Deletion of MEC1 suppresses the replicative senescence of the cdc13-2 mutant in Saccharomyces cerevisiae

Abstract In Saccharomyces cerevisiae, telomerase recruitment to telomeres depends on a direct interaction between Cdc13, a protein that binds single-stranded telomeric DNA, and the Est1 subunit of telomerase. The cdc13-2 allele disrupts telomerase association with telomeres, resulting in progressive telomere shortening and replicative senescence. The Mec1/ATR kinase is both a positive and a negative regulator of telomerase activity and is required for the cell cycle arrest in telomerase-deficient senescent cells. In this study, we find that the deletion of MEC1 suppresses the replicative senescence of cdc13-2. This suppression is dependent on telomerase, indicating that Mec1 antagonizes telomerase-mediated telomere extension in cdc13-2 cells to promote senescence.


Introduction
Telomeres are nucleoprotein structures that protect the ends of chromosomes by preventing natural chromosome ends from being recognized and processed as DNA double-strand breaks (Jain and Cooper 2010). Telomeric DNA consists of G/C-rich DNA repeats, with the G-rich strand extending to form a 3′ single-stranded overhang. Specialized proteins bind to both the double-stranded telomeric repeats and the 3′ single-stranded overhang to ensure proper telomere function. Due to incomplete DNA replication and nucleolytic degradation, telomeres shorten with each cell division. Cells express the enzyme telomerase to extend telomeres and counteract this "end replication problem" (Jain and Cooper 2010). In the budding yeast Saccharomyces cerevisiae, telomerase minimally consists of the protein catalytic subunit Est2 and the noncoding RNA subunit TLC1 (Singer and Gottschling 1994;Lingner et al. 1997). Additional proteins, Est1 and Est3, are required for telomerase activity in vivo and are thought to be involved in telomerase recruitment and/or activation (Wellinger and Zakian 2012). Elimination of any of the Est subunits or TLC1 results in gradual telomere shortening and eventual replicative senescence-the socalled "ever shorter telomeres" (est) phenotype (Lundblad and Szostak 1989;Singer and Gottschling 1994;Lendvay et al. 1996).
Cdc13 is a single-stranded telomeric DNA-binding protein that is essential for telomere end protection and telomerase recruitment (Wellinger and Zakian 2012). These functions are separable. The telomerase recruitment function is mediated by a direct interaction between the recruitment domain (RD) of Cdc13 and Est1 (Pennock et al. 2001). The Cdc13 RD contains 2 conserved Est1-binding motifs called Cdc13 EBM-N and Cdc13 EBM-C (Chen et al. 2018). Mutations in Cdc13 EBM-N abolish the Cdc13-Est1 interaction in vitro, but in vivo cause only a modest reduction in Est1 telomere association and telomere length (Chen et al. 2018). The Cdc13 EBM-N -Est1 telomerase recruitment pathway works in parallel with a second pathway involving Sir4, the yKu complex, and TLC1. Sir4 is recruited to telomeres via the double-strand telomeric DNA-binding protein Rap1 (Moretti et al. 1994). Sir4 interacts with the Yku80 subunit of the yKu complex (Roy et al. 2004), which binds to the tip of a 48-nt hairpin in TLC1 (Peterson et al. 2001;Stellwagen et al. 2003;Chen et al. 2018). Mutations that disrupt the yKu-TLC1 interaction (e.g. tlc1Δ48) cause a modest reduction in telomere length (Peterson et al. 2001). Disrupting both the Cdc13 EBM-N -Est1 and the yKu-TLC1 interactions results in an est phenotype (Chen et al. 2018).
The role of the Cdc13 EBM-C motif is more enigmatic. The wellstudied cdc13-2 allele has a mutation (E252K) located within this motif (Chen et al. 2018) and causes a dramatic reduction in Est1 telomere association in vivo and an est phenotype (Nugent et al. 1996;Chan et al. 2008). However, the mutant Cdc13-2 protein can still interact with Est1 in vitro (Wu and Zakian 2011). Thus, although it is clear that the Cdc13 EBM-C motif is required for a stable association of telomerase to telomeres, it is unclear how exactly it does so (Chen et al. 2018).
The Tel1 and Mec1 PI3K-like DNA damage checkpoint kinases (ATM and ATR in mammals, respectively) are also important for telomerase activity. Strains harboring a mutation in TEL1 maintain short, but stable, telomeres (Greenwell et al. 1995). Mec1 has only a modest role in telomere length homeostasis, but mec1 tel1 double mutants have an est phenotype (Ritchie et al. 1999), which can be suppressed by the expression of either a Cdc13-Est1 or a Cdc13-Est2 fusion protein (Tsukamoto et al. 2001). ATM and ATR are also required for telomere elongation in mouse and human cells (Lee et al. 2015;Tong et al. 2015). In addition to this positive regulatory role, Mec1 also inhibits telomerase at DNA doublestrand breaks through 2 separate mechanisms: (1) via direct phosphorylation of Cdc13 on amino acid S306 (Zhang and Durocher 2010), and (2) via phosphorylation of the helicase and telomerase inhibitor Pif1, which requires the Mec1 downstream kinases Rad53 and Dun1 (Makovets and Blackburn 2009). Mec1 is also required for responding to critically short telomeres and activating a G2/M cell cycle arrest in telomerase-null senescent cells (Enomoto et al. 2002;IJpma and Greider 2003;Abdallah et al. 2009).
We recently discovered that mutation of PIF1 can suppress the est phenotype of the cdc13-2 mutant (Fekete-Szücs et al. 2022). In this study, we show that deletion of MEC1 can also suppress the cdc13-2 est phenotype. Thus, Mec1 acts to inhibit telomerase activity in cdc13-2 strains. Mec1-dependent phosphorylation of Cdc13 and Pif1 is either not required or insufficient for this inhibition.

Yeast strains and plasmids
All yeast strains used in this study are listed in Table 1. Standard yeast genetic and molecular methods were used (Sherman 2002;Amberg et al. 2005). Plasmids pFR95 (pRS415-cdc13-E252K), pYY1 (pRS415-cdc13-S306A), and pYY2 (pRS415-cdc13-E252K, S306A) were created by site-directed mutagenesis of pDD4317 (pRS415-CDC13; Strecker et al. 2017) using primers designed by NEBaseChanger and the Q5 site-directed mutagenesis kit (New England Biolabs, cat. no.: E0554S). The mutations were confirmed by DNA sequencing. Plasmid pEFS4 (pRS415-cdc13-F237A) was generated in the same manner in a previous study (Fekete-Szücs et al. 2022). All mec1Δ strains in this study are accompanied by an sml1Δ mutation, which suppresses mec1Δ lethality by increasing dNTP levels (Zhao et al. 1998). While the effects reported here are likely due to mec1Δ, it should be noted that deletion of SML1 by itself does slightly impact telomere biology (Gupta et al. 2013;Maicher et al. 2017).

Liquid culture senescence assay
Liquid culture senescence assays were performed essentially as previously described (van Mourik et al. 2016). Each senescence assay started with diploid strains that were induced to sporulate. Freshly dissected haploid spores were allowed to form colonies on YPD agar plates after 2 days of growth at 30°C. Cells from these colonies were serially passaged in liquid culture medium at 24-h intervals. For each passage, the cell density of each culture was measured by optical density (calibrated by cell counting using a hemocytometer), and the cultures were diluted back into fresh medium at a cell density of 2 × 10 5 cells/ml. Cell density was plotted as a function of population doublings.

Deletion of MEC1 can bypass the est phenotype of the cdc13-2 mutant
To study the role of Mec1 in cdc13-2 cells, we sporulated CDC13/ cdc13-2 MEC1/mec1Δ sml1Δ/sml1Δ diploid cells and followed the  -11,15/ his3-11,15 leu2-3,112/leu2-3,112 This study growth of the haploid meiotic progeny by serial propagation in liquid cultures for several days. The sml1Δ mutation was necessary to overcome the lethality of mec1Δ (Zhao et al. 1998). We find that cdc13-2 mec1Δ sml1Δ strains do not senesce (Fig. 1a) and maintain short, but stable, telomeres (Fig. 1b). Since Mec1 and Tel1 have partially redundant functions, we tested whether deletion of TEL1 could suppress the cdc13-2 est phenotype. We find that it cannot (Fig. 1c). The suppression of senescence by mec1Δ is telomerase dependent, since it has been previously reported that mec1Δ sml1Δ tlc1Δ triple mutants exhibit an est phenotype (Enomoto  IJpma and Greider 2003). Nevertheless, since the cdc13-2 mutation reduces the association of Est1 with telomeres, it was possible that deletion of MEC1 specifically bypasses the need for Est1 for telomerase activity. To test this idea, we sporulated an EST1/est1Δ MEC1/mec1Δ sml1Δ/sml1Δ diploid strain and monitored the growth of the haploid meiotic progeny (Fig. 1d). We find that est1Δ mec1Δ sml1Δ strains senesce, indicating that deletion of MEC1 cannot bypass the need for Est1. We previously reported that the pif1-m2 mutant, which is depleted for nuclear Pif1 (Schulz and Zakian 1994), can suppress the cdc13-2 est phenotype, but not if the Sir4-yKu-TLC1 pathway is disrupted (Fekete-Szücs et al. 2022). To test whether this was also true for mec1Δ suppression of cdc13-2, we monitored the growth of haploid meiotic progeny derived from the sporulation of a CDC13/cdc13-2 MEC1/mec1Δ SML1/sml1Δ TLC1/tlc1Δ48 diploid strain (Fig. 1e). We find that cdc13-2 mec1Δ sml1Δ tlc1Δ48 strains senesce, suggesting that recruitment of telomerase via the Sir4-yKu-TLC1 pathway is important for telomere maintenance in cdc13-2 mec1Δ sml1Δ cells. However, the tlc1Δ48 mutation leads to a ∼48% reduction in TLC1 abundance (Zappulla et al. 2011), which could also explain the senescence of cdc13-2 mec1Δ sml1Δ tlc1Δ48 strains. Regardless, our findings indicate that telomere length homeostasis of the cdc13-2 mec1Δ sml1Δ strain depends on telomerase. We also observe that cdc13-2 tlc1Δ48 strains senesce more rapidly than cdc13-2 mec1Δ sml1Δ tlc1Δ48 strains, which is consistent with Mec1 being important for inducing senescence (Enomoto et al. 2002;IJpma and Greider 2003).
Mec1 carries out most of its functions through phosphorylation of the effector kinase Rad53 in response to DNA damage or replication stress (Pardo et al. 2017). We therefore tested whether deletion of RAD53 can suppress the cdc13-2 est phenotype. We sporulated a CDC13/cdc13-2 RAD53/rad53Δ sml1Δ/sml1Δ diploid strain and monitored the growth of the haploid meiotic progeny (Fig. 2b). Deletion of RAD53 was unable to suppress the cdc13-2 est phenotype, indicating that Mec1-dependent activation of Rad53 is either not required or insufficient for inducing senescence in cdc13-2 cells.
Mec1 can phosphorylate Cdc13 on amino acid S306, in a Rad53-independent manner, and this phosphorylation is important to inhibit telomerase activity at DNA double-strand breaks (Tseng et al. 2006;Zhang and Durocher 2010). To test whether the cdc13-S306A mutation could suppress the cdc13-2 est phenotype, we combined the cdc13-2 mutation (E252K) with the S306A mutation. We find that cdc13-E252K,S306A mutants senesce (Fig. 2c). To rule out the possibility that Mec1-dependent phosphorylation of both Cdc13 and Pif1 is important to induce senescence in cdc13-2 cells, we monitored the growth of cdc13-E252K,S306A pif1-4A cells (Fig. 2d). While the pif1-4A mutation delays the senescence of cdc13-E252K and cdc13-E252K,S306A cells, it cannot suppress senescence. Thus, Mec1-dependent phosphorylation of Cdc13 and Pif1 is either not required or insufficient for inducing senescence in cdc13-2 cells. Our findings are consistent with previous observations, suggesting that Mec1-dependent phosphorylation of Cdc13 and Pif1 does not inhibit telomerase-mediated extension of native telomeres, only at DNA double-strand breaks (Makovets and Blackburn 2009;Zhang and Durocher 2010).
At present, we do not know what is the relevant Mec1 target or targets responsible for inducing senescence in cdc13-2 cells. Since the tlc1Δ48 mutation causes cdc13-2 mec1Δ sml1Δ cells to senesce, it is possible that Mec1 inhibits the Sir4-yKu-TLC1 telomerase recruitment pathway. Sir4 is among many putative targets of Mec1 identified using mass spectrometry-based approaches (Smolka et al. 2007;Chen et al. 2010;Bastos de Oliveira et al. 2015). Further studies are needed to fully elucidate the role of Mec1 in regulating telomerase activity.

Data availability
Strains and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are included within the article, figures, and tables. Funding Y.Y. was supported by an Abel Tasman Talent Program scholarship. F.R.R.B. was supported by a CONACYT scholarship.

Conflicts of interest
The authors declare no conflict of interest.