts were prepared by bead-beating in lysis buffer and were subjected to immunoprecipitation as described in the Supplemental Material.
Nuclear-cytoplasmic fractionation
Cells were disrupted by Dounce homogenization and fractionated by differential centrifugation. Details are given in the Supplemental Material.
ChIP
ChIP was performed essentially as described (Taggart et al. 2002), with differences as described in the Supplemental Material. DNA was detected by quantitative real-time PCR. Fold enrichment of the HO cut was calculated as [(IPHO)/(inputHO)], with the enrichment of the untagged control strain set to 1. Note that for the untagged and uncross-linked controls, only galactose-induced cultures were analyzed. As a reference, the same samples were also amplified using a different primer set (ARO). Signals from the ARO primers are shown in Supplemental Figure S7.
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E-MAIL elizabeth.blackburn{at}ucsf.edu; FAX (415) 514-2913.
Supplemental material is available at http://www.genesdev.org.
Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.1646208.
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