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The Saccharomyces cerevisiae Sae2 protein negatively regulates DNA damage checkpoint signalling

The Saccharomyces cerevisiae Sae2 protein negatively regulates DNA damage checkpoint signalling scientific report scientificreport The Saccharomyces cerevisiae Sae2 protein negatively regulates DNA damage checkpoint signalling Michela Clerici, Davide Mantiero, Giovanna Lucchini & Maria Pia Longhese Dipartimento di Biotecnologie e Bioscienze, Universita di Milano-Bicocca, Milan, Italy Double-strand breaks (DSBs) elicit a DNA damage response, DNA damage response (Baroni et al, 2004). Finally, the absence resulting in checkpoint-mediated cell-cycle delay and DNA of Sae2 prolongs checkpoint-mediated cell-cycle arrest after UV repair. The Saccharomyces cerevisiae Sae2 protein is known to irradiation (Baroni et al, 2004). This might be due to reduced act together with the MRX complex in meiotic DSB processing, as efficiency in repairing DNA lesions or may reflect a more specific well as in DNA damage response during the mitotic cell cycle. role of Sae2 in checkpoint switch off. Here, we report that cells lacking Sae2 fail to turn off both Mec1- By examining the effects of lack of or excess Sae2 on and Tel1-dependent checkpoints activated by a single irreparable checkpoint response to a single irreparable DSB, we now provide DSB, and delay Mre11 foci disassembly at DNA breaks, indicating evidence that Sae2 negatively regulates signalling to Mec1- that Sae2 may negatively regulate checkpoint signalling by and Tel1-dependent pathways, possibly by modulating MRX modulating MRX association at damaged DNA. Consistently, association to DSBs. high levels of Sae2 prevent checkpoint activation and impair MRX foci formation in response to unrepaired DSBs. Mec1- and Tel1- RESULTS AND DISCUSSION dependent Sae2 phosphorylation is necessary for these Sae2 Sae2 is required for DNA damage checkpoint switch off functions, suggesting that the two kinases, once activated, may Cells carrying a single irreparable DSB undergo checkpoint- regulate checkpoint switch off through Sae2-mediated inhibition mediated cell-cycle block, but then they adapt to the damage, of MRX signalling. decrease Rad53 checkpoint kinase activity and re-enter the cell Keywords: Sae2; checkpoint; MRX; Mec1; Tel1 cycle (Toczyski et al, 1997; Pellicioli et al, 2001). To test whether EMBO reports (2006) 7, 212–218. doi:10.1038/sj.embor.7400593 Sae2 has a role in turning off the checkpoint independently of DNA damage repair, we examined the checkpoint response to an unrepaired DSB in sae2D cells (Lee et al, 1998). A single DSB can INTRODUCTION be generated at the MAT locus of JKM139 derivative strains by Response to double-strand breaks (DSBs) in eukaryotes is expressing the site-specific HO endonuclease gene from a governed by DNA damage checkpoint signal-transduction path- galactose-inducible promoter, and it cannot be repaired by ways, the key players of which belong to a protein kinase family homologous recombination, because the homologous donor including the mammalian ataxia-telangiectasia-mutated (ATM) sequences HML or HMR are deleted (Lee et al, 1998). As shown and ATM- and Rad3-related protein (ATR) and their Saccharo- in Fig 1A, when G1-arrested cell cultures were spotted on myces cerevisiae orthologues Tel1 and Mec1 (Longhese et al, galactose-containing plates, most wild-type JKM139 cells over- 2003). An evolutionarily conserved protein complex, called MRX rode the checkpoint-mediated cell-cycle arrest in 24–32 h, (Mre11–Rad50–Xrs2) in budding yeast, mediates DSB association producing microcolonies with four or more cells, whereas most of both ATM/Tel1 and ATR/Mec1 (Nakada et al, 2003, 2004). isogenic sae2D cells were still arrested at the two-cell dumbbell The S. cerevisiae Sae2 protein is involved in meiotic and stage after 32 h. Moreover, when galactose was added to mitotic DSB processing and in subsets of recombination pathways exponentially growing cell cultures of the same strains, Rad53 together with the MRX complex (Keeney & Kleckner, 1995; phosphorylation, which is required for its activation as a kinase, Rattray et al, 2001; Lobachev et al, 2002; Neale et al, 2002; became detectable as electrophoretic mobility shift in both cell Clerici et al, 2005). Moreover, Sae2 undergoes Mec1- and Tel1- cultures about 2 h after HO induction (Fig 1B). Then, it persisted at dependent phosphorylation that is important for its functions in least for a further 30 h in sae2D cells that did not re-enter the cell cycle, whereas it decreased in wild-type cells after 12–15 h, when most cells resumed cell-cycle progression (Fig 1A,B; data not Dipartimento di Biotecnologie e Bioscienze, Universita` di Milano-Bicocca, shown). Thus, sae2D cells fail to override the DNA damage P.zza della Scienza 2, 20126 Milan, Italy Corresponding author. Tel: þ 39 02 64 48 34 25; Fax: þ 39 02 64 48 35 65; checkpoint response and to resume cell-cycle progression when E-mail: [email protected] DSB repair is prevented. As checkpoint inactivation in the presence of unrepaired DNA lesions occurs independently of Received 13 July 2005; revised 6 October 2005; accepted 3 November 2005; repair pathways (Toczyski et al, 1997), the role of Sae2 in published online 9 December 2005 212 EMBO reports VOL 7 | NO 2 | 2006 &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION Sae2 and checkpoint switch off M. Clerici et al scientificreport 2,5,6,8,9 A wt sae2Δ sae2 1 cell 80 Rad53 2 cells wt 4 cells sae2Δ >4 cells Rad53 2,5,6,8,9 04 8 24 32 04 8 24 32 0 4 8 24 32 Rad53 sae2 Time (h) Time (h) Time (h) wt Rad53 Rad53 wt sae2Δ Rad53 sae2Δ Rad53 2,5,6,8,9 Rad53 2,5,6,8,9 Rad53 sae2 sae2 012468 10 12 15 18 21 24 28 32 Hours after HO induction wt wt exo1Δ sae2Δ sae2Δ exo1Δ Rad53 r7 exo1Δ r6 Rad53 r5 r4 r3 sae2Δ r2 Rad53 sae2Δ exo1Δ r1 Rad53 Uncut sae2Δ tel1Δ HO-cut Rad53 Minutes after HO induction Minutes after HO induction Fig 1 Response to a single irreparable double-strand break in sae2D cells. (A) YEP þ raf G1-arrested cell cultures of wild-type (wt) JKM139 and 2,5,6,8,9 isogenic sae2D and sae2 strains were spotted on galactose-containing plates incubated at 30 1C (time zero). At the indicated time points, 200 cells for each strain were analysed to determine the frequency of single cells and of cells forming microcolonies of two, four or more than four cells. 2,5,6,8,9 (B) Galactose was added at time zero to wild-type JKM139 and isogenic sae2D and sae2 cell cultures exponentially growing in YEP þ raf. Protein extracts from aliquots withdrawn at the indicated times were analysed by western blot with anti-Rad53 antibodies. (C) Wild-type W303 and isogenic 2,5,6,8,9 sae2D and sae2 cell cultures arrested in G1 with a-factor (af) or in G2 with nocodazole (noc) were incubated for 15 min with methylmethane sulphonate (MMS, 0.02%) or bleomycin (bleo, 10 mU/ml), respectively, and then released in YEPD. Protein extracts from samples taken at the indicated times were analysed by western blot with anti-Rad53 antibodies. (D) YEP þ raf nocodazole-arrested cell cultures of wild-type JKM139 and isogenic exo1D, sae2D, sae2D exo1D and sae2D tel1D strains were transferred to YEP þ raf þ gal in the presence of nocodazole at time zero. Genomic DNA prepared from aliquots taken at the indicated times was digested with SspI and separated on alkaline agarose gel. Gel blots were hybridized with a single-stranded RNA probe specific for the MAT locus, which shows HO-cut and uncut fragments of 0.9 and 1.1 kb, respectively. As depicted in 0 0 supplementary Fig S2 online, 5 -to-3 resection progressively eliminates SspI sites located 1.7, 3.5, 4.7, 5.9, 6.5, 8.9 and 15.8 kb centromere-distal from the HO-cut site, producing larger SspI fragments (r1–r7) detected by the probe. The kinetics of resection product accumulation in sae2D tel1D cell cultures (not shown) was undistinguishable from that of sae2D cells. (E) Protein extracts from samples taken at the indicated times during the experiment in (D) were analysed by western blot with anti-Rad53 antibodies. switching off the checkpoint response is probably independent As single-stranded DNA (ssDNA) covered by RPA is one of the of DSB repair. signals activating the checkpoint in response to DSBs (Lee et al, The effects of Sae2 absence were not limited to the checkpoint 1998; Zou & Elledge, 2003), we also monitored the kinetics of triggered by an irreparable HO-induced DSB. In fact, Rad53 both HO-cut and 3 -ended ssDNA formation at the HO-cut site in phosphorylation persisted much longer in sae2D cells treated with galactose-induced cell cultures (Fig 1D) that were blocked in methylmethane sulphonate (MMS) or bleomycin than in wild-type mitosis with nocodazole, to avoid specific cell-cycle effects on cells (Fig 1C), and sae2D cells markedly delayed S-phase DSB processing. The sae2D JKM139 derivative strain showed completion, compared with wild-type cells, after MMS treatment some delay, compared with wild type, in the accumulation of (supplementary Fig S1 online). 3 -ended resection products (r1–r7 in Fig 1D; see supplementary &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 7 | NO 2 | 2006 213 Frequency (%) noc αf 105 bleo MMS 30 30 180 60 (min) (min) Sae2 and checkpoint switch off M. Clerici et al scientificreport Fig S2 online for details), which then remained stable throughout the (Fig 2D) did not alter the ability of the cell to arrest with 2C DNA experiment (Fig 1D), because the HO break could not be repaired content and phosphorylated Rad53 after HO induction (Fig 2E,F). (data not shown). As Rad53 seems to be phosphorylated with Thus, DNA-damage-activated Mec1 and Tel1 might themselves similar kinetics in both wild-type and sae2D cells, even if the latter trigger checkpoint switch off through Sae2 phosphorylation. delayed DSB resection (Fig 1D,E), we asked whether checkpoint activation in the absence of Sae2 could occur independently of Sae2 is required for timely Mre11 removal from DSBs 3 -ended ssDNA generation. Therefore, we further slowed down One of the first proteins recruited to DNA-damaged sites is the accumulation of resection products in sae2D cells by deleting the MRX complex (Lisby et al, 2004), which is also necessary to EXO1 gene, encoding an exonuclease that contributes to DSB trigger Rad53 phosphorylation in response to an HO-induced DSB resection independently of MRX (Llorente & Symington, 2004; even in the absence of Sae2 (Ira et al, 2004; Fig 3C). As Sae2 acts Nakada et al, 2004). As shown in Fig 1D,E, the appearance of together with MRX in meiotic and mitotic DSB resection (Keeney ssDNA intermediates at the HO-cut site was markedly delayed, & Kleckner, 1995; Neale et al, 2002; Clerici et al, 2005) and although not abolished, in nocodazole-arrested galactose-induced belongs to the MRX epistasis group with respect to HO-induced sae2D exo1D cells compared with that in sae2D and exo1D single DSB end resection (supplementary Fig S3 online), Sae2 may mutants, whereas the kinetics of Rad53 phosphorylation was regulate the checkpoint by modulating MRX signalling activity. similar in the single and double mutants, indicating that signals Consistent with this hypothesis, the hypomorphic rad50s mutant, other than ssDNA generation may activate the checkpoint in the which shows the same meiotic and mitotic recombination defects absence of Sae2. These checkpoint signals may be unresected as sae2D cells and which has been proposed to be impaired in DSBs, as Rad53 phosphorylation after HO induction was delayed Rad50–Sae2 interaction (Keeney & Kleckner, 1995; Rattray et al, in sae2D cells lacking the Tel1 kinase (Fig 1E), the activation of 2001; Clerici et al, 2005), showed persistent Rad53 phosphoryla- which does not require ssDNA generation (Usui et al, 2001). tion (Fig 3C) and failed to override the cell-cycle arrest after an If Sae2 negatively regulates the checkpoint, as suggested by HO-induced DSB (data not shown). the above data, excess Sae2 might be expected to counteract As Sae2 lack has been shown to prolong Mre11 foci persistence checkpoint activation. Indeed, when G1-arrested cultures of after g-irradiation or DSBs induced by the I-SceI endonuclease JKM139 derivative strains were spotted on galactose-containing (Lisby et al, 2004), we asked whether the effects caused by the plates, cells carrying a galactose-inducible GAL-SAE2 construct lack of Sae2 on checkpoint switch off correlated with alterations formed microcolonies with more than four cells much faster than in the pattern of Mre11 localization at HO-induced DSBs. To this wild-type cells and similar to mec1D cells (Fig 2A). Moreover, purpose, we analysed Mre11 localization after galactose addition when galactose was added to exponentially growing YEP þ raf cell in nocodazole-arrested wild-type and sae2D JKM139 derivative cultures, most wild-type cells were cell-cycle arrested with 2C strains expressing a fully functional MRE11-MYC-tagged allele DNA content and heavily phosphorylated Rad53 in 2–3 h after from the MRE11 promoter. As shown in Fig 3A,B, Mre11 galactose addition, whereas SAE2-overexpressing cells were localization detected by immunofluorescence was diffusely neither cell-cycle arrested (Fig 2E) nor underwent significant nucleoplasmic in all cell types growing in raffinose, as confirmed Rad53 phosphorylation (Fig 2B,F). This was not due to a failure to by nuclear 4,6-diamidino-2-phenylindole (DAPI) staining, generate 3 -ssDNA at the DSB ends, as accumulation of the whereas 85% of wild-type and sae2D nuclei contained one resection products in nocodazole-arrested galactose-induced Mre11 focus already 30 min after galactose addition. Then, the GAL-SAE2 cells was similar to that in wild-type cells (Fig 2C). Mre11 signal started to decrease in wild-type cells concomitantly A similar impairment of checkpoint activation was also with the appearance of fully phosphorylated Rad53 (Fig 3C), such observed when SAE2 was overexpressed in the presence of the that only 20% of them contained one Mre11 focus after 4 h. cdc5-ad allele, which prevents adaptation to a single unrepaired Conversely, Mre11 foci in sae2D cells were not only of higher DSB (Toczyski et al, 1997). In fact, galactose-induced GAL-SAE2 intensity than those in wild–type cells, but also mostly persisted, cdc5-ad JKM139 derivative cells progressed through cell cycle together with Rad53 phosphorylation, for at least 10 h after HO with unphosphorylated Rad53, whereas otherwise isogenic induction (Fig 3). cdc5-ad cells did not re-enter the cell cycle for at least 32 h MRX persistence at the HO-cut site in sae2D cells may after galactose addition (Fig 2A,B). constitutively signal to the checkpoint. If this was the case, SAE2 Therefore, Sae2 seems to negatively regulate the checkpoint overexpression, which inhibits checkpoint activation, might independently of the ability of the cell to repair the break or to impair Mre11 association to HO-induced DNA breaks. Strikingly, adapt to an unrepaired DSB. less than 40% of SAE2-overexpressing cells showed Mre11 foci Conversely, DNA-damage-induced Mec1- and Tel1-dependent 30 min after HO induction, and most foci disappeared in 4 h in Sae2 phosphorylation, besides being essential for other Sae2 these cells (Fig 3A,B). Consistent with defective Mre11 association functions (Baroni et al, 2004), is required for Sae2-mediated to HO-induced DSBs in Sae2-overproducing cells, 3 -ended checkpoint switch off. In fact, replacement of Sae2 in JKM139 ssDNA accumulated much more slowly in GAL-SAE2 exo1D cells 2,5,6,8,9 with the Sae2 variant, in which alanine substitutions of than in isogenic exo1D, sae2D or mre11D cells (compare Figs 1D serines and threonines in all its [S/T]Q motifs abolish Sae2 and 2C; supplementary Fig S3 online). Moreover, the amount phosphorylation without affecting protein level (Baroni et al, of 3 -ended ssDNA was reduced to the same extent in both 2004; Fig 2D), caused permanent arrest at the two-cell dumbbell GAL-SAE2 exo1D and sae2D exo1D cells compared with that stage and persistent Rad53 phosphorylation after HO induction in GAL-SAE2 cells (compare Figs 1D and 2C), indicating that and MMS or bleomycin treatment (Fig 1A–C). Moreover, unlike Exo1 is mainly responsible for DSB resection in galactose-induced 2,5,6,8,9 GAL-SAE2, GAL-sae2 induction in the JKM139 strain GAL-SAE2 cells. 214 EMBO reports VOL 7 | NO 2 | 2006 &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION Sae2 and checkpoint switch off M. Clerici et al scientificreport wt GAL-SAE2 mec1Δ cdc5-ad GAL-SAE2 cdc5-ad 1 cell 2 cells 4 cells > 4 cells 0 4 8 24 32 0 4 8 24 32 0 4 8 24 32 0 4 8 24 32 0 4 8 24 32 Time (h) Time (h) Time (h) Time (h) Time (h) Hours after HO induction 01 246 8 10 12 15 18 21 24 28 32 wt Rad53 Rad53 GAL-SAE2 cdc5-ad Rad53 Sae2 GAL-SAE2 Rad53 cdc5-ad 2,5,6,8,9 wt GAL-SAE2 GAL-sae2 wt GAL-SAE2 GAL-SAE2 exo1Δ r7 5 r6 3 r5 r4 r3 r2 wt Rad53 r1 GAL-SAE2 Rad53 Uncut 2,5,6,8,9 Rad53 HO-cut GAL-sae2 Hours after HO induction Minutes after HO induction Fig 2 Response to a single irreparable double-strand break in SAE2-overexpressing cells. (A) YEP þ raf G1-arrested cells of wild-type (wt) JKM139 and isogenic GAL-SAE2, cdc5-ad, GAL-SAE2 cdc5-ad and mec1D sml1D strains were spotted on galactose-containing plates incubated at 30 1C (time zero). At the indicated times, 200 cells for each strain were analysed as in Fig 1A. (B) Galactose was added at time zero to wild-type JKM139 and isogenic GAL-SAE2, cdc5-ad and GAL-SAE2 cdc5-ad cell cultures exponentially growing in YEP þ raf. Protein extracts from samples taken at the indicated times were subjected to western blot analysis with anti-Rad53 antibodies. (C) YEP þ raf nocodazole-arrested cell cultures of wild-type JKM139 and isogenic GAL-SAE2 and GAL-SAE2 exo1D strains were transferred to YEP þ raf þ gal in the presence of nocodazole at time zero. Genomic DNA from samples collected at the indicated times was analysed as described in Fig 1D. (D) Cell cultures of JKM139 derivative strains carrying either the SAE2-HA allele 2,5,6,8,9 at the SAE2 chromosomal locus or the GAL-SAE2-HA and GAL-sae2 -HA fusions at the URA3 locus were shifted to YEP þ raf þ gal for 1 h and protein extracts were analysed by western blot with anti-haemagglutinin antibodies. (E,F) Exponentially growing YEP þ raf cell cultures of wild-type 2,5,6,8,9 JKM139 and isogenic GAL-SAE2 and GAL-sae2 strains were transferred to YEP þ raf þ gal at time zero. Aliquots were taken at the indicated times for fluorescence-activated cell sorting analysis (E) or western analysis with anti-Rad53 antibodies (F). As MRX has a nuclease activity (Paull & Gellert, 1998) and Sae2 modulates Mec1- and Tel1-dependent checkpoints Mre11 foci persistence after g-rays in the nuclease-deficient The lack of Sae2 has been shown to trigger activation of a Tel1- mre11-D56N and mre11-H125N mutants is similar to that found dependent DNA damage checkpoint in mec1D cells (Usui et al, in sae2D cells (Lisby et al, 2004), the lack of Sae2 may freeze 2001). However, as the MRX complex is necessary for Mec1- Mre11 at sites of DNA damage by altering its nuclease activity. In dependent Rad53 phosphorylation in response to an HO-induced agreement with this hypothesis, we found that Rad53 phosphor- DSB (Ira et al, 2004) and for association of both Tel1 and Mec1 ylation persisted for at least 28 h after HO induction in to DSBs (Nakada et al, 2003, 2004), Sae2-mediated inhibition the nuclease-deficient mre11-D56N and mre11-H125N cells of MRX signalling activity should influence both Mec1- and (Fig 3C) that did not re-enter the cell cycle (data not shown), Tel1-dependent pathways. whereas it decreased in wild-type cells after 12–15 h (Fig 3C). To test this hypothesis, we used YMV80 derivative strains, in Thus, the lack of Mre11 nuclease activity might constitutively which a single HO cut can be generated on chromosome III by signal to the checkpoint by causing MRX persistence at expressing a galactose-inducible GAL-HO fusion (Vaze et al, HO-induced DSBs. 2002). As shown in Fig 4A,B, Rad53 phosphorylation was below &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 7 | NO 2 | 2006 215 Frequency (%) Hours after HO induction wt GAL-SAE2 2,5,6,8,9 GAL-sae2 Sae2 and checkpoint switch off M. Clerici et al scientificreport wt sae2Δ GAL-SAE2 0 h 1 h 4 h Mre11– MYC DAPI 8 h Hours after HO induction 0 1 2 4 6 8 10 12 15 18 21 24 28 Mre11– MYC DAPI Mre11– MYC DAPI Rad53 wt wt sae2Δ 2,5,6,8,9 sae2 GAL-SAE2 Rad53 sae2Δ 80 rad50s Rad53 Rad53 mre11-H125N Rad53 mre11-D56N mre11Δ Rad53 0 0.5 123468 10 Rad53 sae2Δ mre11Δ Hours after HO induction Fig 3 Mre11 localization at HO breaks in the absence or excess of Sae2. YEP þ raf nocodazole-arrested cell cultures of wild-type (wt) JKM139 and isogenic sae2D and GAL-SAE2 strains, all carrying an MRE11-MYC-tagged allele at the MRE11 locus, were transferred to YEP þ raf þ gal in the presence of nocodazole (time zero). Cell samples taken at the indicated times were processed for staining with 4,6-diamidino-2-phenylindole (DAPI) and anti-Myc antibody indirect immunofluorescence. (A) Representative fields were photographed at the indicated times. (B) Kinetics of Mre11 foci formation were determined by scoring 200 cells for each strain at each time points. (C) Wild-type JKM139 and isogenic sae2D, rad50s, mre11-H125N, mre11-D56N, mre11D and mre11D sae2D strains, exponentially growing in YEP þ raf, were transferred to YEP þ raf þ gal. Protein extracts from samples taken at the indicated times were analysed by western blot with anti-Rad53 antibodies. the detection level in mec1D cells throughout the experiment, Because of the poor Rad53 phosphorylation in response to an whereas it became detectable 2 h after galactose addition and HO-induced DSB in SAE2-overexpressing cells, we investigated the persisted until the end of the experiment in sae2D mec1D cells effects of high levels of Sae2 on Rad53 phosphorylation after UV (Fig 4B), although its amount was not sufficient to slow down irradiation of W303 derivative strains (Fig 4). Both GAL-SAE2 and cell-cycle progression (Fig 4A). Moreover, persistent Rad53 GAL-SAE2 tel1D cells released into cell cycle after UV irradiation phosphorylation was observed in both sae2D and sae2D tel1D in G2 divided nuclei much faster (Fig 4C) and phosphorylated cells, which remained arrested with 2C DNA content (Fig 4A) Rad53 much less efficiently (Fig 4D) than similarly treated tel1D and undivided nuclei (data not shown) for at least 12 h, whereas cells. Moreover, Rad53 phosphorylation after UV irradiation in G1 it started to decrease in wild-type and tel1D cells when they and release into cell cycle (Fig 4E) was reduced in GAL-SAE2 cells re-entered the cell cycle about 9 h after HO induction (Fig 4A,B). compared with that in wild-type cells, whereas it could be detected As DNA-damage-induced Rad53 phosphorylation is completely in mec1D cells 90 min after release and was below the detection abolished in sae2D mec1D tel1D cells (Usui et al, 2001; level in GAL-SAE2 mec1D cells. Thus, also SAE2 overexpression our unpublished observation), this indicates that the lack of affects both Mec1- and Tel1-dependent pathways. Sae2 impairs both Tel1 and Mec1 inactivation in the presence In agreement with the above findings, bleomycin-induced of a DSB. phosphorylation of the Mre11 and Ddc2 proteins, which specifically 216 EMBO reports VOL 7 | NO 2 | 2006 &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION Cells with Mre11 foci (%) Sae2 and checkpoint switch off M. Clerici et al scientificreport A C wt GAL-SAE2 tel1Δ tel1Δ GAL-SAE2 wt sae2Δ mec1Δ sae2Δ sae2Δ mec1Δ tel1Δ 0 30 60 90 120 150 Time after nocodazole release (min) Hours after HO induction D UV 01 23 4 5 6 7 8 910 11 12 exp noc 15 30 45 60 75 90 105 120 150 180 (min) Rad53 wt wt Rad53 Rad53 sae2Δ tel1Δ Rad53 Rad53 mec1Δ Rad53 GAL-SAE2 Rad53 sae2Δ mec1Δ GAL-SAE2 tel1Δ Rad53 Rad53 UV Rad53 sae2Δ tel1Δ exp αf 30 60 75 90 120 150 180 240 270 300 (min) Rad53 wt +bleo+noc +bleo+noc +bleo+noc noc 30 60 90 120 noc 30 60 90 120 noc 30 60 90 120 (min) Rad53 mec1Δ Ddc2 GAL-SAE2 Rad53 Mre11 GAL-SAE2 mec1Δ Rad53 wt sae2Δ GAL-SAE2 Fig 4 Mec1- and Tel1-dependent checkpoints in sae2D and SAE2-overexpressing cells. (A,B) Exponentially growing YEP þ raf cell cultures of wild-type YMV80 and isogenic sae2D, mec1D sml1D, sae2D mec1D sml1D, tel1D and sae2D tel1D strains were transferred to YEP þ raf þ gal to induce HO expression (time zero). Samples withdrawn at the indicated times were used for fluorescence-activated cell sorting analysis of DNA contents (A) and western blot analysis of protein extracts with anti-Rad53 antibodies (B). (C,D) Exponentially growing YEP þ raf cell cultures of wild-type W303 and isogenic tel1D, GAL-SAE2 and GAL-SAE2 tel1D strains were arrested with nocodazole in the presence of galactose for 2 h (noc), UV-irradiated (45 J/m ) and released into cell cycle in YEP þ raf þ gal. Samples were collected at the indicated times to determine the percentage of binucleate cells in unirradiated (open symbols) and UV-irradiated (closed symbols) cultures (C) and to monitor Rad53 phosphorylation from UV-irradiated cell cultures by western analysis (D). (E) Exponentially growing YEP þ raf cell cultures of wild-type W303 and isogenic mec1D sml1D, GAL-SAE2 and GAL-SAE2 mec1D sml1D strains were arrested in G1 with a factor in the presence of galactose for 2 h (af), UV-irradiated (30 J/m ) and released into cell cycle in YEP þ raf þ gal. Protein extracts prepared at the indicated times were analysed by western blot with anti-Rad53 antibodies. (F) YEP þ raf nocodazole-arrested cell cultures of wild-type W303 and isogenic sae2D and GAL-SAE2 strains, all expressing the DDC2-HA3- or MRE11-HA3-tagged alleles from the corresponding endogenous promoters, were transferred in YEP þ raf þ gal containing 10 mU/ml bleomycin and 15 mg/ml nocodazole ( þ bleo þ noc). Protein extracts prepared at the indicated times were subjected to western analysis with anti-haemagglutinin antibodies. depends on Tel1 and Mec1, respectively (Longhese et al, 2003), was METHODS more efficient in sae2D cells than in wild-type cells, whereas it was Yeast strains JKM139 (MATa hmlD hmrD ade1 lys5 leu2-3,112 below the detection level in SAE2-overexpressing cells (Fig 4F). trp1HhisG ura3-52 ho ade3HGAL-HO; Lee et al, 1998), YMV80 Altogether, our data indicate that Sae2 negatively regulates and W303 (MATa or MATa ade2-1 can1-100 his3-11,15 leu2- both Mec1- and Tel1-dependent checkpoint responses, possibly 3,112 trp1-1 ura3, rad5-535) were used to disrupt the SAE2 gene, 2,5,6,8,9 by modulating MRX association to damaged sites. As Mec1- and to replace the SAE2 chromosomal copy with the sae2 2,5,6,8,9 Tel1-dependent Sae2 phosphorylation is necessary for Sae2- allele, or to integrate the GAL-SAE2 and GAL-sae2 fusions mediated checkpoint switch off, it is tempting to speculate that at the URA3 locus. YMV80 is isogenic to YFP17 (MATD hmlD these kinases, once activated, may limit MRX-dependent check- hmrD ade1 lys5 ura3-52 trp1 ho ade3HGAL-HO leu2Hcs), point signalling by phosphorylating Sae2. except for the presence of a LEU2 fragment inserted 25 kb &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 7 | NO 2 | 2006 217 tel1Δ tel1Δ Hours after HO induction Binucleate cells (%) Sae2 and checkpoint switch off M. Clerici et al scientificreport Lisby M, Barlow JH, Burgess RC, Rothstein R (2004) Choreography of the centromere-distal to leu2Hcs (Vaze et al, 2002). Strains JKM139 DNA damage response: spatiotemporal relationships among checkpoint and YMV80 were kindly provided by J. Haber (Brandeis and repair proteins. Cell 118: 699–713 University, Waltham, MA, USA). Llorente B, Symington LS (2004) The Mre11 nuclease is not required for 5 to Supplementary information is available at EMBO reports online 3 resection at multiple HO-induced double-strand breaks. Mol Cell Biol 24: 9682–9694 (http://www.emboreports.org). Lobachev KS, Gordenin DA, Resnick MA (2002) The Mre11 complex is required for repair of hairpin-capped double-strand breaks and prevention of chromosome rearrangements. Cell 108: 183–193 ACKNOWLEDGEMENTS Longhese MP, Clerici M, Lucchini G (2003) The S-phase checkpoint and its We thank J. Haber, J. Diffley, N. Kleckner, L. Symington, D. Toczyski and regulation in Saccharomyces cerevisiae. Mutat Res 532: 41–58 K. Nasmyth for providing yeast strains and antibodies. This work was Nakada D, Matsumoto K, Sugimoto K (2003) ATM-related Tel1 associates supported by grants from Associazione Italiana Ricerca sul Cancro, with double-strand breaks through an Xrs2-dependent mechanism. Genes Dev 16: 1957–1962 Cofinanziamento Ministero dell’lstruzione, dell’Universita` e della Nakada D, Hirano Y, Sugimoto K (2004) Requirement of the Mre11 complex Ricerca/Universita` Milano-Bicocca and European Community’s Human and exonuclease 1 for activation of the Mec1 signaling pathway. Mol Potential Programme HPRN-CT-2002-00238 to M.P.L. and Fondo Cell Biol 24: 10016–10025 Investimenti Ricerca di Base to G.L. Neale MJ, Ramachandran M, Trelles-Sticken E, Scherthan H, Goldman AS (2002) Wild-type levels of Spo11-induced DSBs are required for normal single-strand resection during meiosis. Mol Cell 9: 835–846 0 0 REFERENCES Paull TT, Gellert M (1998) The 3 to 5 exonuclease activity of Mre11 Baroni E, Viscardi V, Cartagena-Lirola H, Lucchini G, Longhese MP (2004) facilitates repair of DNA double-strand breaks. Mol Cell 7: 969–979 The functions of budding yeast Sae2 in the DNA damage response Pellicioli A, Lee SE, Lucca C, Foiani M, Haber JE (2001) Regulation of require Mec1- and Tel1-dependent phosphorylation. Mol Cell Biol 24: Saccharomyces Rad53 checkpoint kinase during adaptation from DNA 4151–4165 damage-induced G2/M arrest. Mol Cell 7: 293–300 Clerici M, Mantiero D, Lucchini G, Longhese MP (2005) The Saccharomyces Rattray AJ, McGill CB, Shafer BK, Strathern JN (2001) Fidelity of mitotic cerevisiae Sae2 protein promotes resection and bridging of double-strand double-strand-break repair in Saccharomyces cerevisiae: a role for break ends. J Biol Chem 280: 38631–38638 SAE2/COM1. Genetics 158: 109–122 Ira G et al (2004) DNA end resection, homologous recombination and Toczyski DP, Galgoczy DJ, Hartwell LH (1997) CDC5 and CKII control DNA damage checkpoint activation require CDK1. Nature 431: adaptation to the yeast DNA damage checkpoint. Cell 90: 1097–1106 1011–1017 Usui T, Ogawa H, Petrini JHJ (2001) A DNA damage response pathway Keeney S, Kleckner N (1995) Covalent protein–DNA complexes at the 5 controlled by Tel1 and the Mre11 complex. Mol Cell 7: 1255–1266 strand termini of meiosis-specific double-strand breaks in yeast. Proc Natl Vaze MB, Pellicioli A, Lee SE, Ira G, Liberi G, Arbel-Eden A, Foiani M, Haber Acad Sci USA 92: 11274–11278 JE (2002) Recovery from checkpoint-mediated arrest after repair of a Lee SE, Moore JK, Holmes A, Umezu K, Kolodner RD, Haber JE (1998) double-strand break requires Srs2 helicase. Mol Cell 10: 373–385 Saccharomyces Ku70, Mre11/Rad50 and RPA proteins regulate Zou L, Elledge SJ (2003) Sensing DNA damage through ATRIP recognition of adaptation to G2/M arrest after DNA damage. Cell 94: 399–409 RPA–ssDNA complexes. Science 300: 1542–1548 218 EMBO reports VOL 7 | NO 2 | 2006 &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png EMBO Reports Springer Journals

The Saccharomyces cerevisiae Sae2 protein negatively regulates DNA damage checkpoint signalling

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Copyright © European Molecular Biology Organization 2006
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10.1038/sj.embor.7400593
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Abstract

scientific report scientificreport The Saccharomyces cerevisiae Sae2 protein negatively regulates DNA damage checkpoint signalling Michela Clerici, Davide Mantiero, Giovanna Lucchini & Maria Pia Longhese Dipartimento di Biotecnologie e Bioscienze, Universita di Milano-Bicocca, Milan, Italy Double-strand breaks (DSBs) elicit a DNA damage response, DNA damage response (Baroni et al, 2004). Finally, the absence resulting in checkpoint-mediated cell-cycle delay and DNA of Sae2 prolongs checkpoint-mediated cell-cycle arrest after UV repair. The Saccharomyces cerevisiae Sae2 protein is known to irradiation (Baroni et al, 2004). This might be due to reduced act together with the MRX complex in meiotic DSB processing, as efficiency in repairing DNA lesions or may reflect a more specific well as in DNA damage response during the mitotic cell cycle. role of Sae2 in checkpoint switch off. Here, we report that cells lacking Sae2 fail to turn off both Mec1- By examining the effects of lack of or excess Sae2 on and Tel1-dependent checkpoints activated by a single irreparable checkpoint response to a single irreparable DSB, we now provide DSB, and delay Mre11 foci disassembly at DNA breaks, indicating evidence that Sae2 negatively regulates signalling to Mec1- that Sae2 may negatively regulate checkpoint signalling by and Tel1-dependent pathways, possibly by modulating MRX modulating MRX association at damaged DNA. Consistently, association to DSBs. high levels of Sae2 prevent checkpoint activation and impair MRX foci formation in response to unrepaired DSBs. Mec1- and Tel1- RESULTS AND DISCUSSION dependent Sae2 phosphorylation is necessary for these Sae2 Sae2 is required for DNA damage checkpoint switch off functions, suggesting that the two kinases, once activated, may Cells carrying a single irreparable DSB undergo checkpoint- regulate checkpoint switch off through Sae2-mediated inhibition mediated cell-cycle block, but then they adapt to the damage, of MRX signalling. decrease Rad53 checkpoint kinase activity and re-enter the cell Keywords: Sae2; checkpoint; MRX; Mec1; Tel1 cycle (Toczyski et al, 1997; Pellicioli et al, 2001). To test whether EMBO reports (2006) 7, 212–218. doi:10.1038/sj.embor.7400593 Sae2 has a role in turning off the checkpoint independently of DNA damage repair, we examined the checkpoint response to an unrepaired DSB in sae2D cells (Lee et al, 1998). A single DSB can INTRODUCTION be generated at the MAT locus of JKM139 derivative strains by Response to double-strand breaks (DSBs) in eukaryotes is expressing the site-specific HO endonuclease gene from a governed by DNA damage checkpoint signal-transduction path- galactose-inducible promoter, and it cannot be repaired by ways, the key players of which belong to a protein kinase family homologous recombination, because the homologous donor including the mammalian ataxia-telangiectasia-mutated (ATM) sequences HML or HMR are deleted (Lee et al, 1998). As shown and ATM- and Rad3-related protein (ATR) and their Saccharo- in Fig 1A, when G1-arrested cell cultures were spotted on myces cerevisiae orthologues Tel1 and Mec1 (Longhese et al, galactose-containing plates, most wild-type JKM139 cells over- 2003). An evolutionarily conserved protein complex, called MRX rode the checkpoint-mediated cell-cycle arrest in 24–32 h, (Mre11–Rad50–Xrs2) in budding yeast, mediates DSB association producing microcolonies with four or more cells, whereas most of both ATM/Tel1 and ATR/Mec1 (Nakada et al, 2003, 2004). isogenic sae2D cells were still arrested at the two-cell dumbbell The S. cerevisiae Sae2 protein is involved in meiotic and stage after 32 h. Moreover, when galactose was added to mitotic DSB processing and in subsets of recombination pathways exponentially growing cell cultures of the same strains, Rad53 together with the MRX complex (Keeney & Kleckner, 1995; phosphorylation, which is required for its activation as a kinase, Rattray et al, 2001; Lobachev et al, 2002; Neale et al, 2002; became detectable as electrophoretic mobility shift in both cell Clerici et al, 2005). Moreover, Sae2 undergoes Mec1- and Tel1- cultures about 2 h after HO induction (Fig 1B). Then, it persisted at dependent phosphorylation that is important for its functions in least for a further 30 h in sae2D cells that did not re-enter the cell cycle, whereas it decreased in wild-type cells after 12–15 h, when most cells resumed cell-cycle progression (Fig 1A,B; data not Dipartimento di Biotecnologie e Bioscienze, Universita` di Milano-Bicocca, shown). Thus, sae2D cells fail to override the DNA damage P.zza della Scienza 2, 20126 Milan, Italy Corresponding author. Tel: þ 39 02 64 48 34 25; Fax: þ 39 02 64 48 35 65; checkpoint response and to resume cell-cycle progression when E-mail: [email protected] DSB repair is prevented. As checkpoint inactivation in the presence of unrepaired DNA lesions occurs independently of Received 13 July 2005; revised 6 October 2005; accepted 3 November 2005; repair pathways (Toczyski et al, 1997), the role of Sae2 in published online 9 December 2005 212 EMBO reports VOL 7 | NO 2 | 2006 &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION Sae2 and checkpoint switch off M. Clerici et al scientificreport 2,5,6,8,9 A wt sae2Δ sae2 1 cell 80 Rad53 2 cells wt 4 cells sae2Δ >4 cells Rad53 2,5,6,8,9 04 8 24 32 04 8 24 32 0 4 8 24 32 Rad53 sae2 Time (h) Time (h) Time (h) wt Rad53 Rad53 wt sae2Δ Rad53 sae2Δ Rad53 2,5,6,8,9 Rad53 2,5,6,8,9 Rad53 sae2 sae2 012468 10 12 15 18 21 24 28 32 Hours after HO induction wt wt exo1Δ sae2Δ sae2Δ exo1Δ Rad53 r7 exo1Δ r6 Rad53 r5 r4 r3 sae2Δ r2 Rad53 sae2Δ exo1Δ r1 Rad53 Uncut sae2Δ tel1Δ HO-cut Rad53 Minutes after HO induction Minutes after HO induction Fig 1 Response to a single irreparable double-strand break in sae2D cells. (A) YEP þ raf G1-arrested cell cultures of wild-type (wt) JKM139 and 2,5,6,8,9 isogenic sae2D and sae2 strains were spotted on galactose-containing plates incubated at 30 1C (time zero). At the indicated time points, 200 cells for each strain were analysed to determine the frequency of single cells and of cells forming microcolonies of two, four or more than four cells. 2,5,6,8,9 (B) Galactose was added at time zero to wild-type JKM139 and isogenic sae2D and sae2 cell cultures exponentially growing in YEP þ raf. Protein extracts from aliquots withdrawn at the indicated times were analysed by western blot with anti-Rad53 antibodies. (C) Wild-type W303 and isogenic 2,5,6,8,9 sae2D and sae2 cell cultures arrested in G1 with a-factor (af) or in G2 with nocodazole (noc) were incubated for 15 min with methylmethane sulphonate (MMS, 0.02%) or bleomycin (bleo, 10 mU/ml), respectively, and then released in YEPD. Protein extracts from samples taken at the indicated times were analysed by western blot with anti-Rad53 antibodies. (D) YEP þ raf nocodazole-arrested cell cultures of wild-type JKM139 and isogenic exo1D, sae2D, sae2D exo1D and sae2D tel1D strains were transferred to YEP þ raf þ gal in the presence of nocodazole at time zero. Genomic DNA prepared from aliquots taken at the indicated times was digested with SspI and separated on alkaline agarose gel. Gel blots were hybridized with a single-stranded RNA probe specific for the MAT locus, which shows HO-cut and uncut fragments of 0.9 and 1.1 kb, respectively. As depicted in 0 0 supplementary Fig S2 online, 5 -to-3 resection progressively eliminates SspI sites located 1.7, 3.5, 4.7, 5.9, 6.5, 8.9 and 15.8 kb centromere-distal from the HO-cut site, producing larger SspI fragments (r1–r7) detected by the probe. The kinetics of resection product accumulation in sae2D tel1D cell cultures (not shown) was undistinguishable from that of sae2D cells. (E) Protein extracts from samples taken at the indicated times during the experiment in (D) were analysed by western blot with anti-Rad53 antibodies. switching off the checkpoint response is probably independent As single-stranded DNA (ssDNA) covered by RPA is one of the of DSB repair. signals activating the checkpoint in response to DSBs (Lee et al, The effects of Sae2 absence were not limited to the checkpoint 1998; Zou & Elledge, 2003), we also monitored the kinetics of triggered by an irreparable HO-induced DSB. In fact, Rad53 both HO-cut and 3 -ended ssDNA formation at the HO-cut site in phosphorylation persisted much longer in sae2D cells treated with galactose-induced cell cultures (Fig 1D) that were blocked in methylmethane sulphonate (MMS) or bleomycin than in wild-type mitosis with nocodazole, to avoid specific cell-cycle effects on cells (Fig 1C), and sae2D cells markedly delayed S-phase DSB processing. The sae2D JKM139 derivative strain showed completion, compared with wild-type cells, after MMS treatment some delay, compared with wild type, in the accumulation of (supplementary Fig S1 online). 3 -ended resection products (r1–r7 in Fig 1D; see supplementary &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 7 | NO 2 | 2006 213 Frequency (%) noc αf 105 bleo MMS 30 30 180 60 (min) (min) Sae2 and checkpoint switch off M. Clerici et al scientificreport Fig S2 online for details), which then remained stable throughout the (Fig 2D) did not alter the ability of the cell to arrest with 2C DNA experiment (Fig 1D), because the HO break could not be repaired content and phosphorylated Rad53 after HO induction (Fig 2E,F). (data not shown). As Rad53 seems to be phosphorylated with Thus, DNA-damage-activated Mec1 and Tel1 might themselves similar kinetics in both wild-type and sae2D cells, even if the latter trigger checkpoint switch off through Sae2 phosphorylation. delayed DSB resection (Fig 1D,E), we asked whether checkpoint activation in the absence of Sae2 could occur independently of Sae2 is required for timely Mre11 removal from DSBs 3 -ended ssDNA generation. Therefore, we further slowed down One of the first proteins recruited to DNA-damaged sites is the accumulation of resection products in sae2D cells by deleting the MRX complex (Lisby et al, 2004), which is also necessary to EXO1 gene, encoding an exonuclease that contributes to DSB trigger Rad53 phosphorylation in response to an HO-induced DSB resection independently of MRX (Llorente & Symington, 2004; even in the absence of Sae2 (Ira et al, 2004; Fig 3C). As Sae2 acts Nakada et al, 2004). As shown in Fig 1D,E, the appearance of together with MRX in meiotic and mitotic DSB resection (Keeney ssDNA intermediates at the HO-cut site was markedly delayed, & Kleckner, 1995; Neale et al, 2002; Clerici et al, 2005) and although not abolished, in nocodazole-arrested galactose-induced belongs to the MRX epistasis group with respect to HO-induced sae2D exo1D cells compared with that in sae2D and exo1D single DSB end resection (supplementary Fig S3 online), Sae2 may mutants, whereas the kinetics of Rad53 phosphorylation was regulate the checkpoint by modulating MRX signalling activity. similar in the single and double mutants, indicating that signals Consistent with this hypothesis, the hypomorphic rad50s mutant, other than ssDNA generation may activate the checkpoint in the which shows the same meiotic and mitotic recombination defects absence of Sae2. These checkpoint signals may be unresected as sae2D cells and which has been proposed to be impaired in DSBs, as Rad53 phosphorylation after HO induction was delayed Rad50–Sae2 interaction (Keeney & Kleckner, 1995; Rattray et al, in sae2D cells lacking the Tel1 kinase (Fig 1E), the activation of 2001; Clerici et al, 2005), showed persistent Rad53 phosphoryla- which does not require ssDNA generation (Usui et al, 2001). tion (Fig 3C) and failed to override the cell-cycle arrest after an If Sae2 negatively regulates the checkpoint, as suggested by HO-induced DSB (data not shown). the above data, excess Sae2 might be expected to counteract As Sae2 lack has been shown to prolong Mre11 foci persistence checkpoint activation. Indeed, when G1-arrested cultures of after g-irradiation or DSBs induced by the I-SceI endonuclease JKM139 derivative strains were spotted on galactose-containing (Lisby et al, 2004), we asked whether the effects caused by the plates, cells carrying a galactose-inducible GAL-SAE2 construct lack of Sae2 on checkpoint switch off correlated with alterations formed microcolonies with more than four cells much faster than in the pattern of Mre11 localization at HO-induced DSBs. To this wild-type cells and similar to mec1D cells (Fig 2A). Moreover, purpose, we analysed Mre11 localization after galactose addition when galactose was added to exponentially growing YEP þ raf cell in nocodazole-arrested wild-type and sae2D JKM139 derivative cultures, most wild-type cells were cell-cycle arrested with 2C strains expressing a fully functional MRE11-MYC-tagged allele DNA content and heavily phosphorylated Rad53 in 2–3 h after from the MRE11 promoter. As shown in Fig 3A,B, Mre11 galactose addition, whereas SAE2-overexpressing cells were localization detected by immunofluorescence was diffusely neither cell-cycle arrested (Fig 2E) nor underwent significant nucleoplasmic in all cell types growing in raffinose, as confirmed Rad53 phosphorylation (Fig 2B,F). This was not due to a failure to by nuclear 4,6-diamidino-2-phenylindole (DAPI) staining, generate 3 -ssDNA at the DSB ends, as accumulation of the whereas 85% of wild-type and sae2D nuclei contained one resection products in nocodazole-arrested galactose-induced Mre11 focus already 30 min after galactose addition. Then, the GAL-SAE2 cells was similar to that in wild-type cells (Fig 2C). Mre11 signal started to decrease in wild-type cells concomitantly A similar impairment of checkpoint activation was also with the appearance of fully phosphorylated Rad53 (Fig 3C), such observed when SAE2 was overexpressed in the presence of the that only 20% of them contained one Mre11 focus after 4 h. cdc5-ad allele, which prevents adaptation to a single unrepaired Conversely, Mre11 foci in sae2D cells were not only of higher DSB (Toczyski et al, 1997). In fact, galactose-induced GAL-SAE2 intensity than those in wild–type cells, but also mostly persisted, cdc5-ad JKM139 derivative cells progressed through cell cycle together with Rad53 phosphorylation, for at least 10 h after HO with unphosphorylated Rad53, whereas otherwise isogenic induction (Fig 3). cdc5-ad cells did not re-enter the cell cycle for at least 32 h MRX persistence at the HO-cut site in sae2D cells may after galactose addition (Fig 2A,B). constitutively signal to the checkpoint. If this was the case, SAE2 Therefore, Sae2 seems to negatively regulate the checkpoint overexpression, which inhibits checkpoint activation, might independently of the ability of the cell to repair the break or to impair Mre11 association to HO-induced DNA breaks. Strikingly, adapt to an unrepaired DSB. less than 40% of SAE2-overexpressing cells showed Mre11 foci Conversely, DNA-damage-induced Mec1- and Tel1-dependent 30 min after HO induction, and most foci disappeared in 4 h in Sae2 phosphorylation, besides being essential for other Sae2 these cells (Fig 3A,B). Consistent with defective Mre11 association functions (Baroni et al, 2004), is required for Sae2-mediated to HO-induced DSBs in Sae2-overproducing cells, 3 -ended checkpoint switch off. In fact, replacement of Sae2 in JKM139 ssDNA accumulated much more slowly in GAL-SAE2 exo1D cells 2,5,6,8,9 with the Sae2 variant, in which alanine substitutions of than in isogenic exo1D, sae2D or mre11D cells (compare Figs 1D serines and threonines in all its [S/T]Q motifs abolish Sae2 and 2C; supplementary Fig S3 online). Moreover, the amount phosphorylation without affecting protein level (Baroni et al, of 3 -ended ssDNA was reduced to the same extent in both 2004; Fig 2D), caused permanent arrest at the two-cell dumbbell GAL-SAE2 exo1D and sae2D exo1D cells compared with that stage and persistent Rad53 phosphorylation after HO induction in GAL-SAE2 cells (compare Figs 1D and 2C), indicating that and MMS or bleomycin treatment (Fig 1A–C). Moreover, unlike Exo1 is mainly responsible for DSB resection in galactose-induced 2,5,6,8,9 GAL-SAE2, GAL-sae2 induction in the JKM139 strain GAL-SAE2 cells. 214 EMBO reports VOL 7 | NO 2 | 2006 &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION Sae2 and checkpoint switch off M. Clerici et al scientificreport wt GAL-SAE2 mec1Δ cdc5-ad GAL-SAE2 cdc5-ad 1 cell 2 cells 4 cells > 4 cells 0 4 8 24 32 0 4 8 24 32 0 4 8 24 32 0 4 8 24 32 0 4 8 24 32 Time (h) Time (h) Time (h) Time (h) Time (h) Hours after HO induction 01 246 8 10 12 15 18 21 24 28 32 wt Rad53 Rad53 GAL-SAE2 cdc5-ad Rad53 Sae2 GAL-SAE2 Rad53 cdc5-ad 2,5,6,8,9 wt GAL-SAE2 GAL-sae2 wt GAL-SAE2 GAL-SAE2 exo1Δ r7 5 r6 3 r5 r4 r3 r2 wt Rad53 r1 GAL-SAE2 Rad53 Uncut 2,5,6,8,9 Rad53 HO-cut GAL-sae2 Hours after HO induction Minutes after HO induction Fig 2 Response to a single irreparable double-strand break in SAE2-overexpressing cells. (A) YEP þ raf G1-arrested cells of wild-type (wt) JKM139 and isogenic GAL-SAE2, cdc5-ad, GAL-SAE2 cdc5-ad and mec1D sml1D strains were spotted on galactose-containing plates incubated at 30 1C (time zero). At the indicated times, 200 cells for each strain were analysed as in Fig 1A. (B) Galactose was added at time zero to wild-type JKM139 and isogenic GAL-SAE2, cdc5-ad and GAL-SAE2 cdc5-ad cell cultures exponentially growing in YEP þ raf. Protein extracts from samples taken at the indicated times were subjected to western blot analysis with anti-Rad53 antibodies. (C) YEP þ raf nocodazole-arrested cell cultures of wild-type JKM139 and isogenic GAL-SAE2 and GAL-SAE2 exo1D strains were transferred to YEP þ raf þ gal in the presence of nocodazole at time zero. Genomic DNA from samples collected at the indicated times was analysed as described in Fig 1D. (D) Cell cultures of JKM139 derivative strains carrying either the SAE2-HA allele 2,5,6,8,9 at the SAE2 chromosomal locus or the GAL-SAE2-HA and GAL-sae2 -HA fusions at the URA3 locus were shifted to YEP þ raf þ gal for 1 h and protein extracts were analysed by western blot with anti-haemagglutinin antibodies. (E,F) Exponentially growing YEP þ raf cell cultures of wild-type 2,5,6,8,9 JKM139 and isogenic GAL-SAE2 and GAL-sae2 strains were transferred to YEP þ raf þ gal at time zero. Aliquots were taken at the indicated times for fluorescence-activated cell sorting analysis (E) or western analysis with anti-Rad53 antibodies (F). As MRX has a nuclease activity (Paull & Gellert, 1998) and Sae2 modulates Mec1- and Tel1-dependent checkpoints Mre11 foci persistence after g-rays in the nuclease-deficient The lack of Sae2 has been shown to trigger activation of a Tel1- mre11-D56N and mre11-H125N mutants is similar to that found dependent DNA damage checkpoint in mec1D cells (Usui et al, in sae2D cells (Lisby et al, 2004), the lack of Sae2 may freeze 2001). However, as the MRX complex is necessary for Mec1- Mre11 at sites of DNA damage by altering its nuclease activity. In dependent Rad53 phosphorylation in response to an HO-induced agreement with this hypothesis, we found that Rad53 phosphor- DSB (Ira et al, 2004) and for association of both Tel1 and Mec1 ylation persisted for at least 28 h after HO induction in to DSBs (Nakada et al, 2003, 2004), Sae2-mediated inhibition the nuclease-deficient mre11-D56N and mre11-H125N cells of MRX signalling activity should influence both Mec1- and (Fig 3C) that did not re-enter the cell cycle (data not shown), Tel1-dependent pathways. whereas it decreased in wild-type cells after 12–15 h (Fig 3C). To test this hypothesis, we used YMV80 derivative strains, in Thus, the lack of Mre11 nuclease activity might constitutively which a single HO cut can be generated on chromosome III by signal to the checkpoint by causing MRX persistence at expressing a galactose-inducible GAL-HO fusion (Vaze et al, HO-induced DSBs. 2002). As shown in Fig 4A,B, Rad53 phosphorylation was below &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 7 | NO 2 | 2006 215 Frequency (%) Hours after HO induction wt GAL-SAE2 2,5,6,8,9 GAL-sae2 Sae2 and checkpoint switch off M. Clerici et al scientificreport wt sae2Δ GAL-SAE2 0 h 1 h 4 h Mre11– MYC DAPI 8 h Hours after HO induction 0 1 2 4 6 8 10 12 15 18 21 24 28 Mre11– MYC DAPI Mre11– MYC DAPI Rad53 wt wt sae2Δ 2,5,6,8,9 sae2 GAL-SAE2 Rad53 sae2Δ 80 rad50s Rad53 Rad53 mre11-H125N Rad53 mre11-D56N mre11Δ Rad53 0 0.5 123468 10 Rad53 sae2Δ mre11Δ Hours after HO induction Fig 3 Mre11 localization at HO breaks in the absence or excess of Sae2. YEP þ raf nocodazole-arrested cell cultures of wild-type (wt) JKM139 and isogenic sae2D and GAL-SAE2 strains, all carrying an MRE11-MYC-tagged allele at the MRE11 locus, were transferred to YEP þ raf þ gal in the presence of nocodazole (time zero). Cell samples taken at the indicated times were processed for staining with 4,6-diamidino-2-phenylindole (DAPI) and anti-Myc antibody indirect immunofluorescence. (A) Representative fields were photographed at the indicated times. (B) Kinetics of Mre11 foci formation were determined by scoring 200 cells for each strain at each time points. (C) Wild-type JKM139 and isogenic sae2D, rad50s, mre11-H125N, mre11-D56N, mre11D and mre11D sae2D strains, exponentially growing in YEP þ raf, were transferred to YEP þ raf þ gal. Protein extracts from samples taken at the indicated times were analysed by western blot with anti-Rad53 antibodies. the detection level in mec1D cells throughout the experiment, Because of the poor Rad53 phosphorylation in response to an whereas it became detectable 2 h after galactose addition and HO-induced DSB in SAE2-overexpressing cells, we investigated the persisted until the end of the experiment in sae2D mec1D cells effects of high levels of Sae2 on Rad53 phosphorylation after UV (Fig 4B), although its amount was not sufficient to slow down irradiation of W303 derivative strains (Fig 4). Both GAL-SAE2 and cell-cycle progression (Fig 4A). Moreover, persistent Rad53 GAL-SAE2 tel1D cells released into cell cycle after UV irradiation phosphorylation was observed in both sae2D and sae2D tel1D in G2 divided nuclei much faster (Fig 4C) and phosphorylated cells, which remained arrested with 2C DNA content (Fig 4A) Rad53 much less efficiently (Fig 4D) than similarly treated tel1D and undivided nuclei (data not shown) for at least 12 h, whereas cells. Moreover, Rad53 phosphorylation after UV irradiation in G1 it started to decrease in wild-type and tel1D cells when they and release into cell cycle (Fig 4E) was reduced in GAL-SAE2 cells re-entered the cell cycle about 9 h after HO induction (Fig 4A,B). compared with that in wild-type cells, whereas it could be detected As DNA-damage-induced Rad53 phosphorylation is completely in mec1D cells 90 min after release and was below the detection abolished in sae2D mec1D tel1D cells (Usui et al, 2001; level in GAL-SAE2 mec1D cells. Thus, also SAE2 overexpression our unpublished observation), this indicates that the lack of affects both Mec1- and Tel1-dependent pathways. Sae2 impairs both Tel1 and Mec1 inactivation in the presence In agreement with the above findings, bleomycin-induced of a DSB. phosphorylation of the Mre11 and Ddc2 proteins, which specifically 216 EMBO reports VOL 7 | NO 2 | 2006 &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION Cells with Mre11 foci (%) Sae2 and checkpoint switch off M. Clerici et al scientificreport A C wt GAL-SAE2 tel1Δ tel1Δ GAL-SAE2 wt sae2Δ mec1Δ sae2Δ sae2Δ mec1Δ tel1Δ 0 30 60 90 120 150 Time after nocodazole release (min) Hours after HO induction D UV 01 23 4 5 6 7 8 910 11 12 exp noc 15 30 45 60 75 90 105 120 150 180 (min) Rad53 wt wt Rad53 Rad53 sae2Δ tel1Δ Rad53 Rad53 mec1Δ Rad53 GAL-SAE2 Rad53 sae2Δ mec1Δ GAL-SAE2 tel1Δ Rad53 Rad53 UV Rad53 sae2Δ tel1Δ exp αf 30 60 75 90 120 150 180 240 270 300 (min) Rad53 wt +bleo+noc +bleo+noc +bleo+noc noc 30 60 90 120 noc 30 60 90 120 noc 30 60 90 120 (min) Rad53 mec1Δ Ddc2 GAL-SAE2 Rad53 Mre11 GAL-SAE2 mec1Δ Rad53 wt sae2Δ GAL-SAE2 Fig 4 Mec1- and Tel1-dependent checkpoints in sae2D and SAE2-overexpressing cells. (A,B) Exponentially growing YEP þ raf cell cultures of wild-type YMV80 and isogenic sae2D, mec1D sml1D, sae2D mec1D sml1D, tel1D and sae2D tel1D strains were transferred to YEP þ raf þ gal to induce HO expression (time zero). Samples withdrawn at the indicated times were used for fluorescence-activated cell sorting analysis of DNA contents (A) and western blot analysis of protein extracts with anti-Rad53 antibodies (B). (C,D) Exponentially growing YEP þ raf cell cultures of wild-type W303 and isogenic tel1D, GAL-SAE2 and GAL-SAE2 tel1D strains were arrested with nocodazole in the presence of galactose for 2 h (noc), UV-irradiated (45 J/m ) and released into cell cycle in YEP þ raf þ gal. Samples were collected at the indicated times to determine the percentage of binucleate cells in unirradiated (open symbols) and UV-irradiated (closed symbols) cultures (C) and to monitor Rad53 phosphorylation from UV-irradiated cell cultures by western analysis (D). (E) Exponentially growing YEP þ raf cell cultures of wild-type W303 and isogenic mec1D sml1D, GAL-SAE2 and GAL-SAE2 mec1D sml1D strains were arrested in G1 with a factor in the presence of galactose for 2 h (af), UV-irradiated (30 J/m ) and released into cell cycle in YEP þ raf þ gal. Protein extracts prepared at the indicated times were analysed by western blot with anti-Rad53 antibodies. (F) YEP þ raf nocodazole-arrested cell cultures of wild-type W303 and isogenic sae2D and GAL-SAE2 strains, all expressing the DDC2-HA3- or MRE11-HA3-tagged alleles from the corresponding endogenous promoters, were transferred in YEP þ raf þ gal containing 10 mU/ml bleomycin and 15 mg/ml nocodazole ( þ bleo þ noc). Protein extracts prepared at the indicated times were subjected to western analysis with anti-haemagglutinin antibodies. depends on Tel1 and Mec1, respectively (Longhese et al, 2003), was METHODS more efficient in sae2D cells than in wild-type cells, whereas it was Yeast strains JKM139 (MATa hmlD hmrD ade1 lys5 leu2-3,112 below the detection level in SAE2-overexpressing cells (Fig 4F). trp1HhisG ura3-52 ho ade3HGAL-HO; Lee et al, 1998), YMV80 Altogether, our data indicate that Sae2 negatively regulates and W303 (MATa or MATa ade2-1 can1-100 his3-11,15 leu2- both Mec1- and Tel1-dependent checkpoint responses, possibly 3,112 trp1-1 ura3, rad5-535) were used to disrupt the SAE2 gene, 2,5,6,8,9 by modulating MRX association to damaged sites. As Mec1- and to replace the SAE2 chromosomal copy with the sae2 2,5,6,8,9 Tel1-dependent Sae2 phosphorylation is necessary for Sae2- allele, or to integrate the GAL-SAE2 and GAL-sae2 fusions mediated checkpoint switch off, it is tempting to speculate that at the URA3 locus. YMV80 is isogenic to YFP17 (MATD hmlD these kinases, once activated, may limit MRX-dependent check- hmrD ade1 lys5 ura3-52 trp1 ho ade3HGAL-HO leu2Hcs), point signalling by phosphorylating Sae2. except for the presence of a LEU2 fragment inserted 25 kb &2006 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 7 | NO 2 | 2006 217 tel1Δ tel1Δ Hours after HO induction Binucleate cells (%) Sae2 and checkpoint switch off M. Clerici et al scientificreport Lisby M, Barlow JH, Burgess RC, Rothstein R (2004) Choreography of the centromere-distal to leu2Hcs (Vaze et al, 2002). Strains JKM139 DNA damage response: spatiotemporal relationships among checkpoint and YMV80 were kindly provided by J. Haber (Brandeis and repair proteins. Cell 118: 699–713 University, Waltham, MA, USA). Llorente B, Symington LS (2004) The Mre11 nuclease is not required for 5 to Supplementary information is available at EMBO reports online 3 resection at multiple HO-induced double-strand breaks. Mol Cell Biol 24: 9682–9694 (http://www.emboreports.org). Lobachev KS, Gordenin DA, Resnick MA (2002) The Mre11 complex is required for repair of hairpin-capped double-strand breaks and prevention of chromosome rearrangements. Cell 108: 183–193 ACKNOWLEDGEMENTS Longhese MP, Clerici M, Lucchini G (2003) The S-phase checkpoint and its We thank J. Haber, J. Diffley, N. Kleckner, L. Symington, D. Toczyski and regulation in Saccharomyces cerevisiae. Mutat Res 532: 41–58 K. Nasmyth for providing yeast strains and antibodies. This work was Nakada D, Matsumoto K, Sugimoto K (2003) ATM-related Tel1 associates supported by grants from Associazione Italiana Ricerca sul Cancro, with double-strand breaks through an Xrs2-dependent mechanism. Genes Dev 16: 1957–1962 Cofinanziamento Ministero dell’lstruzione, dell’Universita` e della Nakada D, Hirano Y, Sugimoto K (2004) Requirement of the Mre11 complex Ricerca/Universita` Milano-Bicocca and European Community’s Human and exonuclease 1 for activation of the Mec1 signaling pathway. Mol Potential Programme HPRN-CT-2002-00238 to M.P.L. and Fondo Cell Biol 24: 10016–10025 Investimenti Ricerca di Base to G.L. Neale MJ, Ramachandran M, Trelles-Sticken E, Scherthan H, Goldman AS (2002) Wild-type levels of Spo11-induced DSBs are required for normal single-strand resection during meiosis. 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Journal

EMBO Reports – Springer Journals

Published: Feb 1, 2006

Keywords: Sae2; checkpoint; MRX; Mec1; Tel1

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