TEN1 Is Essential for CDC13-Mediated Telomere Capping

Telomere binding proteins protect chromosome ends from degradation and mask chromosome termini from checkpoint surveillance. In Saccharomyces cerevisiae, Cdc13 binds single-stranded G-rich telomere repeats, maintaining telomere integrity and length. Two additional proteins, Ten1 and Stn1, interact with Cdc13 but their contributions to telomere integrity are not well defined. Ten1 is known to prevent accumulation of aberrant single-stranded telomere DNA; whether this results from defective end protection or defective telomere replication is unclear. Here we report our analysis of a new group of ten1 temperature-sensitive (ts) mutants. At permissive temperatures, ten1-ts strains display greatly elongated telomeres. After shift to nonpermissive conditions, however, ten1-ts mutants accumulate extensive telomeric single-stranded DNA. Cdk1 activity is required to generate these single-stranded regions, and deleting the EXO1 nuclease partially suppresses ten1-ts growth defects. This is similar to cdc13-1 mutants, suggesting ten1-ts strains are defective for end protection. Moreover, like Cdc13, our analysis reveals Ten1 promotes de novo telomere addition. Interestingly, in ten1-ts strains at high temperatures, telomeric single-stranded DNA and Rad52-YFP repair foci are strongly induced despite Cdc13 remaining associated with telomeres, revealing Cdc13 telomere binding is not sufficient for end protection. Finally, unlike cdc13-1 mutants, ten1-ts strains display strong synthetic interactions with mutations in the POLα complex. These results emphasize that Cdc13 relies on Ten1 to execute its essential function, but leave open the possibility that Ten1 has a Cdc13-independent role in DNA replication.GENOME stability is critically dependent upon functional telomeres. DNA ends that lack telomeres, or that have dysfunctional telomeres, are metabolized by DNA repair processes; without an appropriate repair template, such chromosome ends can be resected or joined inappropriately with other chromosome ends. Thus, genomic integrity can be significantly compromised by telomere dysfunction, particularly in proliferating cells where cycles of instability may ensue due to creation of dicentric chromosomes (Bailey and Murnane 2006). Protein complexes that bind to the duplex and single-stranded telomere repeats are key for stabilizing the chromosome ends (de Lange 2005). In proliferating cells, this job is complicated not only because the terminal chromatin must be opened during the process of chromosome replication, but also because additional processes that metabolize DNA ends are active. For example, while nonhomologous end joining processes are preferentially used in repair of DNA double-strand breaks in G1, homologous recombination is preferentially used for this repair in S and G2 (Ferreira and Cooper 2004; Zierhut and Diffley 2008). Given these complexities, it is not surprising that our molecular understanding of how telomere proteins protect chromosomes ends is incomplete.Budding yeast has been useful for dissecting how cells correctly metabolize their chromosome ends. In Saccharomyces cerevisiae, the terminal DNA comprises approximately 300 bp of TG_1-3/C_1-3A sequences, ending with a short single-stranded overhang of the G-rich repeats. This 3′ overhang is ∼12–14 nucleotides, although during the late S/G2 phase of the cell cycle, it becomes longer, >30 nucleotides in length (Wellinger et al. 1993b; Dionne and Wellinger 1996; Larrivee et al. 2004). Central among factors that prevent inappropriate telomere degradation in S. cerevisiae is Cdc13, a protein that binds to single-stranded telomere G-rich repeats (Garvik et al. 1995; Lin and Zakian 1996; Nugent et al. 1996). Reducing Cdc13 function through either the cdc13-1 temperature sensitive (ts) allele or the cdc13-td conditional null (degron) allele results in telomere C-strand loss, with degradation continuing into the subtelomeric chromosomal regions (Garvik et al. 1995; Vodenicharov and Wellinger 2006). Correspondingly, homologous recombination at chromosome termini increases in cdc13-1 strains (Carson and Hartwell 1985; Garvik et al. 1995). The loss of Cdc13 unmasks the telomeres, provoking activation of the DNA damage checkpoint (Weinert and Hartwell 1993; Garvik et al. 1995). This protective role of Cdc13 is most likely its essential function.A thorough, mechanistic understanding of how Cdc13 mediates chromosome end protection is hampered in part because the activities responsible for the loss of the telomere C strand are not fully known. At normal telomeres, the Mre11-Rad50-Xrs2 complex has a role regulating resection required for telomere addition, whereas the Exo1 nuclease, Rad9 and Rad24 checkpoint proteins each influence the resection process at uncapped telomeres (Lydall and Weinert 1995; Maringele and Lydall 2002; Larrivee et al. 2004; Zubko et al. 2004). The 5′-to-3′ resection of both normal and uncapped telomeres is regulated by the activity of Cdk1, the yeast cyclin-dependent kinase (Frank et al. 2006; Vodenicharov and Wellinger 2006). Similar to the activities that promote 5′-to-3′ degradation of DNA ends at double-strand breaks (Aylon et al. 2004; Ira et al. 2004), the activities that lead to telomere resection are active in late S and G2 cell cycle phases (Wellinger et al. 1993a, 1996; Marcand et al. 2000; Vodenicharov and Wellinger 2006). Interestingly, Cdc13 is required to prevent degradation at telomeres only in proliferating cells and not when cells are blocked in stationary phase (Vodenicharov and Wellinger 2006). Additional factors, such as the S. cerevisiae Rap1 protein, prevent chromosome fusions by nonhomologous recombination during the G1 phase of the cell cycle (Pardo and Marcand 2005; Marcand et al. 2008).At least two additional proteins, Stn1 and Ten1, aid the capping role of Cdc13. Like CDC13, both STN1 and TEN1 are essential, and loss of their function leads to excessive single-stranded telomeric DNA (Grandin et al. 1997, 2001; Petreaca et al. 2007). STN1 was originally identified as a high copy suppressor of cdc13-1 temperature sensitivity (Grandin et al. 1997), and TEN1 was similarly isolated as a dosage suppressor of stn1-13 (Grandin et al. 2001). Combining either the cdc13-1 allele with stn1 mutations or the ten1-31 allele with stn1-13 is lethal (Grandin et al. 2001; Petreaca et al. 2007). The essential nature of these genes makes it difficult to clearly differentiate whether these genes operate in the same, or in parallel pathways to protect telomeres. A compelling argument that Cdc13, Stn1, and Ten1 likely function in a common pathway is that, in addition to these genetic interactions, Stn1 and Ten1 proteins interact with one another both in vivo and in vitro (Grandin et al. 2001; Gao et al. 2007), and each associates with Cdc13 in the yeast two-hybrid assay (Grandin et al. 1997, 2001; Petreaca et al. 2007). From these data, Cdc13, Stn1, and Ten1 are suggested to function as a single complex that mediates chromosome end protection in S. cerevisiae. Such a complex would share some similarities with the single-stranded DNA binding complex RPA (Gao et al. 2007). Whether these proteins normally operate exclusively as a heterotrimeric complex is still not entirely clear. Stn1 and Ten1 can make contributions to capping that are independent of Cdc13, as shown in experiments where overproducing the Stn1 essential domain with Ten1 replaced the essential function of Cdc13 (Petreaca et al. 2006). In addition, while the Schizosaccharomyces pombe Stn1 and Ten1 homologs are critical for telomere protection, they do not interact with Pot1, the single-stranded telomere binding protein that is also critical for telomere capping (Martin et al. 2007).The role of Ten1 in maintaining both telomere integrity and length homeostasis is not understood. It has been assumed that Stn1 and Ten1 play the same role as Cdc13 in maintaining telomere integrity, namely, preventing inappropriate terminal resection. However, whether this is in fact the case is not entirely clear. For one, disrupting the DNA replication machinery can give rise to an excess of terminal single-stranded DNA, although in this case, the ssDNA accumulation is attributed to a failure to synthesize the lagging DNA strand rather than removing a block to telomere resection (Diede and Gottschling 1999; Adams Martin et al. 2000). Although both Cdc13 and Stn1 are thought to act as capping proteins, each can interact with Polα subunits (Qi et al. 2003; Grossi et al. 2004; Petreaca et al. 2006), making it important to evaluate Ten1 function more carefully. Our goal here was to compare how Cdc13 and Ten1 promote chromosome end protection, first by testing whether Ten1 acts to prevent telomere resection from activities comparable to those that degrade telomeres in cdc13-1, and second by determining the impact of ten1 dysfunction upon Cdc13. The cdc13-1 allele has been extremely useful in analyzing the CDC13 essential function; TEN1 analysis has been hindered by a lack of equivalent genetic reagents. Here we have created a collection of ten1-ts alleles useful for probing the essential role of TEN1. Analysis of these alleles, which show constitutive telomere elongation, reveals that Ten1 promotes telomere capping with a similar cell cycle dependency as Cdc13, protecting ends during the period in which mitotic forms of Cdk1 are active. Critically, by showing that single-stranded DNA is generated in ten1-ts strains under conditions where semi-conservative replication is complete, we conclude that Ten1 truly can function as a capping protein. Moreover, the ten1-ts strains fail to restrain degradation of chromosome ends and induce formation of Rad52 repair foci, despite the association of wild-type Cdc13 with telomeres, indicating not only that Cdc13 binds telomeres independent of Ten1 function, but also that Cdc13 telomere localization is not sufficient for end protection. Finally, although the ten1-ts capping-deficient phenotypes parallel cdc13-1, only the ten1-ts strains are highly sensitive to impaired POL1 function, leaving open the possibility that TEN1 function additionally impacts terminal replication.