Telomeres, specialized nucleoprotein structures located at the end of linear chromosomes in eukaryotic cells, are crucial for maintaining genomic stability and protecting chromosomal ends from being perceived as DNA breaks (Wellinger and Zakian, 2012). In the budding yeast Saccharomyces cerevisiae, telomeric DNA consists of approximately ~300 ± 75 base pairs of C_1-3A/TG_1-3 repeats with a 3' G-rich single-stranded overhang (Wellinger and Zakian, 2012). Adjacent to the telomeric TG_1-3 repeats, there are subtelomeric repeat elements known as X- and Y’-elements, which vary between telomeres, as well as strains (Chan and Tye, 1983a; Chan et al., 1983b; Louis, 1995). The Y’-elements, immediately internal to the telomeric repeats, are present as a tandem array of 0–4 copies, they fall into two major size classes, 6.7 kb Y’-long (Y’-L) and 5.2 kb Y’-short (Y’-S) (Chan and Tye, 1983a; Chan et al., 1983b). Y’-elements are highly conserved with only ~2% divergence between strains (Louis and Haber, 1992). One entire Y’-element contains two large open-reading frames (ORFs), an ARS consensus sequence (ACS), and a STAR element (subtelomeric anti-silencing regions) consisting of binding sites for Tbf1 and Reb1 (Chan and Tye, 1983a; Chan et al., 1983b; Fourel et al., 1999; Louis and Haber, 1992). The X-element, a much more heterogeneous sequence abutting Y’-elements or telomeric repeats, contains the 473 bp ‘core X’ sequence and the subtelomeric repeats (STRs) A, B, C, and D (Louis and Haber, 1991; Louis et al., 1994). The STRs are found in some chromosome ends, while the ‘core X’ sequence is shared by all chromosomes. Recent long-read sequencing shows that subtelomeric regions display high evolutionary plasticity and are rich in various structure variants such as reciprocal translocations, transpositions, novel insertions, deletions, and duplications (O’Donnell et al., 2023).

Telomeric DNA elongation primarily relies on telomerase, an enzyme comprising a reverse transcriptase, an RNA component, and accessory factors (Palm and de Lange, 2008; Wellinger and Zakian, 2012). In S. cerevisiae, the telomerase holoenzyme consists of the reverse transcriptase Est2, the RNA template TLC1, and accessory factors Est1, Est3, Pop1/Pop6/Pop7 proteins (Lemieux et al., 2016; Lendvay et al., 1996; Lundblad and Szostak, 1989; Singer and Gottschling, 1994). In the absence of telomerase, homologous recombination can take place to replicate telomeres, resulting in telomerase-deficient ‘survivors’ (Lundblad and Blackburn, 1993; Teng and Zakian, 1999). These survivors are broadly categorized into Type I and Type II based on distinct telomere structures (Lundblad and Blackburn, 1993; Teng and Zakian, 1999). Type I survivors possess tandem amplified Y’-elements (both Y’-L and Y’-S) and very short TG_1-3 tracts, indicating that Y’-elements serve as substrates for homologous recombination. Type II survivors display long heterogeneous TG_1-3 tracts. On solid medium, approximately 90% of the survivors are Type I, while 10% are Type II (Teng et al., 2000). However, in liquid culture, Type II survivors grow faster and eventually dominate the population (Teng and Zakian, 1999). The proteins required for ype I and II survivor formation appear to be different. Type I survivors depend on Rad51, Rad54, Rad55, Rad57, and Pif1 (Chen et al., 2001; Hu et al., 2013; Le et al., 1999). while the formation of Type II survivors requires the Mre11/Rad50/Xrs2 (MRX) complex, KEOPS complex, Rad59, Sgs1, and Rad6, most of which are critical for DNA resection (Chen et al., 2001; He et al., 2019; Hu et al., 2013; Johnson et al., 2001; Le et al., 1999; Huang et al., 2001; Nicolette et al., 2010; Teng et al., 2000; Wellinger and Zakian, 2012; Wu et al., 2017). Although Type I and II pathways are working independently, Kockler et al. found that the proteins involved in each pathway can work together via two sequential steps and contribute to a unified ALT (alternative lengthening of telomeres) process (Kockler et al., 2021).

The amplification of Y’-elements represents a significant feature of telomere recombination in telomerase-null Type I survivors (Lundblad and Blackburn, 1993; Teng and Zakian, 1999), and as a result, extrachromosomal Y’ circular DNAs have been observed in Type I survivors (Larrivée and Wellinger, 2006). Additionally, Y’-element acquisition has been observed in the initiation step of pre-senescence, suggesting a potential role for Y’-elements in Type II survivor formation (Churikov et al., 2014). Furthermore, Y’-elements are mobilized through a transposition-like RNA-mediated process involving Ty1 activity in telomerase-negative survivors (Maxwell et al., 2004). Y’-elements also express potential DNA helicases, Y’-Help, in telomerase-null survivors (Yamada et al., 1998). Thus, Y’-elements play a significant role as donors in homologous recombination-mediated telomere maintenance. The functions of X-elements, on the other hand, are less clear. The ‘core X’ sequence consists of an ACS element and, in most cases, an Abf1 binding site (Louis, 1995), and acts as a protosilencer (Lebrun et al., 2001). In contrast, STRs and Y’-STAR possess anti-silencing properties that limit the spreading of heterochromatin (Fourel et al., 1999). Interestingly, a previous study demonstrated that telomeres with Χ-only ends (containing only X-elements) were more efficiently elongated compared to those with X-Y’ ends (containing both X- and Y’-elements) in tel1Δ rif1Δ strains (Craven and Petes, 1999). Moreover, subtelomeric elements (including X-elements) and associated factors like Reb1 and Tbf1 antagonize telomere anchoring at the nuclear envelope (Hediger et al., 2006). However, considering that X-elements are present in all telomeres while Y’-elements are not, the specific functions of X- and Y’-elements in genome integrity after the evolution of telomerase have long been a subject of questioning (Jäger and Philippsen, 1989; Zakian and Blanton, 1988).

In wild-type yeast strain BY4742, there are 8 Y’-S and 11 Y’-L elements at the 32 telomere loci. Additionally, each telomere locus contains one X-element. The genetic deletion of all X- and Y’-elements to directly investigate the roles of X- and Y’-elements in genome integrity is a challenging and complex task. In this study, we utilized recently reported chromosome-fused budding yeast strains (Shao et al., 2018) to eliminate both X- and Y’-elements completely. This approach allows us to reinvestigate the roles of X- and Y’-elements at telomeres.

The wild-type yeast strain BY4742, commonly used in laboratories, possesses 19 Y’-elements at 17 telomere loci and 32 X-elements at 32 telomere loci. This abundance of Y’-elements and X-elements poses challenges for loss-of-function studies, highlighting the need for a strain lacking all Y’-elements and X-elements. Fortunately, we have previously constructed the single-chromosome yeast strain SY14, which contains only one copy of Y’-element and two copies of X-element (Shao et al., 2018), and could have been an ideal tool. However, the telomerase-null survivors of SY14 mainly bypassed senescence through chromosomal circularization, providing limited insights into the roles of Y’- and X-elements in telomere maintenance (Wu et al., 2020). Therefore, in this study, we employed the SY12 strain, which has three chromosomes, to investigate the functions of Y’- and X-elements at telomeres (Figure 2A, left panel).

We constructed the SY12^YΔ, SY12^XYΔ+Y, and SY12^XYΔ strains, which lack the Y’-element, X-elements, and both X- and Y’-elements, respectively (Figure 3A). Surprisingly, the SY12^YΔ, SY12^XYΔ, and SY12^XYΔ+Y strains exhibited minimal defects in cell proliferation, genotoxic sensitivity, and telomere homeostasis (Figures 3 and 4). These results demonstrate, for the first time, that both X- and Y’-elements are dispensable for cellular functions. Thus, the SY12^YΔ, SY12^XYΔ, and SY12^XYΔ+Y strains established in this study, with their simplified telomere structures, are valuable resources for telomere biology research.

Subtelomeric regions are known to be highly variable and often contain species-specific homologous DNA sequences. In the case of fission yeast, subtelomeric regions consist of subtelomeric homologous (SH) and telomere-distal sequences. Previous studies have shown that SH sequences in fission yeast do not significantly impact telomere length, mitotic cell growth, or stress responses. However, they do play a role in buffering against the spreading of silencing signals from the telomere (Tashiro et al., 2017). Though the ‘core X’ sequence acts as a protosilencer (Lebrun et al., 2001), the X-STRs and Y’-STAR possess anti-silencing properties that limit the spreading of heterochromatin in budding yeast (Fourel et al., 1999), the telomere position effect remains effective in the strains that lack both X- and Y’-elements (Figure 4B). Given the remarkable differences in both sequence and size between the subtelomeric regions of budding yeast and fission yeast, it is difficult to compare the extent to which subtelomeric elements affect telomere silencing.

Amplification of Y’-element(s) is a characteristic feature of canonical Type I survivors. Type I survivors emerged in SY12 strain, indicating that multiple Y’-elements in tandem are not strictly required for Type I recombination (Figure 2D). Interestingly, the telomerase-null SY12^YΔ and SY12^XYΔ cells, lacking Y’-elements, failed to generate Type I survivors but could generate Type II survivors, indicating that the acquisition of Y’-elements is not a prerequisite for Type II survivor formation (Figures 5C and 6B). These observations support the notion that Type I and Type II survivors form independently, although both may utilize a common alternative telomere-lengthening pathway (Kockler et al., 2021). Moreover, a subset of SY12 tlc1Δ, SY12^YΔ tlc1Δ, SY12^XYΔ+Y tlc1Δ, and SY12^XYΔ tlc1Δ cells could escape senescence and become survivors through microhomology-mediated intra-chromosomal end-to-end fusion (chromosome circularization) (Figures 2D, 5C, and 6A and B, labeled in blue). Notably, the survivors with all circular chromosomes were readily recovered from the telomerase-null SY11 to SY14, but not SY1 to SY10 cells (Figure 1). Several reasons could account for this. First, a smaller number of telomeres provides fewer recombination donors and acceptors, resulting in less efficient inter-chromosomal homologous recombination (e.g., TG_1-3 tracts recombination or Y’-element acquisition). Second, the continuously shortened telomeres of linear chromosomes may trigger another round of senescence, while survivors with circular chromosomes do not encounter end-replication problems and therefore exhibit greater stability. Third, the presence of homologous sequences at both chromosome ends appears to be a minimum requirement for microhomology-mediated intra-chromosomal end-to-end fusion. With fewer homologous sequences, the probability of chromosome circularization decreases, and with more chromosomes, the likelihood of circularizing each chromosome within a cell diminishes. Fourth, in cells with fewer telomeres, intra-chromosomal telomere fusions are more likely to occur, while lethal inter-chromosomal fusions are competed out. However, we can speculate that in telomerase-null cells with eroded chromosome ends, stochastic repair mechanisms such as homologous recombination, microhomology-mediated end joining, and inter- and intra-chromosomal fusions operate simultaneously. Only those survivors that maintain a relatively stable genome and robust growth can be experimentally recovered.

S. cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast) are the most commonly used laboratory systems, separated by approximately 1 Gya (billion years ago) according to molecular-clock analyses (Hedges, 2002). Despite both species having genomes are both over 12 megabases in length, haploid S. cerevisiae contains 16 chromosomes, while S. pombe has only 3 chromosomes (Forsburg, 2005). The telomerase-independent mechanisms for maintaining chromosome ends differ between these two yeasts. In budding yeast, homologous recombination is the primary mode of survival in telomerase-deficient cells, resulting in the generation of Type I or Type II survivors (McEachern and Haber, 2006). Telomerase- and recombination-deficient cells occasionally escape senescence through the formation of palindromes at chromosome ends in the absence of EXO1 (Maringele and Lydall, 2004). Fission yeast cells lacking telomerase can also maintain their chromosome termini by recombining persistent telomere sequences, and survivors with all intra-circular chromosomes (Nakamura et al., 1998) or intermolecular fusions (Tashiro et al., 2017; Wang and Baumann, 2008) have been observed. In our research, some SY12 tlc1Δ cells, which have three chromosomes, also bypassed senescence by circularizing their chromosomes (Figure 2D), suggesting that a lower chromosome number increases the likelihood of recovering survivors containing circular chromosomes.

While most eukaryotes employ telomerase for telomere replication, some eukaryotes lack telomerase and utilize recombination as an alternative means to maintain telomeres (Biessmann and Mason, 1997). In Drosophila, telomeres are replicated through a retrotransposon mechanism (Levis et al., 1993; Louis, 2002). The structure and distribution of Y’-elements in S. cerevisiae suggest their origin from a mobile element (Jäger and Philippsen, 1989; Louis and Haber, 1992), and Y’-elements can be mobilized through a transposition-like RNA-mediated process (Maxwell et al., 2004). In telomerase-deficient yeast cells, homologous recombination can acts as a backup mechanism for telomere replication (Lundblad and Blackburn, 1993), and the reintroduction of telomerase efficiently inhibits telomere recombination and dominates telomere replication (Chen et al., 2009; Peng et al., 2015; Teng and Zakian, 1999), These findings suggest that subtelomeric region amplification mediated by recombination and/or transposition may represent ancient telomere maintenance mechanisms predating the evolution of telomerase (de Lange, 2004). Therefore, subtelomeric X- and Y’-elements might be considered as evolutionary ‘fossils’ in the S. cerevisiae genome, and their elimination has little impact on telomere essential functions and genome stability.

All data generated or analysed during this study are included in the manuscript and supporting files; source data files have been provided for all figures.

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Author details

1. Can Hu

   The State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

2. Xue-Ting Zhu

   The State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

3. Ming-Hong He

   The State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China

   Contribution

   Writing – review and editing

   Competing interests

   No competing interests declared

4. Yangyang Shao

   Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

5. Zhongjun Qin

   Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

6. Zhi-Jing Wu

   1. The State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China
   2. Key Laboratory of Systems Health Science of Zhejiang Province, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

   Contribution

   Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review and editing

   For correspondence

   zhijing.wu@utsouthwestern.edu

   Competing interests

   No competing interests declared

7. Jin-Qiu Zhou

   1. The State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China
   2. Key Laboratory of Systems Health Science of Zhejiang Province, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

   Contribution

   Conceptualization, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   jqzhou@sibcb.ac.cn

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1986-8611

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