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Sep 25, 2014 - recover unless they had been incubated in the presence of rapa- mycin (Figure 1G). ... These data suggest that TORC1 inhibition may preserve cell .... Cdc5 activity would drive adaptation even in the presence of rapamycin.
Article

High Nutrient Levels and TORC1 Activity Reduce Cell Viability following Prolonged Telomere Dysfunction and Cell Cycle Arrest Graphical Abstract

Authors Julia Klermund, Katharina Bender, Brian Luke

Correspondence [email protected]

In Brief In the face of irreparable DNA damage, cells eventually overcome the DNA damage checkpoint and continue to divide (checkpoint adaptation). Checkpoint escape can lead to chromosomal aberrations and may have detrimental outcomes. Klermund et al. now find that the propensity to adapt depends on nutritional status and hence TORC1 activity. They show that TORC1 inhibition prevents checkpoint adaptation and therefore maintains cell viability following chronic telomere dysfunction.

Highlights Inhibition of TORC1 activity prevents adaptation to the DNA damage checkpoint Cells that have not adapted stay viable and retain their proliferative potential TORC1 inhibition prevents inactivation of the checkpoint via Cdc5 regulation The ability of rapamycin to extend yeast lifespan requires a functional checkpoint

Klermund et al., 2014, Cell Reports 9, 324–335 October 9, 2014 ª2014 The Authors http://dx.doi.org/10.1016/j.celrep.2014.08.053

Cell Reports

Article High Nutrient Levels and TORC1 Activity Reduce Cell Viability following Prolonged Telomere Dysfunction and Cell Cycle Arrest Julia Klermund,1 Katharina Bender,1 and Brian Luke1,* 1Zentrum

fu¨r Molekulare Biologie der Universita¨t Heidelberg (ZMBH), DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2014.08.053 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

SUMMARY

Cells challenged with DNA damage activate checkpoints to arrest the cell cycle and allow time for repair. Successful repair coupled to subsequent checkpoint inactivation is referred to as recovery. When DNA damage cannot be repaired, a choice between permanent arrest and cycling in the presence of damage (checkpoint adaptation) must be made. While permanent arrest jeopardizes future lineages, continued proliferation is associated with the risk of genome instability. We demonstrate that nutritional signaling through target of rapamycin complex 1 (TORC1) influences the outcome of this decision. Rapamycin-mediated TORC1 inhibition prevents checkpoint adaptation via both Cdc5 inactivation and autophagy induction. Preventing adaptation results in increased cell viability and hence proliferative potential. In accordance, the ability of rapamycin to increase longevity is dependent upon the DNA damage checkpoint. The crosstalk between TORC1 and the DNA damage checkpoint may have important implications in terms of therapeutic alternatives for diseases associated with genome instability. INTRODUCTION Checkpoints prevent cell cycle progression in the presence of damaged DNA (Zhou and Elledge, 2000), thereby allowing time for repair to occur while simultaneously preventing lethal/ pathogenic genetic alterations from being propagated into subsequent generations. Consistently, checkpoints provide a barrier to cancer development (Kastan and Bartek, 2004; Sperka et al., 2012). Double-strand breaks (DSBs) are particularly toxic DNA lesions because of their propensity to promote genome rearrangements. The response to a DSB is initiated by exonuclease-mediated resection of the newly exposed DNA ends, which generates 30 single-stranded (ss) DNA that becomes bound by the replication protein A complex (RPA) (Dewar and Lydall, 2012; Garvik et al., 1995; Lee et al., 1998; Mimitou and Symington, 2008). In budding yeast (S. cerevisiae), RPA recruits Ddc2 (ATRIP) and 324 Cell Reports 9, 324–335, October 9, 2014 ª2014 The Authors

Mec1 (ATR/ATM), which activate the downstream effectors Chk1 and Rad53 (CHK2) to elicit a G2/M arrest (Sanchez et al., 1999; Weinert et al., 1994). Chk1 contributes to this arrest by stabilizing Pds1 (securin), which prevents Esp1 (separase) from cleaving the Scc1 subunit of cohesin and as a result hinders the onset of anaphase A (Agarwal et al., 2003; Ciosk et al., 1998). Rad53, in addition to contributing to Pds1 stabilization, inactivates the polo-like kinase, Cdc5 (PLK1), a positive regulator of multiple mitotic events, including spindle elongation (Zhang et al., 2009) and the mitotic exit network (MEN) (Hu et al., 2001). Telomeres make up the ends of linear chromosomes and are structurally similar to DSBs. In contrast to DSBs, they are neither subject to repair nor do they activate the DNA damage checkpoint (de Lange, 2009). Telomeres terminate in a 30 ss overhang, which in S. cerevisiae is bound by the CST (Cdc13-Stn1-Ten1) complex that caps and protects chromosome ends (Garvik et al., 1995; Giraud-Panis et al., 2010; Grandin et al., 2001). Upon CST inactivation, ssDNA accumulates due to 50 strand resection by the exonuclease, Exo1 (Maringele and Lydall, 2002). Similar to DSB processing, the accumulation of telomeric ssDNA elicits a Mec1/Rad53-orchestrated DNA damage response (DDR), which prevents subsequent mitotic events (Garvik et al., 1995; Lydall and Weinert, 1995; Zubko et al., 2004). When DSBs and dysfunctional telomeres are repaired, the checkpoint signal is attenuated allowing mitotic progression and passage into the succeeding G1, a process referred to as recovery (Cle´menson and Marsolier-Kergoat, 2009; van Vugt and Medema, 2004; Vaze et al., 2002). Although repair and recovery represent the preferred outcome following DNA damage, an alternative solution exists. In the presence of irreparable DNA damage/telomere dysfunction, budding yeast eventually inactivate the DNA damage checkpoint and proceed through mitosis despite damage persistence—a phenomenon known as checkpoint adaptation (Pellicioli et al., 2001; Sandell and Zakian, 1993; Toczyski et al., 1997). In a screen to identify regulators of adaptation, the polo-like kinase Cdc5 emerged as a promoting factor. Cells expressing an adaptation-defective mutant allele of CDC5 (cdc5-ad) fail to adapt (Toczyski et al., 1997), whereas the overexpression of Cdc5 accelerates adaptation (Donnianni et al., 2010; Lopez-Mosqueda et al., 2010; Vidanes et al., 2010). Although adaptation offers cells the opportunity to recommence proliferation more quickly in the face of

irreparable damage, it is associated with the risk of increased genome instability and chromosome loss (Galgoczy and Toczyski, 2001; Sandell and Zakian, 1993). Target of rapamycin complex 1 (TORC1) controls cell growth. In nutrient-rich conditions, TORC1 is active and promotes anabolic pathways via growth-promoting transcription programs, translation initiation, and ribosome biogenesis, while at the same time inhibiting catabolic processes such as autophagy (Loewith and Hall, 2011). Rapamycin is a lipophilic macrocyclic lactone that inhibits TORC1 (Zheng et al., 1995; Wullschleger et al., 2006) and hence mimics nutrient starvation. TORC1 activity promotes the transition from G1 to S as rapamycin-treated cells accumulate in G1 (Barbet et al., 1996). Recent evidence, however, has demonstrated that TORC1 also influences the G2/M transition by regulating Cdc5 localization (Nakashima et al., 2008). Moreover, it was shown that the induction of autophagy is sufficient to prevent checkpoint adaptation in response to a single irreparable DSB, likely due to the inability to localize Esp1 to the nucleus (Dotiwala et al., 2013; Eapen and Haber, 2013; Zheng et al., 1995). Surprisingly, low (nontoxic) doses of rapamycin are able to rescue the lethality associated with prolonged CDC13 inactivation (Qi et al., 2008). However, the mechanistic details regarding how TORC1 inhibition rescues cell viability remain elusive. Here we demonstrate that rapamycin prevents lethality associated with chronic telomere dysfunction by preventing checkpoint adaptation and hence inhibiting mitotic progression. We demonstrate that rapamycin acts via multiple parallel pathways, including autophagy induction, as previously demonstrated (Dotiwala et al., 2013; Eapen and Haber, 2013), as well as the inhibition of Cdc5 activity. In addition, we show that the longevity-promoting effect of rapamycin in S. cerevisiae is, to an extent, dependent on an intact DNA damage checkpoint. We propose that high nutrient levels, which activate TORC1 and in turn promote cell growth, are detrimental in the face of DNA damage, as they encourage mitotic progression. These results indicate that TOR inhibitors and dietary control may have potential benefits in disorders associated with DNA damage and genome instability. RESULTS Rapamycin Prevents Checkpoint Adaptation following Telomere Dysfunction To induce telomere dysfunction in yeast, we inactivated a temperature-sensitive (ts) allele of the CDC13 gene (cdc13-1) via incubation at its restrictive temperature of 37 C, which leads to the activation of the DDR and eventual loss of cell viability (Garvik et al., 1995). Cell death was monitored by the uptake of SYTOX Green, as previously described (Murakami et al., 2012). After 24 hr at 37 C, cell viability was compromised in cdc13-1 cells, but not in isogenic wild-type cells (Figures 1A and 1C). Strikingly, rapamycin prevented cell death associated with CDC13 inactivation (Figures 1A and 1C), as previously described (Qi et al., 2008). Neither CDC13 inactivation nor rapamycin treatment affected cell viability following a shorter (6 hr) incubation at 37 C, suggesting that rapamycin’s protective effects are specific to prolonged telomere dysfunction (Figures 1B and 1C). Consis-

tent with a previous report, rapamycin had no influence on the accumulation of single-stranded telomeric DNA either at the 6 or 24 hr time point (Figure S1A) following CDC13 inactivation, indicating that the increased viability is not due to enhanced telomere protection (Qi et al., 2008). cdc13-1 cells eventually undergo checkpoint adaptation following prolonged incubation at 37 C (Toczyski et al., 1997). In our experimental conditions, cdc13-1 haploid cells held at the restrictive temperature for 6 hr arrested uniformly at the G2/M border with 2N DNA content, as expected (Figure 1D). Following prolonged telomere uncapping (24 hr), adaptation could be observed as cells with 1N (G1 cells) and sub-1N (dead cells) DNA content became apparent, indicating cell cycle progression despite the presence of telomere dysfunction (Figure 1D). We observed dephosphorylation of the checkpoint kinase Rad53 in adapted cells (24 hr at 37 C), along with degradation of the mitotic cyclin Clb2 and accumulation of the cyclindependent kinase inhibitor Sic1, indicative of exit from mitosis and entry into the following G1 (Figure 1E). Strikingly, the addition of rapamycin prevented checkpoint adaptation with cells remaining uniformly arrested at the G2/M border with phosphorylated Rad53, high levels of Clb2, and no detectable Sic1 (Figures 1D and 1E). Recovery was assessed by placing cdc13-1 cells back to their permissive temperature of 23 C and analyzing cell viability once telomere function was restored. cdc13-1 cells that had experienced 6 hr of telomere dysfunction formed colonies similar to wild-type cells in a recovery assay (Figure 1F). In contrast, cdc13-1 cells that had undergone adaptation were unable to recover unless they had been incubated in the presence of rapamycin (Figure 1G). cdc13-1 cells that were kept at the permissive temperature of 23 C for 6 and 24 hr (temperature control cells) did not experience compromised viability (Figures S1B and S1C). To ensure that the rapamycin-mediated rescue was not a result of a selection-bias for suppressors of the cdc13-1 mutation, we reinoculated the rescued rapamycin-treated cells and subjected them to a second round of incubation at 37 C. Indeed, these cells could not recover unless they were also treated with rapamycin in the repeat experiment, ruling out the possibility that genetic suppressors accounted for the rapamycin rescue (Figure S1D). The deletion of TOR1 phenocopied the viability effects of rapamycin on cdc13-1 cells (Figure S1E), whereas the deletion of RAS2 (activator of the cyclic AMP pathway) did not (Figure S1F), demonstrating TORC1 specificity. Rapamycin and TOR1 deletion also rescued cell death associated with another CST ts mutant (stn1-13) (Figure S1G). Although active TORC1 signaling is a potent activator of translation, we were not able to phenocopy the cdc13-1 rescue effects when the general translation inhibitor, cyclohexamide, was substituted for rapamycin at a concentration (0.1 mg/ml) that prevented cell growth to an equal extent as 3 nM rapamycin (Figure S1H; data not shown). We ruled out the effects of TORC1 inhibition on the G1/S transition by prearresting cdc13-1 cells at the G2/M border prior to the addition of rapamycin (Figure S1I) and found that the rapamycin-mediated lethality rescue was not altered (Figure S1J). Finally, the deletion of SAN1, encoding the nuclear E3 ubiquitin ligase, did not rescue cdc13-1 in our experimental conditions (Figure S1K), excluding the notion that Cell Reports 9, 324–335, October 9, 2014 ª2014 The Authors 325

Figure 1. Rapamycin Prevents Checkpoint Adaptation in cdc13-1 Cells

(A and B) Following an overnight culture in YPD at 23 C, cells were diluted and shifted to 37 C for the indicated times in the presence or absence of rapamycin. SYTOX Green staining of dead cells followed by FACS scanning determined cell viability. Numbers in the boxes represent % of dead (top) and viable (bottom) cells, for one representative experiment (FSC = forward scatter). (C) Quantification of (A) and (B), with data represented as the means ± SEM of multiple biological replicates (wild-type: 6 hr, n = 4; 24 hr, n = 5; cdc13-1: 6 hr, n = 6; 24 hr, n = 7). The p value was calculated using an unpaired Student’s t test (****p < 0.0001). (D) Cells from the above experiments were prepared for DNA FACS scan to determine ploidy at the indicated time points. The peaks corresponding to 1N (G1 cells) and 2N (G2/M cells) are indicated. (E) Western blot showing phosphorylation of Rad53, as well as Clb2 and Sic1 levels. (F and G) After 6 (F) and 24 hr (G) at the nonpermissive temperature, the indicated genotypes were spotted as 10-fold serial dilutions on YPD plates and incubated for 3 days at the permissive temperature of 23 C to assess recovery capacity.

rapamycin may be promoting the accumulation of the cdc13-1 gene product as has been previously observed in SAN1 deletion cells (Gardner et al., 2005). 326 Cell Reports 9, 324–335, October 9, 2014 ª2014 The Authors

These data suggest that TORC1 inhibition may preserve cell viability following prolonged telomere dysfunction by preventing checkpoint adaptation.

Figure 2. The Sch9 Kinase Checkpoint Adaptation

Regulates

(A) Recovery capacity was assessed by allowing cells to regrow at 23 C after 24 hr at 37 C as in Figure 1G. (B) Following an overnight culture in YPD, the indicated strains were diluted in the presence of 1NM-PP1 and subsequently shifted to 37 C for 24 hr before assessing recovery as in Figure 1G. (C and D) DNA FACS scan (C) and western blotting (D) depict a failure to adapt in the absence of the Sch9 kinase. (E) Cells transformed with vector control, wild-type SCH9, or rapamycin nonresponsive SCH92D3E were grown overnight in SD-Ura and diluted in YPD in the presence or absence of rapamycin before being shifted to 37 C for 24 hr. Viability staining (as in Figure 1A) was performed on the indicated strains, with data represented as the means ± SEM of three independent experiments; p values were calculated using a one-way ANOVA (****p < 0.0001, ***p = 0.0005, **p = 0.0062).

Inhibition of the Sch9 Branch of TORC1 Signaling Prevents Adaptation A major effector of TORC1 is the AGC kinase, Sch9, which is phosphorylated and activated by TORC1 (Urban et al., 2007). To determine whether rapamycin prevents adaptation through Sch9 inhibition, we created cdc13-1 sch9 mutants and assessed recovery following prolonged CDC13 inactivation. Similar to rapamycin addition, the deletion of SCH9 prevented cell death following prolonged telomere dysfunction (Figure 2A). This effect was recapitulated in an SCH9 analog-sensitive allele (sch9as) (Figure 2B), where kinase activity is inhibited by the ATP analog, 1NM-PP1 (Huber et al., 2011) (see Figures S2A and S2B for temperature control experiment). The deletion of SCH9 prevented both cell cycle progression (Figure 2C) and Rad53 dephosphorylation (Figure 2D), albeit the latter not as efficiently as with rapamycin (Figure 1E), suggesting that Sch9 is not the only target of rapamycin responsible for maintaining Rad53 activity. In order to test this notion more rigorously, we performed viability analysis as in Figure 1C. In accordance with enhanced recovery (Figure 2A), the deletion of SCH9 rescued cdc13-1 cells in the absence of rapamycin, and this effect was enhanced in the presence of rapamycin suggestive of an SCH9-independent pathway (Figure 2E). To confirm this, we expressed either wild-type SCH9 or an allele that is no longer inhibited by rapamycin (SCH92D3E) (Urban et al., 2007) as the only copy of SCH9 in cdc13-1 cells. In both cases, rapamycin was able to rescue cell viability (Figure 2E), confirming the presence of SCH9-independent compensatory pathways. In summary, the inhibition of TORC1 and its effector Sch9 prevents checkpoint adaptation and maintains Rad53 in an active state. However, other parallel pathways exist, independent of Sch9 inhibition, which can promote cell viability upon rapamycin addition.

Rapamycin Prevents Adaptation in an Autophagy-Independent Manner The ectopic activation of autophagy (via the ATG13-8SA allele) prevents adaptation following the induction of a DSB (Dotiwala et al., 2013; Eapen and Haber, 2013). We observed that ATG13-8SA expression also prevents cell death in cdc13-1 cells after 24 hr at 37 C (Figures 3A and S3A). The recovery was dependent on the presence of Mec1 (Figure 3A), as previously described (Dotiwala et al., 2013), where it was postulated that a transient checkpoint-dependent burst of autophagy contributes to cell cycle arrest. Although cells expressing ATG138SA do not adapt, they still experience Rad53 dephosphorylation (Dotiwala et al., 2013), suggesting that autophagy induction and Sch9 inhibition prevent adaptation in mechanistically distinct manners. We assessed the contribution of autophagy to the rapamycin-mediated inhibition of adaptation by monitoring adaptation capacity in either atg5 or atg1 mutants, which are autophagy defective (Tsukada and Ohsumi, 1993). After 24 hr at the restrictive temperature, rapamycin improved the viability of cdc13-1 atg5 cells, albeit not to the same extent as in cdc13-1 single mutants (Figure 3B). Analysis of DNA content demonstrated that rapamycin prevented adaptation even in the absence of autophagy (Figure 3C). Consistent with the notion that autophagy induction is not required to maintain Rad53 phosphorylation (Dotiwala et al., 2013), Rad53 dephosphorylation was still prevented by rapamycin in cdc13-1 atg5 cells (Figure 3D). Rapamycin impaired both Clb2 degradation and Sic1 accumulation in the presence and absence of autophagy when prolonged telomere dysfunction was induced (Figure 3D). Moreover, rapamycin treatment of autophagy-deficient cells that had experienced long-term CDC13 inactivation rendered them nearly fully recovery competent, as shown for the deletions of ATG5 (Figures 3E and S3B temperature control) and ATG1 (Figure S3C). We assessed autophagy activity in cdc13-1 atg5 mutants using the GFP-Atg8 readout, where Cell Reports 9, 324–335, October 9, 2014 ª2014 The Authors 327

Figure 3. Rapamycin Prevents Adaptation in the Absence of Autophagy (A) Cells harboring the indicated plasmids were grown in raffinose-containing media before being diluted in medium containing 2% galactose to induce ATG138SA and shifted to 37 C for 24 hr. Cells were subsequently spotted as in Figure 1G. (B) Viability staining was performed on the indicated strains, with data represented as the means ± SEM of multiple biological replicates (wild-type n = 3, atg5 n = 3, cdc13-1 n = 4, cdc13-1 atg5 n = 4); p values were calculated using a one-way ANOVA (****p < 0.0001, **p = 0.0036, ns = not significant). (C and D) DNA FACS scan (C) and western blot analysis (D) demonstrate that checkpoint adaptation is prevented by rapamycin even in cdc13-1 atg5 cells. (E and F) The indicated strains were spotted onto YPD and allowed to recover at 23 C as described in Figure 1G. (G) Cells transformed with either vector control, wild-type SCH9, or rapamycin nonresponsive SCH92D3E were grown overnight in SD-Ura and diluted in YPD medium in the presence or absence of rapamycin before being shifted to 37 C for 24 hr. Viability staining was performed on the indicated strains, with data represented as the means ± SEM of three independent experiments; p values were calculated using a one-way ANOVA (****p < 0.0001, ns = not significant).

the release of free GFP serves as an indicator for the conversion of autophagosomes to autolysosomes (Klionsky, 2011), and observed that cdc13-1 atg5 mutants were completely defective for autophagy in the presence and absence of rapamycin (Figures S3D and S3E). Importantly, the viability rescue 328 Cell Reports 9, 324–335, October 9, 2014 ª2014 The Authors

of cdc13-1 cells by the SCH9 deletion is independent of autophagy (Figures 3F and Figure S3F temperature control) in accordance with the absence of autophagy induction in sch9 mutants (Yorimitsu et al., 2007). The above data (Figures 2 and 3) indicate that rapamycin prevents adaptation via at least

Figure 4. Rapamycin Prevents Adaptation via the Mec1-Rad53 DNA Damage Checkpoint (A) DNA FACS scan of the indicated strains as in Figure 1D. (B–E) Recovery was assessed by assessing regrowth at 23 C following the incubation at 37 C as in Figure 1G.

two independent pathways: Sch9 inhibition (Rad53 dependent) and autophagy induction (Rad53 independent). To determine if these were the only pathways involved, we combined the deletion of ATG5 with the rapamycin-resistant SCH92D3E allele. Surprisingly, we found that cdc13-1 atg5 cells expressing SCH92D3E could still be rescued by rapamycin (Figure 3G), suggesting that in addition to Sch9 inhibition and autophagy induction, rapamycin controls other compensatory TORC1 effectors that contribute to the inhibition of checkpoint adaptation. Rapamycin Requires Mec1 and Rad53 to Prevent Adaptation Since Sch9 inhibition prevents Rad53 dephosphorylation (Figure 2D) and robustly improves the survival of cdc13-1 cells, we hypothesized that an intact DNA damage checkpoint would be critical for rapamycin to prevent adaptation. Indeed, rapamycin could not prevent the accumulation of G1 cells following prolonged telomere uncapping in cells carrying the checkpointdefective rad53-11 allele (Weinert et al., 1994) (Figure 4A). Furthermore, the ability of rapamycin to improve recovery potential following chronic telomere dysfunction was largely (but not completely) compromised in both rad53-11 and mec1 mutants (Figures 4B, 4C, and S4A temperature control). Importantly, cdc13-1 atg1 rad53-11 mutants could also be rescued, to an extent, by rapamycin treatment (Figure 4D), confirming the notion that the regulation of multiple TORC1 effectors is able to promote adaptation in a compensatory manner. In contrast to the partial rapamycin rescue we see in cdc13-1 rad53-11 cells, loss of Rad53 function completely abolished the rescue associated with Sch9 inhibition (Figure 4E). In summary, these data confirm the notion that rapamycin largely prevents checkpoint adaptation via the maintenance of the Mec1/Rad53 checkpoint (through Sch9 inhibition) but suggest that other TORC1 effectors, in addition to autophagy induction, are also at play.

Rapamycin Inhibits Rad53 Dephosphorylation via Cdc5 Regulation CDC5 was one of the first genes implicated in promoting checkpoint adaptation (Toczyski et al., 1997). The overexpression of Cdc5 stimulates Rad53 dephosphorylation and accelerates adaptation (Donnianni et al., 2010; Vidanes et al., 2010). Since the maintenance of Rad53 activity is critical for rapamycinmediated prevention of checkpoint adaptation (Figures 4A and 4B), we tested whether Cdc5 activity may be decreased following TORC1 inactivation. Following CDC13 inactivation and subsequent metaphase arrest, Bfa1 (a bona fide Cdc5 target) migrates as a higher molecular weight form due to Cdc5-dependent phosphorylation (Figure 5A) (Hu et al., 2001; Valerio-Santiago et al., 2013). In the presence of rapamycin, Bfa1 phosphorylation was lost both in the presence and absence of autophagy, suggesting reduced Cdc5 activity (Figure 5A). Consistent with our previous genetic data, the deletion of SCH9 also had reduced Cdc5 activity. Analysis of DNA content via flow cytometry (Figure S5A) combined with the confirmation that Pds1 was stable (Figure 5A), confirmed that differences in cell cycle stage were not responsible for the altered Cdc5 activity. These data suggest that rapamycin, via Sch9 inhibition, reduces Cdc5 activity in the presence of dysfunctional telomeres, which may be directly responsible for the adaptation defect. If reduced Cdc5 activity is responsible for the rapamycinmediated adaptation defect, we reasoned that increasing Cdc5 activity would drive adaptation even in the presence of rapamycin. We shifted cdc13-1 cells harboring a galactose inducible CDC5 vector to the nonpermissive temperature of 30 C. Following 2 hr at the nonpermissive temperature, both galactose and nocodazole were added to induce Cdc5 expression and simultaneously prevent cells from progressing to the following G1 phase. Rapamycin was added to half of the culture, whereas the other half was exposed to an equal volume of the vehicle control, DMSO (Figure 5B). Already after 2 hr of Cdc5 Cell Reports 9, 324–335, October 9, 2014 ª2014 The Authors 329

Figure 5. Cdc5-Induced Adaptation Is Prevented by TORC1 Inhibition (A) Western blot showing the phosphorylation status of Bfa1. Cells from an overnight culture in YPD at 23 C were diluted and shifted to 37 C for 3 hr in the presence or absence of rapamycin. (B) Scheme of the experimental setup for (C) and (D). Overnight cultures were diluted and shifted to 30 C or 37 C. Galactose was added to a concentration of 2%, and the cultures split into a DMSO or rapamycin-treated culture. Time points were taken as indicated. (C) Western blot of cdc13-1 cells harboring a Gal-inducible Cdc5 overexpression plasmid treated as in (B), showing phosphorylation of Rad53, as well as Cdc5 expression and Pgk1 as a loading control. Nocodazole was added to a final concentration of 15 mg/ml. (D) Following the scheme in (B), the indicated strains were spotted onto YPD and allowed to recover at 23 C as in Figure 1G. (E) Cell viability staining was performed on the indicated strains, with data represented as the means ± SEM of multiple biological replicates (wild-type n = 3, cdc5ad n = 3, cdc13-1 n = 5, cdc13-1 cdc5-ad n = 5); p values were calculated using a one-way ANOVA (****p < 0.0001). (F) Recovery was assessed by allowing cells to regrow at 23 C as in Figure 1G. (G) DNA FACS scan of the indicated strains as in Figure 1D. (H) Western blot analysis demonstrates that checkpoint adaptation is prevented in cdc13-1 cdc5-ad cells without rapamycin treatment. Gal, galactose; Noc, nocodazole.

overexpression (4 hr time point), Rad53 was dephosphorylated (Figure 5C). This rapid dephosphorylation was not prevented in the presence of rapamycin (Figure 5C), suggesting that rapamycin is preventing adaptation via the inhibition of a Cdc5-mediated process. In agreement, Cdc5 overexpression severely impaired the recovery capacity associated with cdc13-1 cells that had been exposed to rapamycin (Figures 5D and S5B temperature control). 330 Cell Reports 9, 324–335, October 9, 2014 ª2014 The Authors

The cdc5-ad allele has been characterized as being adaptation defective because of a single amino acid mutation (L251W) (Jin and Wang, 2006; Toczyski et al., 1997). Although CDC5 is an essential gene, cells harboring the cdc5-ad allele are not compromised in other aspects of cell growth, suggesting that the -ad allele is adaptation specific. The presence of the cdc5-ad allele significantly increased both the viability and recovery of cdc13-1 cells following a 24 hr incubation at 37 C

even in the absence of rapamycin (i.e., phenocopying the rapamycin effects) (Figures 5E, 5F, and S5C temperature control). The cdc5-ad allele prevented adaptation in terms of cell cycle progression as well as the maintenance of Rad53 phosphorylation (Figures 5G and 5H). Therefore, the rapamycin-mediated regulation of the DNA damage checkpoint via Cdc5 appears to be important in cells that arrest in G2/M due to telomere dysfunction. The further increase in viability upon rapamycin addition in cdc13-1 cdc5-ad cells (Figure 5E) is consistent with multiple TORC1 effector pathways (in addition to autophagy induction) being involved. Indeed, the viability of cdc13-1 cdc5-ad atg5 cells was still increased upon rapamycin addition (Figure S5D). A major effector pathway downstream of TORC1 and parallel to Sch9 is regulated by the phosphatase inhibitor, Tap42. We inactivated Tap42 function by using the tap42-11 allele at 37 C, thereby mimicking rapamycin addition (Di Como and Arndt, 1996; Yan et al., 2006) and found that this was also able to rescue cdc131 cells (Figure S5E). Future work will focus on understanding mechanistic details of the tap42-11-mediated rescue. Importantly, we found that many cell division cycle ‘‘cdc’’ mutants that display a G2/M arrest point (Weinert and Hartwell, 1993) were rescued through rapamycin addition and that this was not a specific effect associated with telomere dysfunction. Indeed, cdc14-1, cdc15-2, cdc16-1, cdc17-1, and cdc20-1 mutants all had increased viability (to variable extents) and improved recovery in the presence of rapamycin (Figures S5F and S5G; note that cdc17-1 mutants do not require Rad9 for arrest at 38 C (Weinert and Hartwell, 1993). In contrast to cdc13-1 mutants, the cdc5-ad allele was not able to recue viability in cdc14-1, cdc15-2, cdc16-1, and cdc17-1 cells, while only a slight rescue could be detected in cdc20-1 mutants, suggesting that Cdc5-mediated adaptation may be specific for telomere dysfunction/DNA damage (Figure S5H), as previously proposed (Valerio-Santiago et al., 2013). The induction of autophagy, however, was able to rescue lethality in cdc15-2, cdc16-1, and cdc20-1 mutants (Figure S5I; note that cdc13-1 cells are more viable in the presence of galactose). Importantly, the rapamycin-mediated rescue of ‘‘cdc’’ mutants was not specific to a G2/M arrest, as cdc4-1, cdc7-4, and cdc2-2 mutants, which arrest in either G1 (cdc4-1) or early S can also be rescued, to variable extents, by inhibiting TORC1 (Figure S5J). Lifespan Extension via Rapamycin Requires the DNA Damage Checkpoint We investigated which nutrient sources, when depleted, would improve the viability of cells with chronic telomere dysfunction. Caloric restriction (0.1% glucose) (Figure 6A) as well as leucine or uracil starvation (Figure 6B) and growth in the presence of a nonfermentable carbon source (ethanol) (Figure 6C) were all able to improve the recovery of cdc13-1 cells (see Figures S6A–S6C for temperature control). Growth in ethanol prevented cdc13-1 cells from adapting by keeping Rad53 in a phosphorylated and active state (Figure S6D), suggesting that nutrient deprivation prevents checkpoint adaptation in a manner similar to TORC1 inhibition by rapamycin. Rapamycin extends lifespan in a variety of model organisms ranging from yeast to mouse (Cornu et al., 2013; Harrison

et al., 2009; Longo et al., 2012). Similarly, caloric restriction and other forms of nutrient deprivation can also delay agingassociated processes (Anderson and Weindruch, 2012; Katewa and Kapahi, 2010). Our results suggest that rapamycin and nutrient deprivation may promote longevity by preventing checkpoint adaptation, which would in turn prevent the accumulation of DNA damage and genetic alterations over time. To test this hypothesis, we inoculated wild-type and rad53-11 cells in the presence or absence of rapamycin and assayed cell viability via SYTOX Green staining over time to assess chronological aging. As expected, rapamycin decreased the rate of aging in wild-type cells (Figure 6D). Strikingly, rapamycin was unable to prevent the loss of viability associated with aging when the DNA damage checkpoint was impaired in rad53-11 cells (Figure 6D). The rapamycin antiaging effects, at early time points, were not dependent on the induction of autophagy, as the rate of aging was still decreased by rapamycin in atg5 mutant cells (Figure 6E). Although the DMSO (control)-treated rad53-11 cells aged similar to wild-type cells over the first 4 days (Figure 6D), the loss of the checkpoint led to increased aging rates at later time points, which was prevented to an extent by rapamycin (Figure S5E). Together, these data suggest that rapamycin prevents chronological aging, at least in part, through reinforcement of the DNA damage checkpoint. DISCUSSION Dysfunctional telomeres activate the DNA damage checkpoint, which prevents mitotic progression and ensures that damaged chromosomes are not segregated to daughter cells. Indeed, the ablation of a single telomere from a yeast chromosome results in increased loss of the affected chromosome, an effect that is exacerbated when the DNA damage checkpoint is impaired (Sandell and Zakian, 1993). Upon DNA damage, Rad53 (CHK2) inhibits mitosis through inactivation of the mitotic kinase Cdc5 (Sanchez et al., 1999) (Figure 6F). Inactivation of Cdc5 hinders mitotic progression via (1) blocking the elongation of the mitotic spindle during anaphase B (Zhang et al., 2009) and (2) preventing activation of the MEN (Valerio-Santiago et al., 2013). Following repair, the checkpoint is inactivated due to the dephosphorylation of Rad53 and other checkpoint targets, which allows mitotic progression, referred to as recovery (Harrison and Haber, 2006; Keogh et al., 2006; Leroy et al., 2003). When damage is irreparable, Cdc5 eventually reacquires activity and inactivates Rad53 (Figure 6F, dashed line 1), which further relieves Cdc5 inhibition and permits mitotic progression despite the presence of damaged DNA (checkpoint adaptation) (Figure 6F, dashed line 2) (Pellicioli et al., 2001; Sandell and Zakian, 1993; Toczyski et al., 1997; van Vugt and Medema, 2004). Cdc5 regulation is critical for adaptation, as the cdc5-ad allele prevents adaptation (Toczyski et al., 1997), and increased Cdc5 levels accelerate it (Donnianni et al., 2010; Vidanes et al., 2010). Similar to yeast Cdc5, PLK1 has been shown to drive adaptation following DNA damage in vertebrates (Syljua˚sen et al., 2006; van Vugt and Medema, 2004; Yoo et al., 2004). TORC1 activity has been proposed to promote Cdc5 function (Nakashima et al., 2008). Therefore, cdc13-1 cells held at nonpermissive temperature are confronted with conflicting Cell Reports 9, 324–335, October 9, 2014 ª2014 The Authors 331

Figure 6. Rapamycin Requires Rad53 to Extend Lifespan (A–C) Cells were grown overnight in YPD before being diluted in the different nutrient conditions as indicated and shifted to 37 C for 24 hr. Recovery capacity was assessed by allowing cells to regrow at 23 C as in Figure 1G on YPD. (D and E) Overnight cultures in YPD were diluted to OD600 0.1 in SCD medium and incubated at 30 C. Samples were taken every day for cell viability staining as in Figure 1A. Graphs represent the means ± SEM of multiple biological replicates (n = 4). WT, wild-type. (F) Model depicting the pathways by which rapamycin inhibits checkpoint adaptation following prolonged DNA damage (see Discussion).

signals in terms of whether or not to proceed through mitosis (i.e., the DNA damage checkpoint is antimitotic, while high nutrient conditions via TORC1/Cdc5 function are promitotic). The checkpoint dominates in the short term, as cells remain arrested at the G2/M border (Figure 1D). The prevalence of the checkpoint ensures that cells remain in a viable state and upon repair are recovery competent (Figure 1F). When telomere dysfunction is prolonged, the continued signaling from TORC1 eventually prevails, promoting Cdc5-mediated checkpoint adaptation despite the presence of damage (Figure 6F). The consequences of checkpoint adaptation include cell death (Figure 1A), genome instability (Galgoczy and Toczyski, 2001), and chromosome loss (Sandell and Zakian, 1993). In the presence of high-nutrient conditions and irreparable DNA damage, it may be more beneficial for a cell to adapt in the face of damage to maximize propagation potential in nutrient-rich conditions than to risk permanent arrest and even332 Cell Reports 9, 324–335, October 9, 2014 ª2014 The Authors

tual cellular senescence. In contrast, when nutrients are limiting, it is likely more beneficial to remain arrested with DNA damage than to risk proceeding through mitosis with DNA damage in a nutrient-poor environment. We speculate that cells have evolved such signaling crosstalk to maximize overall survival. Indeed, it has recently been established that crosstalk also exits between the DNA damage checkpoint and the PKA pathway (Searle et al., 2004, 2011). Multiple TORC1 effectors can prevent adaptation, including Sch9 inhibition, autophagy induction, and presumably Tap42 inactivation (Figure 6F). Sch9 inhibition decreases Cdc5 activity (Figure 5A) and hence prevents Rad53 inactivation (Figures 2D and 6F). Autophagy induction does not prevent adaptation through Rad53 activity (Figure 3D) and likely prevents mitosis by inhibiting other mitotic events as previously proposed (Dotiwala et al., 2013). Since the deletion of SCH9 does not prevent Rad53 dephosphorylation as completely as either rapamycin

addition or the presence of the cdc5-ad allele, we speculate that Tap42 may also regulate Cdc5 activity as depicted in Figure 6F (gray lines), although we cannot exclude the possibility that other mitotic processes may be targeted. It has been reported that PP2A (a Tap42-regulated phosphatase) is required for the nuclear accumulation of Cdc5 (Nakashima et al., 2008). Therefore, the combined inhibition of Sch9 and Tap42 may be required for the complete inactivation of Cdc5. Tap42 has also been demonstrated to negatively regulate autophagy (Yorimitsu et al., 2009), which likely accounts for a portion of the tap42-11-mediated rescue of cdc13-1 mutants (Figure 6F). Many temperature-sensitive ‘‘cdc’’ mutants (Weinert and Hartwell, 1993) that arrest in a checkpoint-dependent manner at various cell cycle stages have increased viability in the presence of rapamycin (Figure S5F). We speculate that high TORC1 activity overrides checkpoints and promotes downstream cell cycle events before the successful completion of critical upstream events. Importantly, only cdc13-1 cells are rescued by the presence of the cdc5-ad allele (Figure S5H), whereas many (but not all) of the G2/M mutants can be rescued through autophagy induction (Figure S5I). Therefore, TORC1 activity may also be responsible for checkpoint adaptation in the absence of DNA damage; however, the regulation of Rad53 via Cdc5 activity is specific for dysfunctional telomeres. Reducing TORC1 activity has the ability to extend healthy lifespan (Alic and Partridge, 2011; Bjedov and Partridge, 2011). Furthermore, the inhibition of both TORC1 and Sch9 improves genome integrity and hence longevity in aged budding yeast (Madia et al., 2007; Madia et al., 2008, 2009). We have demonstrated that the increased longevity phenotype imparted by rapamycin is, at least in part, dependent on the DNA damage checkpoint (Figure 6D). We interpret these data to suggest that TORC1 inactivation prevents cell cycle progression when spontaneous DNA lesions/dysfunctional telomeres arise during aging (Figure 6F). In the presence of rapamycin, only proper repair followed by recovery would allow cells to proceed through mitosis. This would have the overall effect of decreasing ageassociated genome instability. TORC1 inhibition may therefore provide relevant therapeutic alternatives for diseases associated with genome instability and age. Indeed, sgs1 mutants, which recapitulate many phenotypes associated with Werner and Bloom syndrome, are rescued in terms of rapid aging, mutation frequency, and gross chromosomal rearrangements upon deletion of SCH9 (Madia et al., 2008). EXPERIMENTAL PROCEDURES Yeast Strains and Plasmids Strains and plasmids used in this study are listed in Tables S1 and S2, respectively. Haploid strains were derived from the listed diploids in Table S1. Adaptation Assay An overnight culture at 23 C of the indicated strains in yeast extract peptone dextrose (YPD) medium was diluted to OD600 0.3. Rapamycin (BioChemica) diluted in DMSO was added to the indicated final concentration, and the corresponding volume of DMSO was added to the control flasks. Cells were shifted to 37 C, and time points were taken after 6 and 24 hr. Samples were taken for spotting assays, FACS viability staining, FACS DNA staining, and protein extraction for western blotting (see Supplemental Experimental Procedures).

FACS Viability Staining The viability staining protocol was modified from Murakami et al. (2012). In detail, culture volumes corresponding to 0.68 OD600 units were collected by centrifugation at 7,000 rpm for 1 min. Pelleted cells were washed in 50 mM sodium citrate (pH 7.4) and spun down at 7,000 rpm for 1 min. Cells were then resuspended in 1 ml 50 mM sodium citrate (pH 7.4) containing 0.5 mM SYTOX Green (Life Technologies). Cells were kept dark, sonicated briefly, and immediately analyzed on a BD FACSCanto II flow cytometer using the following filters and settings: forward scatter (FSC) and side scatter (SSC) were detected with a 488 nm laser with detector settings of 350 and 230 V, respectively; SYTOX Green was detected with a 502 nm long-pass filter and 530/30 nm band-pass filter at 370 V. In each run, 20,000 events per sample were analyzed. Data collection and analysis were performed using the BD FACSDiva software and the FlowJo v.10.0.6 (Miltenyi Biotec) software. Statistical Analysis of Viability Staining For statistical analysis of cell viability, a gate was set using the FlowJo v.10.0.6 (Miltenyi Biotec) software that contained R98% viable cells for the wild-type cell population in the DMSO sample after 24 hr at 37 C. This gate also included 100% of unstained cells (no SYTOX Green) and was applied to all further measurements. Since the measurements of the percentage of viable cells had different standard deviations (SDs) in the different experimental settings, the data were transformed by calculating the logarithm to base 10 of (100%  viable cells [in%])/100%. When comparing only two factors, an unpaired Student’s t test was performed on the transformed data. When four factors were analyzed, two-way ANOVA with factors ‘‘genotype’’ and ‘‘treatment’’ and interaction was performed on the transformed data. Comparison of the four groups was then done by one-way ANOVA and subsequent pairwise comparisons with Bonferroni adjustment for multiple comparisons. Chronological Lifespan Measurements The chronological lifespan assay protocol was adapted from Fabrizio and Longo (2007). An overnight culture of the indicated strains in YPD was diluted to OD600 = 0.1 in 20 ml SDC medium (SD complete + 43 His, Leu, Met, Ura) in 100 ml flasks. Rapamycin (BioChemica) diluted in DMSO was added to a final concentration of 10 nM, and the corresponding volume of DMSO was added to the control flasks. Cells were incubated at 30 C, shaking at 230rpm, and samples were taken for viability measurements every day. The viability staining protocol was used as described above, with the following adjustments to circumvent the poor pelleting in synthetic medium: cells were spun for 2 min at 7,000 rpm. The wash step was omitted, and the cells were directly resuspended in 50 mM sodium citrate (pH 7.4) containing 0.5 mM SYTOX Green (Life Technologies). For flow cytometry and western blotting protocols, please see the Supplemental Experimental Procedures. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, six figures, and two tables and can be found with this article online at http:// dx.doi.org/10.1016/j.celrep.2014.08.053. ACKNOWLEDGMENTS We acknowledge Monika Langlotz for FACS support; Marco Foiani, Elmar Schiebel, Ted Weinert, Gislene Pereira, Robbie Loewith, and Charlie Boone for reagents; and Andre´ Maicher and Arianna Lockhart for comments on the manuscript. J.K. has been supported by an HBIGS fellowship. This work was supported by the DFG (SFB 1036) to B.L. and the Excellence Frontiers Fonds. J.K. and B.L. designed experiments. J.K and K.B. carried out experiments and performed data analysis. J.K. and B.L. wrote the manuscript. Received: April 13, 2014 Revised: August 8, 2014 Accepted: August 22, 2014 Published: September 25, 2014

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