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# Transient telomere uncapping triggers telomeric and subtelomeric rearrangements

Liébaut Dudragne, Clotilde Garrido, Oana Ilioaia, Juliana Silva Bernardes, Zhou Xu

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Liébaut Dudragne, Clotilde Garrido, Oana Ilioaia, Juliana Silva Bernardes, Zhou Xu. Transient telomere uncapping triggers telomeric and subtelomeric rearrangements. 2025. hal-05371178

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1 **Title:** Transient telomere uncapping triggers telomeric and subtelomeric rearrangements

2 **Authors:** Liébaut Dudragne¹,², Clotilde Garrido¹,², Oana Iliaia¹,², Juliana Silva Bernardes³, Zhou Xu¹,²

3

4 **Affiliations:**

5 ¹ Sorbonne Université, CNRS, Laboratory of Computational, Quantitative and Synthetic Biology, CQSB, F-

6 75005 Paris, France.

7 ² Sorbonne Université, CNRS, Inserm, Institut de Biologie Paris-Seine, IBPS, F-75005 Paris, France.

8 ³ Sorbonne Université, CNRS, UMR 7144, Adaptation & Diversity in the Marine Environment, Station

9 Biologique de Roscoff (SBR), 29680 Roscoff, France.

10

11 **Correspondence:**

12 juliana.silva_bernardes@sorbonne-universite.fr

13 zhou.xu@sorbonne-universite.fr

14

15 **Keywords:** telomere, subtelomere, yeast, genome rearrangement, long-read sequencing

16
1 Abstract

2 Telomeres are nucleoprotein structures that cap the extremities of eukaryotic linear chromosomes, thus
3 preventing them from being detected as DNA damage. Telomere uncapping poses a profound threat to
4 genome integrity, yet the immediate consequences of transient uncapping remain unclear. In
5 *Saccharomyces cerevisiae*, the Cdc13-Stn1-Ten1 complex caps telomeres and limits resection, which
6 would otherwise lead to DNA damage checkpoint activation. Here, using the temperature-sensitive
7 *cdc13-1* allele, we demonstrate that even transient telomere uncapping induces extensive genomic
8 rearrangements within a few cell cycles, despite a functional DNA damage checkpoint. Two distinct
9 rearrangement signatures were observed in cells surviving transient uncapping: one characterized by the
10 reorganization and recombination of the subtelomeric region, mostly involving the Y' elements, and the
11 other exhibiting massively elongated telomeres up to 10 kb, corresponding to a ~30-fold increase. Long-
12 read sequencing revealed that the genomic instability was confined to the subtelomere and telomere
13 regions, and evidenced Y' element loss/amplification, terminal duplication of chromosome ends, and
14 telomeric-circle-driven amplification of telomere repeats. Rearrangements unfold over multiple
15 generations and require the homologous recombination factor Rad52 and the Polδ subunit Pol32, which
16 is essential for break-induced replication. The recombination proteins Rad51 and Rad59 also contribute
17 to the rearrangements in partially independent pathways. Remarkably, survivors with elongated
18 telomeres demonstrate robust resistance to subsequent telomere uncapping, in a Rad52-dependent
19 manner. Our findings provide novel insights into the consequences of transient telomere uncapping for
20 genome stability, a process that might contribute to subtelomere and telomere dynamics and evolution.
# 1 Introduction

Protection of chromosome ends is critical for genome integrity. In most Eukaryotes, it is ensured by specialized repetitive DNA sequences, known as telomeres, that prevent chromosome ends from being recognized and processed as DNA damage (de Lange, 2018; Jain & Cooper, 2010). However, telomeres shorten at each cell division due to the end-replication problem. This process is compensated for by the recruitment of telomerase, an enzyme capable of elongating telomeres and maintaining them at a steady-state length distribution (Wellinger & Zakian, 2012). In *Saccharomyces cerevisiae*, telomeric sequences are composed of ~350 bp of degenerated TG₁⁻₃ repeats with a short (5-15 nt) 3' single-stranded overhang (Larrivée et al., 2004; Soudet et al., 2014).

In yeast cells lacking telomerase, telomeres continue shorten to eventually reach a critical length at which point cells activate the DNA damage checkpoint and stop dividing, in a process called replicative senescence (Enomoto et al., 2002; IJpma & Greider, 2003; Lundblad & Szostak, 1989). Genome instability, especially near chromosome ends, has been shown to increase during this phase (Coutelier et al., 2018; Hackett et al., 2001; Hackett & Greider, 2003). In prolonged telomerase-negative cultures, rare cells can escape senescence at an estimated frequency of ~10⁻⁵ per generation by engaging in alternative, recombination-based pathways to maintain their chromosome ends (Chen et al., 2001; Kockler et al., 2021; Le et al., 1999; Lundblad & Blackburn, 1993; Teng et al., 2000; Teng & Zakian, 1999). These post-senescence survivors emerge through two distinct homology-dependent mechanisms: type I survivors amplify the subtelomeric Y' elements, whereas type II survivors elongate telomeric repeats, a mechanism also found in ALT cancer cells (Cesare & Reddel, 2010). Both types of survivors depend on Rad52, which is required for all types of recombination in yeast, and Pol32, a subunit of polymerase δ involved in break-induced replication (BIR) (Lundblad & Blackburn, 1993; Lydeard et al., 2007). Other genetic requirements for both types have been investigated in depth. For example, type I and type II survivors depend on the
recombination factors Rad51 and Rad59, respectively (Chen et al., 2001; Teng et al., 2000). Although the type I and type II pathways have long been thought to be independent, a recent work proposed a unified pathway in which Rad51 is required for the formation of precursors, followed by Rad51-independent but Rad59-dependent maturation into stable survivors (Kockler et al., 2021). Thus, telomere shortening leads to genome instability and rearrangements in telomerase-independent survivors, which in turn bypass the need for telomerase to maintain telomeres.

Telomere instability can also be induced when telomere protection fails. Telomeres are normally capped by the binding of specific proteins. Defects in these telomere-capping proteins or their binding have been shown to cause inappropriate repair events, such as telomere fusions or recombination involving telomeric or subtelomeric sequences (Fellerhoff et al., 2000; Garvik et al., 1995; Grandin, 2001; Grandin & Charbonneau, 2003; Marcand et al., 2008; Mieczkowski et al., 2003; Zubko & Lydall, 2006). These proteins include Rap1 and its cofactors Rif1, Rif2 and the Sir complex, the Ku complex, as well as the CST (Cdc13-Stn1-Ten1) complex (Wellinger & Zakian, 2012). Cdc13 is a key telomeric factor given its triple role in telomere maintenance: it assists telomere replication together with Stn1 and Ten1, promotes telomere elongation by recruiting telomerase and prevents excessive degradation of the 5' end by nucleases (Churikov et al., 2013). The *cdc13-1* allele is a temperature-sensitive mutant widely used to study telomere capping (Garvik et al., 1995; Hartwell & Smith, 1985; Mersaoui & Wellinger, 2019). The incubation of the *cdc13-1* mutant at restrictive temperatures (≥ 32°C) leads to resection of the 5' end and accumulation of single-stranded DNA (ssDNA) at telomeres, which activates the DNA damage checkpoint (Garvik et al., 1995; Lydall & Weinert, 1995). Permanent incubation of *cdc13-1* cells at restrictive temperature leads to cell death, despite a limited number of cell divisions allowed by adapting to the checkpoint in the first 24 hours (Lee et al., 1998; Sandell & Zakian, 1993; Toczyski et al., 1997). Cells resistant to permanent telomere uncapping can only be selected if the resection or checkpoint pathways are also altered (Grandin, 2001; Grandin & Charbonneau, 2003, 2013; Zubko & Lydall, 2006), suggesting that cells with
1 deprotected telomeres are eliminated in conditions with functional DNA processing and checkpoint, thus
2 safeguarding genome integrity. For example, checkpoint-deficient *mec3Δ cdc13-1* cells incubated at an
3 intermediate temperature of 29°C tolerated telomere deprotection and, after several passages,
4 generated Cdc13-independent survivors with very long telomeres akin to those of telomerase-negative
5 type II survivors (Grandin, 2001). Similar survivors were also generated in *cdc13-1 exo1Δ* mutants grown
6 at 36°C, and at a higher frequency in the checkpoint-deficient *cdc13-1 exo1Δ rad9Δ* mutant (Zubko &
7 Lydall, 2006).

8 Overall, Cdc13 dysfunction alone, without additional mutations in checkpoint- or resection-related genes,
9 has not been shown to lead to the emergence of cells with rearranged telomere structures. In addition,
10 previous studies of the long-term consequences of permanent telomere deprotection might have missed
11 the direct molecular response to telomere deprotection. Here, instead, we investigated the early effects
12 of telomere deprotection on genome stability in the presence of a functional checkpoint. To do so, we use
13 an experimental protocol where telomeres are transiently deprotected and cells are allowed to recover,
14 thus enabling us to characterize the full spectrum of molecular consequences, including events that would
15 have been counterselected if telomeres were kept deprotected permanently. We find that genomic
16 instability arises earlier than anticipated, even in the presence of a functional checkpoint, and is limited
17 to the telomere and subtelomere regions. Using long-read sequencing and telomere-to-telomere genome
18 assembly, we characterized Rad52- and Pol32-dependent rearrangements of the subtelomeric Y'
19 elements and telomere elongation up to 8-10 kb. Strikingly, we discovered that very long telomeres
20 together with Rad52 protect cells from a second telomere uncapping event, suggesting that this telomeric
21 nucleoproteic structure acts as a Cdc13-independent protective cap. Our results provide new insights into
22 telomere-driven genomic instability and underscore the impact of transient telomere dysfunction on
23 chromosome integrity.

24
1 **Results**

2 **Transient telomere uncapping impairs cell survival**

3 To evaluate the direct molecular and cellular response to telomeric uncapping, we employed the
4 temperature-sensitive cdc13-1 allele to induce a transient uncapping. We exposed the cdc13-1 strain to
5 the restrictive temperature (32°C) for various durations. The cells were first grown overnight in rich liquid
6 medium at the permissive temperature (23°C), then plated on solid YPD media and incubated at 32°C for
7 a defined time "t" (0–72 h) to induce telomere uncapping (Fig. 1A). The cells were then returned to the
8 permissive temperature to assess recovery and survival. The cells tolerated uncapping for up to 6 h
9 without a noticeable decrease in viability, but the survival frequency decreased significantly for t ≥ 12 h
10 (Fig. 1B and C). Some rare colonies formed even after uninterrupted incubation at 32°C for 72 h,
11 establishing a lower limit for cell survival to cdc13-1-induced telomeric uncapping at 32°C. The survival
12 frequency for t = 24 h was 5.5-fold greater than for t = 72 h (Fig. 1C).

13 We conclude that transient Cdc13 dysfunction at telomeres impairs cell survival in a time-dependent
14 manner. To investigate the molecular changes induced by transient uncapping, we selected the 24-h-long
15 uncapping condition for further analysis.
<figure><img src="image_3.png" /><figcaption>**Figure 1. Effect of transient telomere uncapping on survival.**</figcaption></figure>

(A) Scheme of the experimental protocol: *cdc13-1* cells were first grown at permissive temperature (23°C) before plating and incubation at restrictive temperature (32°C) for a duration *t*. The plate was then returned to 23°C for recovery and growth.

Individual colonies were picked for further analysis.

(B) Spot assay showing the growth of *cdc13-1* cells on YPD rich media after *t* hours of telomere uncapping.

(C) Colony formation frequency measured using the protocol shown in (A). A control plate was maintained at 23°C for normalization. The error bars correspond to the standard error of the mean; n = 7 (24 h) and n = 4 (72 h) independent experiments.

Cells exposed to telomere uncapping for 24 h show extensive genomic rearrangements

To study the impact of transient *cdc13-1*-mediated uncapping on genomic stability, we selected and grew colonies formed from cells that survived 24 h at 32°C. We investigated large-scale genomic rearrangements in these survivors using Pulsed Field Gel Electrophoresis (PFGE), which enables the resolution of whole chromosomes on agarose gels. Among the 7 analyzed colonies, 6 presented significant shifts in chromosome length, corresponding to changes of at least tens of thousands of base pairs (Fig. 2A).
Regions adjacent to the telomeric repeats typically contain Telomere-Associated Sequences (TASs), namely X and Y' elements. Y' elements, present in zero, one, or multiple tandem copies separated by short interstitial telomeric sequences, exist in two main size classes: long (6.7 kb) and short (5.2 kb). The X element, which is found at all extremities in laboratory strains, varies in sequence and size. To test whether telomeres or TASs are involved in the observed instability, we employed Terminal Restriction Fragment (TRF) Southern blotting, which probes telomere sequences in Xhol-restricted fragments to inform on characteristic features of the telomere structure. In the control cdc13-1 strain grown at 23°C, telomeres associated with Y'-containing subtelomeres ("Y' telomeres" in Fig. 2B) migrated to form a smear near 1.2 kb, corresponding to ~300 bp of telomeric repeats and a ~900 bp segment defined by the Xhol site in the Y' element. In contrast, telomeres at X-only subtelomeres ('X telomeres' in Fig. 2B) migrated as longer fragments owing to more distal Xhol restriction sites. Additionally, interstitial telomere sequences between Y' elements allowed probing of tandem Y' repeats ("Y' bands" in Fig. 2B). Out of 31 clones that survived transient telomere uncapping, 6 exhibited very long and heterogeneous telomeres (c8, c12, c13, c20, c21, and c27, in Fig. 2B and Supp. Fig. S1A), a pattern that resembles that observed in telomerase-independent, recombination-dependent, type II survivors (Lundblad & Blackburn, 1993). Almost all other survivors showed visible alterations in Y'-specific-band intensity and/or loss of X-associated telomeric bands, indicative of putative Y' amplification or acquisition at X-only subtelomeres, respectively. Y' amplification and acquisition are also observed in type I post-senescent survivors, although to a much greater extent where no X band is retained and the Y' signal is strongly amplified. Notably, we observed no telomere shortening in any clone, which is consistent with telomerase being active (Fig. 2B). We referred to these uncapping-induced rearrangement patterns as type-II-like (T-II-L) survivors for the survivors with lengthened telomeres and Y'-associated survivors (YAS) for those only exhibiting altered Y' organization.

24
1 **Genomic instability unravels over multiple cell divisions independently of checkpoint adaptation**

2 The PFGE profiles of several clones presented blurred bands or bands of different intensities, suggesting
3 intraclonal heterogeneity (Fig. 2A). To test this possibility, 3 primary clones were subcloned, and 4
5 subclones of each were reanalyzed by PFGE. All 3 primary clones exhibited subclonal heterogeneity in
6 their PFGE patterns, revealing that genomic instability persisted during several cell divisions (Fig. 2C and
7 indicative of at least 2 rounds of divisions with ongoing rearrangements. Whether these cell divisions
8 occurred while telomeres were still uncapped or after returning to 23°C is unclear.

9 Indeed, after prolonged activation (~8 h) of the DNA damage checkpoint, cells can undergo checkpoint
10 adaptation (Lee et al., 1998; Sandell & Zakian, 1993; Toczyski et al., 1997), resuming cell cycle despite
11 unrepaired DNA damage and allowing for a few cell divisions while telomeres are uncapped. This process
12 is associated with increased genomic instability (Coutelier et al., 2018; Galgoczy & Toczyski, 2001). To
13 assess the role of checkpoint adaptation both in genomic instability and in intraclonal heterogeneity, we
14 introduced the adaptation-deficient *cdc5-ad* allele of *CDC5*, encoding the Polo kinase essential for late cell
15 cycle events and for adaptation (Toczyski et al., 1997). *cdc5-ad cdc13-1* cells that survived 24 h of telomere
16 uncapping exhibited levels of genome rearrangements and intraclonal heterogeneity comparable to the
17 *CDC5 cdc13-1* strain (Supp. Fig. S1C and D). Since *cdc5-ad cdc13-1* cells do not divide while at 32°C, this
18 finding indicates that genomic instability could persist over several divisions after telomere protection is
19 re-established. To circumvent this intraclonal heterogeneity, all subsequent analyses were conducted
20 using subclones of survivor colonies.
<figure><img src="image_4.png" /><figcaption>Figure 2. Transient telomere uncapping leads to chromosomal instability and changes in telomere and subtelomere structure.</figcaption></figure>

(A) PFGE of 7 *cdc13-1* survivor clones as well as a *cdc13-1* control strain grown constantly at 23°C. Compared to the control strain, 6 out of the 7 survivor clones exhibited apparent chromosome size shifts, marked by orange arrows.

(B) TRF Southern blot analysis of 14 *cdc13-1* survivor clones and a control strain. The genomic DNAs were cut with the *Xhol* enzyme and a telomeric probe was used.

(C) PFGE of 4 subclones of clone 3 from (A) and (B), derived from single cells grown at 23°C. The orange boxes indicate bands with distinct migration patterns in the 4 subclones.

Genomic rearrangements are confined to telomeric regions

Given the important size shifts observed on the PFGE gels, we wondered whether the observed genomic instability was confined to the telomeric regions. To comprehensively assess the localization of the rearrangements, we performed Oxford Nanopore long-read sequencing on the genomic DNA of 9 survivors as well as the *cdc13-1* control strain, which did not experience telomere uncapping. The longest and highest quality Nanopore reads were assembled, and the assemblies were manually inspected to correct any assembly mistake in telomeric regions. This procedure yielded highly contiguous, high quality telomere-to-telomere genome assemblies (Supp. Table 2).
1 To investigate the rearrangements in telomeric regions, we first focused on the dynamics of Y' elements.

2 To track them accurately, Y' elements identified in the control strain genome assembly were labeled and

3 clustered based on sequence similarity (Fig. 3A). We identified 34 Y' elements found at 20 out of 32

4 extremities, with 0 to 6 tandem Y' elements per extremity (Supp. Data 1). Among the 34 Y' elements, we

5 detected 20 unique sequence variants that could be grouped into 11 clusters. Two elements were

6 classified as the same variant if their sequences were identical within the expected margin of assembly

7 errors. Clustering analysis recovered the two main size families of Y' elements—short and long—along

8 with finer sub-clusters, revealing the presence of outliers alongside large groups (Fig. 3A). Interestingly,

9 although Y' elements are typically classified as either 5.2 kb or 6.7 kb in size, we observed that Y' short

10 elements ranged from 5,062 bp to 5,592 bp (mean: 5,470 bp), Y' long elements ranged from 6,484 bp to

11 6,933 bp (mean: 6,670 bp), and one element exhibited an intermediate size of 5,981 bp.

12 Using this labeling and clustering approach, we quantified the number of Y' elements associated with

13 rearrangement events in the clones surviving transient uncapping (Fig. 3B and C). On average, 8.3 ± 3.7

14 (mean ± SD) Y' elements were lost and 18 ± 8.9 were gained in each survivor clone, affecting 7.4 ± 3.1

15 extremities, and far exceeding what could be confidently detected by TRF Southern blot analysis. These

16 events included loss or gain of a known Y' element, tandem amplification of existing elements, and most

17 interestingly, emergence of new Y' elements whose sequences did not correspond to any element present

18 in the control (Fig. 3C and D, Supp. Data 2). The frequency of these Y' structure alterations was significantly

19 higher at Y' telomeres than at X telomeres (28% vs. 15%; p-value = 0.007, Mann-Whitney test). We found

20 that at least 84% of the new Y' elements could be explained as mosaics of 2 or more other Y' elements,

21 suggesting that most of them originated from recombination events between existing Y' elements rather

22 than mutation accumulation. Interestingly, type-II-like survivors exhibited levels of Y' rearrangements

23 comparable to Y'-associated survivors (YAS: 7.3 ± 3.3 extremities affected, T-II-L: 7.7 ± 2.5 extremities

24 affected; mean ± SD).
1 We also examined whether genome rearrangements extended beyond the X and Y' elements into more
2 centromere-proximal regions. We found 10 cases of terminal duplications from one chromosome end to
3 another, spanning between 850 bp and 89 kb of sequences centromere-proximal to the X element across
4 5 clones (Fig. 3B). In 2 cases, 2 events were found at the same extremity, with one duplication contained
5 within the other. By mapping these regions against the control genome, we found that these events
6 consistently involved junctions in regions of homology between the donor and recipient chromosome
7 arms, the size of which ranged from several hundreds to several thousands of base pairs. Based on the
8 presence of homology and on the pattern of terminal duplication, these events likely resulted from break-
9 induced replication (BIR).

10 Finally, we used the variant callers Sniffles2 (Smolka et al., 2024) and PAV (Ebert et al., 2021) to identify
11 other structural rearrangements (>50 bp) that might have occurred in the rest of the genome. No event
12 was detected aside from those described above, indicating that genome rearrangements were confined
13 to telomere-proximal regions.
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>Figure 3. Subtelomeric Y' elements are rearranged following telomere uncapping.</figcaption></figure>

(A) Similarity matrix and clustering of Y' elements of the control strain. A similarity measure was computed for each pair of Y' sequences and used for subsequent hierarchical clustering (dendrogram shown above the matrix). The colors correspond to the different clusters (n = 11 clusters). The bar plot on the right side indicates the corresponding lengths of the Y' sequences. See *Supp. Data 1* for the sequences of all 34 Y' elements.

(B) Map of alterations at all chromosome extremities. The labelling of Y' elements was used to detect changes of Y' structure at a given telomere (orange square or triangle), and duplications in core genome sequences indicate probable BIR events (blue
1 square or triangle). YAS = Y'-associated survivors, T-II-L: Type-II-like survivors. Extremities with more than 5 Y' elements in the
2 reference could not always be resolved with high confidence and are thus marked as "undetermined".

3 (C) Summary table of the total number of indicated Y' changes in a survivor clone.

4 (D) Visual representation of an illustrative subset of modified Y' structures. Three extremities are displayed as examples of typical
5 modifications of subtelomere structure found in the indicated clones, as compared with the control: Y' losses, gains, tandem
6 duplications, and new Y' sequence emergence. The long telomere sequences of type-II-like survivors (c20, c21 and c27) are
7 apparent. See Supp. Data 2 for the representation of all extremities across all sequenced clones.

8

9 **Type II-like survivors exhibit tandem repeat amplification of telomeres**

10 To study the impact of uncapping on telomere sequences, telomere length distributions were measured
11 from the sequencing reads of all sequenced survivors (Garrido et al., 2025). In Y'-associated survivors, no
12 significant change in telomere length distribution was observed in any clone (Fig. 4A), which is consistent
13 with the results of TRF Southern blot analysis (Fig. 2B and Supp. Fig. S1A). In contrast, we confirmed that
14 type-II-like strains presented very heterogeneous and long telomeres of up to 10 kb (Fig. 4B and C, and
15 Supp. Fig. S2A-C). Telomere length distributions in type-II-like survivors presented multiple peaks,
16 explained by the heterogeneity in chromosome extremity-specific average lengths, including a major peak
17 aggregating 4-7 extremities with an average telomere length < 500 bp (Fig. 4C and Supp. Fig. S2B-C),
18 indicating that not all telomeres were elongated equally.

19 Telomere lengthening in telomerase-negative post-senescence survivors is attributed to recombination
20 events and rolling circle amplification of telomeric circles (t-circles) (Aguilera et al., 2022; Larrivée &
21 Wellinger, 2006; Lin et al., 2005; Natarajan & McEachern, 2002). Telomerase in budding yeast adds
22 imperfect repeats and creates a degenerated telomere motif (Forstemann, 2000). As a consequence, long
23 enough telomere sequences generated by telomerase are unique and can be identified unambiguously.
24 In contrast, recombination between telomeres or rolling circle amplification is expected to yield 2 or more
1 copies of identical sequences. We thus looked for tandem amplification of telomeric sequences. In the
2 control strain, we found a few tandem sequences of 2 copies for repeats of < 45 bp. These tandem repeats
3 could either be signatures of previous events of recombination at telomeres or instances in which
4 telomerase stochastically added 2 identical sequences in tandem. To specifically capture signatures of
5 telomere uncapping, we excluded all repeats shorter than 50 bp from our analysis of type-II-like survivors.
6 We found that the 3 sequenced type-II-like strains contained many perfect tandem repeats at telomeres.
7 While we detected relatively short sequences (< 100 bp) that were repeated 3 times or fewer in tandem,
8 some repeats were longer than 600 bp and others were found in arrays of up to 9 copies (Fig. 4D and
9 Suppl. Fig. S2D). We found multiple raw Nanopore reads supporting these tandem repeats, thus excluding
10 assembly errors as a potential explanation for these observations. Interestingly, several of the same
11 repeated sequences were found at multiple telomeres, often beginning at different positions within the
12 repeat, strongly suggesting that these tandem repeats might have resulted from t-circle-mediated
telomere amplification (Supp. Fig. S2D). Depending on the clone, between 12% and 22% of the total
13 telomeric DNA was part of perfect tandem repeats, which is likely an underestimate given that we only
14 allowed the detection of perfect matches, highlighting the importance of the inferred t-circle-mediated
15 amplification in response to telomere uncapping.
16
<figure><img src="image_7.png" /><figcaption>Figure 4. Telomeres are elongated heterogeneously in T-II-L survivors and contain large perfect tandem repeats.</figcaption></figure>

(A) Telomere length distributions of YAS strains compared to the control.

(B) Telomere length distribution of T-II-L survivors. The left y-axis is used for survivors, whereas the right y-axis is used for the control strain. The x-axis is truncated at 8000 bp.

(C) Boxplots representing the telomere length distribution at each chromosome extremity. The control strain (left) displays extremity-specific mean telomere lengths of approximately 300-400 bp with limited variations between extremities (see also Supp. Fig. S2A), whereas survivor c27 shows heterogeneously elongated telomeres with great variations between extremities (see Supp. Fig. S2B-C for other T-II-L survivors). The blue boxplots correspond to distributions with a mean telomere length < 500 bp.

(D) Arrays of perfect tandem telomere repeats detected in the genome assemblies of T-II-L survivors and the control strain are represented in a 2-D plot showing the number of repeats and the length of each repeat.
1 **Telomere-uncapping-induced rearrangements are RAD52- and POL32-dependent.**

2 The Y' rearrangement events and telomere recombination signatures we observed suggest a key role of
3 homologous recombination (HR) pathways. To investigate the involvement of HR factors, we deleted
4 RAD52 and POL32 in the cdc13-1 strain. We exposed cdc13-1 rad52Δ and cdc13-1 pol32Δ cells to 24 h of
5 telomere uncapping followed by recovery at 23°C. A mild ~2-fold decrease in survival in cdc13-1 rad52Δ
6 and a ~10-fold decrease in survival in cdc13-1 pol32Δ compared to wild-type were observed (Supp. Fig.
7 3A). TRF Southern blot analysis showed that none of the rad52Δ and pol32Δ survivor clones presented
8 either Y'-associated rearrangements or telomere elongation (Fig. 5A and B). We then performed
9 Nanopore sequencing and genome assembly on survivor clones of both genotypes to better characterize
10 potential rearrangements that might have escaped detection by Southern blot. The genomes of the cdc13-
11 1 rad52Δ and cdc13-1 pol32Δ strains almost perfectly matched the control one in telomeric regions, and
12 the rare alterations affecting Y' elements were all Y' losses (Fig. 5E-F). In one pol32Δ survivor clone, we
13 found that chromosome III circularized using HML and HMR for homology. Circularization of chromosome
14 III has previously been described in the context of mating-type interconversion events and recombination
15 between mating-type loci (Klar et al., 1983; Strathern et al., 1979). Interestingly, while the cdc13-1 rad52Δ
16 control strain and survivors showed modest variations in telomere length, telomeres in cdc13-1 pol32Δ
17 were already longer at 23°C, consistent with a previous report (Gatbonton et al., 2006), and telomere-
18 uncapping-induced survivors displayed highly variable telomere lengths (Fig. 5A and B, Supp. Fig. 3B and
19 C). Overall, these results indicate that telomeric and subtelomeric rearrangements strongly depend on
20 Rad52 and Pol32, while survival to transient telomere uncapping is partially independent of Rad52 and
21 Pol32.

22
1 Rad51 and Rad59 are partially involved in Y' recombination

2 To further dissect the molecular pathways underlying Y'-associated and type-II-like rearrangements, we
3 tested the roles of Rad51 and Rad59. We exposed the cdc13-1 rad51Δ and cdc13-1 rad59Δ mutants to
4 transient telomere uncapping and observed a ~2-fold decrease in survival in both mutants, further
5 suggesting a protective role of homologous recombination at uncapped telomeres (Supp. Fig. S3A).

6 Next, we examined the structural changes in survivors using TRF Southern blotting. Only 1 out of 27
7 *rad51Δ* survivors and 0 out of 16 in *rad59Δ* survivors displayed a type-II-like pattern (Fig. 5C and D). While
8 the sample size limited the statistical power, these results suggest the involvement of Rad51 and Rad59
9 in type-II-like formation (p-value = 0.11 and 0.08, respectively, Fischer's exact test). Furthermore, the
10 effects of both proteins became clearer when type-II-like signature and X band alterations were
11 considered simultaneously: in *cdc13-1*, 18 out of 30 clones displayed alterations in TRF Southern blot,
12 whereas only 5 out of 26 *cdc13-1 rad51Δ* clones (p-value = 0.002, Fischer's exact test) and 4 out of 16 in
13 *rad59Δ cdc13-1* clones (p-value = 0.03, Fischer's exact test) were affected. Additionally, Nanopore
14 sequencing and analysis of genome assemblies of a subset of clones confirmed a strong decrease in Y'
15 recombination frequency in *rad51Δ*, whereas the effect in *rad59Δ* appeared milder (Fig. 5E and F). We
16 then constructed the *rad51Δ rad59Δ* double mutant and found a very low frequency of modified
17 subtelomeres, which was lower than that of each single mutant and at a level similar to that of *rad52Δ*,
18 based on TRF Southern blot analysis and Nanopore sequencing (Fig. 5E and F, and Supp. Fig. S3F). The few
19 remaining Y' alterations were all Y' losses, which did not necessarily depend on recombination. In contrast,
20 only 2 out of 10 Y' alterations in *rad51Δ* were losses.

21 Intriguingly, 2 *rad51Δ* survivors exhibited a marked reduction in telomere length (Fig. 5C and Supp. Fig.
22 S3D). Telomere length distribution analysis of clone c9 revealed a decrease from the expected 350 bp to
23 ~200 bp across all chromosome ends (Supp. Fig. S3D), suggesting a potential change in global telomere
1 length regulation in this survivor. In contrast, the telomere length distributions of the *rad59Δ* and *rad59Δ*

2 *rad51Δ* survivors were not altered compared to control (Fig. 5D and Supp. Fig. S3E).

3

4 Taken together, these findings suggest that Rad51 and Rad59 both contribute to the telomeric and

5 subtelomeric rearrangements found in Y'-associated and type-II-like survivors, and function in at least

6 partially independent pathways.

7
<figure><img src="image_8.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_13.png" /></figure>
1 **Figure 5. RAD52 and POL32 are essential for telomere-uncapping-induced rearrangements, while RAD51 and RAD59 are**

2 **partially required.**

3 (A-D) TRF Southern blots of cdc13-1 *pol32Δ* (A), *cd13-1 rad52Δ* (B), *cdc13-1 rad51Δ* (C), and *cdc13-1 rad59Δ* (D) survivor colonies.

4 Cultures from the same strains that were maintained at 23°C were used as controls.

5 (E) Map of chromosome extremity alterations as in Fig. 3B, for sequenced survivor clones of the indicated genotype. A
6 chromosome circularization event in one *pol32Δ* survivor is indicated as black squares. See Supp. Data 2 for the representation
7 of all extremities across all sequenced clones.

8 (F) Fractions of extremities with altered sequence or structure in the indicated strains. A statistically significant decrease was
9 observed for the *rad52Δ*, *pol32Δ*, *rad51Δ* and *rad51Δ rad59Δ* mutants (Student's t-test, *: p-value < 0.05).

10

11 **Type-II-like survivors are resistant to a second telomere uncapping**

12 To test whether recombination at telomeres and subtelomeres allows yeast cells to resist telomere
13 uncapping, we challenged 4 Y'-associated survivors and 3 type-II-like survivors with a second 24-h
14 incubation at 32°C. While both survivor types grew normally at 23°C, only the Y'-associated survivors
15 displayed a loss of viability comparable to that of the original *cdc13-1* control after the second stress (Fig.
16 6A). In contrast, all 3 type-II-like survivors maintained robust growth after 24 h at 32°C, showing little to

17 no survival defect. We wondered whether the DNA damage checkpoint pathway in type-II-like survivors
18 might be dysfunctional, thus supporting cell growth despite telomere uncapping. The type-II-like survivor
19 cells arrested in G2/M after 3 h at 32°C to a similar extent as the initial *cdc13-1* strain did, as quantified
20 by microscopy (Supp. Fig. S4), indicating DNA damage checkpoint activation in response to telomere
21 uncapping.

22 To assess whether recombination contributed to this observed resistance, we deleted RAD52 in the 3
23 previously analyzed type-II-like survivor strains. As we observed that long telomeres in type-II-like strains
24 shortened over successive passages (Supp. Fig. S5A), we accounted for the cell divisions occurring during
1 the transformation procedure to delete RAD52 by passaging the control RAD52 parents 3 times. Strikingly,

2 the *rad52Δ* strains completely lost resistance to telomere uncapping (Fig. 6B), indicating that

3 recombination was required for this protective effect. Importantly, this loss was not attributable to a

4 reduction in telomere length, as TRF Southern blotting showed that the *rad52Δ* mutants retained long

5 telomeres comparable to those of their parental strains (Fig. 6C and Supp. Fig. S5B).

6 Notably, the RAD52 type-II-like strains shown in Fig. 6B exhibited reduced survival compared to those in

7 Fig. 6A, which was particularly apparent for clone c20. Since the former were passaged 3 additional times,

8 we attributed this decreased viability to telomere shortening that occurred during the 50-75 divisions,

9 including potentially sporadic events of rapid telomere deletion. After 11 passages, the gradual telomere

10 shortening led to less defined migrating bands and heterogeneity in the population of cells (Fig. S5A).

11 Collectively, these findings demonstrate that, together, Rad52 and long telomeres protect against a

12 second uncapping.

13
<figure><img src="image_14.png" /><figcaption>Figure 6. T-II-L survivors show RAD52-dependent ability to resist second uncapping.</figcaption></figure>

(A) Spot assay showing survival and growth either at a constant 23°C (left) or after a second 24-h telomere uncapping (right) for the indicated strains.

(B) Same as (A) for the indicated strains. "RAD52 T-II-L (3 p.)" c20, c21 and c27 correspond to the same strains as in (A) but were passaged 3 more times.

(C) TRF Southern blots of the indicated strains. The type-II-like clones c20, c21 and c27 were analyzed once they were isolated and after 3 additional passages ("3 p."). RAD52 was deleted in the same clones, and the transformation and selection procedure required the equivalent of 3 passages. See Supp. Fig. S5B for reordered lanes grouped by clone.
# 1 Discussion

2 How the genome is affected by telomere uncapping remains an outstanding question in the telomere field. Long-term survivors of permanent telomere uncapping have been previously selected after many 3 passages and in the context of mutated checkpoint or DNA processing pathways, thus offering little 4 information regarding the immediate molecular and cellular response to telomere uncapping in 5 checkpoint-proficient conditions. Using a transient uncapping experimental scheme combined with 6 molecular methods and long-read Nanopore sequencing, we provide a comprehensive characterization 7 of the genome rearrangements following telomere uncapping, their genetic requirements and their 8 potential functional role in protecting against further telomere deprotection. 9

10

# 11 Early onset of genomic instability even with a functional checkpoint

12 Our experimental scheme allowed us to show that genome rearrangement can arise as early as during the 13 first cell cycle after telomere uncapping, as suggested by the presence of sharp shifted PFGE bands (Fig. 14 2A) indicating rearrangements present in the whole clonal population. Genomic heterogeneity in cdc13- 15 1 cdc5-ad survivor subclones indicates that genome instability persists for at least two cell divisions after 16 uncapping is relieved and cells resume their cell cycle. We thus propose that transient telomere uncapping 17 initiates a cascade of recombination events, with the first rearrangements arising within a few hours and 18 others unfolding during the recovery phase in the progeny. Importantly, rearrangements occurred in 19 checkpoint-proficient cells, suggesting that they did not result from uncontrolled cell cycling with broken 20 DNA, mis-segregated chromosomes, or other abnormal structures, but rather from a canonical response 21 to ssDNA exposed at uncapped telomeres. The use of a checkpoint-proficient background in our study 22 stands in contrast with previous works where Cdc13-independent survivors and telomere elongation 23 could be observed after extensive incubation at restrictive temperatures only if checkpoint or DNA
1 processing pathways are simultaneously mutated (Grandin, 2001; Grandin & Charbonneau, 2003, 2013;

2 Zubko & Lydall, 2006).

3 Therefore, transient telomere deprotection alone can be a source of genome instability, a finding with
4 important ramifications for understanding the potential contribution of partial and transient telomere
deprotection during the initial steps of tumorigenesis when checkpoints are potentially functional.

6

7 **Mechanisms underlying telomere-uncapping-driven rearrangements**

8 Since transient-telomere-uncapping-driven telomeric rearrangements are dependent on RAD52 and
9 POL32, we propose that homologous recombination and BIR are the main molecular mechanisms
10 underlying their formation. Additionally, both Rad51 and, to a lesser degree, Rad59 are important for Y'-
11 associated rearrangements, especially amplifications. However, the Rad51 requirement is partial, since
12 tandem amplification of up to 8 Y' elements could still be observed in cdc13-1 rad51Δ survivors, which
13 stands in contrast to the strictly Rad51-dependent Y' amplification observed in telomerase-negative type
14 I survivors (Chen et al., 2001; Teng et al., 2000). Interestingly, the fraction of type-II-like survivors
15 measured in the cdc13-1 rad51Δ strain is not greater than in the cdc13-1 strain —if anything, they tend to
16 be less frequent in the absence of RAD51—, another major difference compared to telomerase-negative
type II survivors, which are strongly increased in the absence of RAD51. We found no case of type-II-like
18 telomere elongation in cdc13-1 rad59Δ survivors, suggesting that, as for telomerase-negative survivors,
19 Rad59 might promote Rad52-mediated annealing and recombination between TG1_3 sequences (Churikov
20 et al., 2014; Kockler et al., 2021), possibly through a Rad51-independent BIR pathway that relies on
21 microhomologies (Ira & Haber, 2002). The additive effect of deleting RAD51 and RAD59 indicates that they
22 act on independent pathways, at least partially.
1 With respect to telomere elongation in type-II-like survivors, our detailed sequence analyses strongly 2 suggest the use of t-circles as templates for rolling circle amplification, resulting in perfect tandem repeats 3 of specific motifs of 50-600 bp. Overall, t-circle-mediated telomere elongation accounts for ~10-20% of all 4 telomere sequences in type-II-like survivors and most likely more. Interestingly, t-circles also contribute 5 to telomere elongation in telomerase-negative type II survivors (Aguilera et al., 2022; L arrivée & 6 Wellinger, 2006), suggesting a common mechanism for generating very long telomeres regardless of the 7 initial triggering signal.

8 Y'-associated survivors represent the most frequent survivor outcome of transient telomere uncapping,
9 and we propose that extensive resection following telomere uncapping favors Y' rearrangements owing
to the homology length these elements provide and to the presence of internal telomeric sequences, as
10 well as their organization as tandem repeats at some subtelomeres. When the homology used is within
11 the Y' element itself, new hybrid Y' elements can emerge, suggesting that this process might participate
12 in Y' element evolution and diversification. Telomere elongation would occur less frequently and later
13 after telomere uncapping, but not always, since we observed rearrangement patterns involving > 1 kb-
14 long telomere sequences interspersed between Y' elements (Fig. 3D), suggesting cases where telomere
15 elongation precedes the invasion into an interstitial telomeric sequence and subsequent Y'
16 rearrangements.
17

18 Overall, extensively resected uncapped telomeres engage in multiple homology-dependent mechanisms,
19 resulting in Y'-associated rearrangements, telomere elongation, and even more distal subtelomere
20 duplication and translocation. While some similarities can be drawn with the mechanisms and the genetic
21 requirements of post-senescence telomerase-independent survivors, significant differences in the
22 response to telomere uncapping and critically short telomeres are also observed and might be due to
23 differences in the initial length and state of the telomeres, their processing, and the signaling pathways
24 they activate.
1

2

Survival to transient telomere uncapping is partially dependent on homologous recombination

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

Loss of viability is observed when telomeres are uncapped for more than 6 h. While the exact causes of cell death remain unclear, survival partially depends on recombination factors and is associated with subtelomere and telomere rearrangements. However, altered Y' element organization in Y'-associated survivors, the most frequent rearrangement pattern, does not confer any selective advantage in response to a second round of transient telomere uncapping. We thus speculate that recombination factors might directly promote survival by engaging molecularly with chromosome extremities, regardless of the outcome of the molecular transaction. For example, by directly interacting with ssDNA, Rad52, Rad51 and Rad59 might stabilize the end structure and limit excessive checkpoint activation, thereby acting as a protective cap for the telomeres.

On the other hand, we demonstrate that the massively elongated telomeres in type-II-like survivors strongly promote survival to a second telomere uncapping event. This finding is consistent with previous studies that used permanent telomere uncapping protocols, often together with additional mutations in checkpoint or resection pathways (Grandin, 2001; Grandin & Charbonneau, 2003; Zubko & Lydall, 2006), where survivor cells systematically displayed very long and heterogeneous telomeres. However, we show that an active RAD52-dependent homologous recombination pathway is required for type-II-like cells to survive a second transient telomere uncapping. A parallel can be drawn with observations that deletion of RAD52 in type II telomerase-negative survivors very quickly leads to loss of viability, despite the presence of long telomeres (Teng & Zakian, 1999). This finding is consistent with our model, which proposes that the presence of recombination factors at telomeres or their activity is important for promoting survival to telomere uncapping. In type-II-like survivors, as compared to the parental strain, the very long telomeres would provide much more homology for Rad52-dependent annealing, thus
1 promoting survival. Conceptually, we thus propose that recombination factors or the molecular structures

2 formed by long telomeres undergoing recombination serve as an alternative cap for telomeres when

3 Cdc13 is dysfunctional.

4

5 Material and Methods

6 **Yeast strains**

7 All strains are from the W303 background (ura3-1 trp1-1 leu23,112 his3-11,15 can1-100) corrected for
8 RAD5 and ADE2 (Supp. Table 1). Most strains carry the cdc13-1 allele and were grown routinely at the
9 permissive temperature of 23°C in YPD (yeast extract, peptone, dextrose) media. Deletion strains were
10 created using PCR-based methods as described in (Longtine et al., 1998). Point mutations were introduced
11 using Cas9-mediated gene targeting as described in (Anand et al., 2017).

12

13 **Transient uncapping survival assay**

14 Cells were inoculated in YPD and grown overnight at 23°C. The culture was then diluted and grown in
15 exponential phase until it reached a concentration of 1-2 x 10⁷ cells/mL. Between 5 x 10⁵ and 5 x 10⁶ cells
16 were plated on solid YPD media preheated at 32°C and then kept at 32°C for 24 h before the temperature
17 was shifted back to 23°C until colony formation (~3 days). Another plate was inoculated with ~500 cells of
18 the same liquid culture and kept at 23°C to measure plating efficiency and to be used for normalization.
19 The number of cells plated was dependent on the expected survival frequency of each strain and was
20 chosen in order to yield between 50 and 300 individual colonies. The colonies on the plate that was placed
21 transiently at 32°C and on the plate kept at 23°C were then counted to calculate the survival frequency.
1 Colonies randomly selected for further investigation were subsequently subcloned and then grown at

2 23°C.

3

4 **Genomic DNA extraction**

5 Two genomic DNA extraction protocols were used in this study for subsequent Nanopore sequencing.

6 Details of the extraction protocol used in each strain can be found in Supp. Table 2. For the first protocol,

7 QIAGEN Genomic-tip 100/G was used. A total of 5 x 10⁹ cells from an overnight liquid culture were

8 pelleted, washed with water, and then resuspended in 4 mL of Y1 buffer with 400 U of lyticase for 1 h at

9 30°C. The resulting spheroblasts were pelleted and resuspended in 5 mL of G2 buffer with 1 mg of RNase

10 A and 1 mg of proteinase K for 1 h at 50°C. The lysate was centrifuged at 12000 g for 15 min at 4°C, and

11 the supernatant was then passed through a Qiagen column according to the manufacturer's instructions.

12 For the second protocol, 1-2 x 10⁹ cells from an overnight liquid culture were pelleted, washed with 500

13 µL of spheroblasting solution (1 M sorbitol, 50 mM KPO₄, 10 mM EDTA), resuspended in 500 µL

14 spheroblasting solution supplemented with 5 µL of β-mercaptoethanol and 1.25 mg of zymolyase 20T and

15 incubated for 30 min at 37°C. Spheroblasts were pelleted at 2500 g for 3 min, resuspended in 500 µL of

16 lysis solution (0.1 M Tris HCL at pH 8.0, 50 mM EDTA, 0.5 M NaCl, 1% PVP40, 2.5% SDS) with 1.5 mg of

17 RNase A, and then incubated for 45 min at 50°C. 1 mL of TE and 500 µL of 5 M potassium acetate were

18 added and gently mixed in before 2 centrifugation steps of 10 min at 9500 g at 4°C to clarify the

19 supernatant. DNA was precipitated from the supernatant with an equal volume of isopropanol and

20 washed with 70% cold ethanol before final elution in 40 µL of H₂O.

21
## 1 Pulsed field gel electrophoresis

2 Agarose plugs containing 2 x 10⁸ yeast cells were prepared as described in (Török et al., 1993) and sealed

3 in a 0.5X TBE (0.89 M Tris, 0.89 M boric acid, and 20 mM EDTA) 1% Seakem GTC agarose gel. PFGE was

4 run on the CHEF-DRII system (Bio-Rad) with the following program: 6 V/cm for 10 h with a switching time

5 of 60 sec followed by 6 V/cm for 17 h with a switching time of 90 sec. The reorientation angle was set to

6 120° throughout the entire run.

7

## 8 Library preparation and sequencing

9 The sequencing libraries were prepared following Oxford Nanopore Technologies (ONT) protocols for

10 genomic DNA without preamplification and using LSK109 or LSK110 kits. Genomic DNA was enriched for

11 long fragments using the SRE kit (PacBio). DNA libraries were sequenced on R9.4 or R10.4 Nanopore flow

12 cells in either a MinION Mk1C or PromethION P2 sequencer, respectively, with default parameters on the

13 MinKNOW operating software. For each run, 500 ng of DNA was loaded and sequenced for 8–24 h. In case

14 of DNA reloading, the flow cell was washed using ONT's wash kit following the instructions.

15

## 16 TRF Southern blot

17 Genomic DNA was extracted from cultures using a standard phenol:chloroform:isoamyl (25:24:1)

18 purification procedure and isopropanol precipitation. A sample of 2 µg of genomic DNA was digested with

19 Xhol, and the products were ethanol-precipitated, resuspended in loading buffer (purple gel loading dye

20 6X, New England Biolabs), and resolved on a 1.1% agarose gel for 6 h at 3.6 V/cm. The gel was then soaked

21 in a denaturation bath (0.4 M NaOH, 1 M NaCl) for 20 min and transferred by capillary action to a charged

22 nylon membrane (Hybond XL, GE Healthcare). A telomere-specific oligonucleotide probe (5'-
1 CCCACCACACACACCCACACCC-3') biotinylated at both ends was used for hybridization. After hybridization

2 of the probe, the membrane was washed 3×5 min in wash buffer (58 mM Na2HPO4, 17 mM NaH2PO4, 68

3 mM NaCl, 0.1% SDS). The membrane was next processed for detection with 3 successive incubations (5,

4 5 and 30 min) in blocking buffer (Thermo Scientific, Nucleic Acid Detection Blocking Buffer) before a 30

5 min incubation with alkaline phosphatase-conjugated streptavidin (Invitrogen) diluted in blocking buffer

6 (0.4 g/mL). The membrane was then washed again 3 × 5 min in wash buffer, incubated for 2 × 2 min in

7 assay buffer (0.1 M Tris, 0.1 M NaCl pH 9.5) and for 5 min in CDP-Star substrate (Applied Biosystems)

8 before being imaged with a Bio-Rad ChemiDoc Touch device by chemiluminescence.

9

10 Whole genome assemblies

11 Data from sequencing was basecalled using Guppy with model dna_r9.4.1_450bps_sup.cfg for R9 data

12 and Dorado for with model dna_r10.4.1_e8.2_400bps_sup@v5.0.0 for R10 data. Reads were filtered using

13 Filtlong to keep only the best 500-700 Mb using the filtering parameters --length_weight 6 --

14 mean_q_weight 3 --window_q_weight 1 -min_length 1000. The genomes were assembled using Canu 2.2

15 and Flye 2.9.1, using standard parameters for Canu and the option --nano-hq for Flye, then polished using

16 one round of Racon v1.5.0 followed by 2 rounds of Medaka (v1.11.3 for R9 data, v2.0.1 for R10 data). The

17 output assemblies were mapped against the S288C reference genome for contig name annotation, and

18 short contigs (<70 kb) and contigs corresponding to mitochondrial DNA were removed. To obtain the final

19 assembly, reads were mapped against Canu and Flye assemblies, telomeric regions were manually

20 inspected using Tablet, and the best contigs were retained for each chromosome. Trimming of contigs

21 was manually performed when overassembly at telomeres was detected through loss of coverage. The

22 statistics of each sample for the reads and assemblies can be found in Supp. Table 2.

23
1 **Annotation of telomeric regions**

2 Telomeres were detected on assemblies using Telofinder (O'Donnell 2023)

3 (https://github.com/GillesFischerSorbonne/telofinder). X elements were detected in the assemblies using

4 LRSDAY 1.7.2 (Yue & Liti, 2018). Y' elements were detected by aligning known Y' elements from the

5 reference genome against the considered assembly using BLAST 2.12.0, and then taking the union of all

6 alignments whose lengths were greater than 200 bp.

7

8 **Y' element labelling and clustering**

9 Because Nanopore sequencing makes most errors in homopolymers, the detected Y' sequences were
10 transformed by condensing all homopolymers of length >4 into homopolymers of length 4. Doing so
11 increased robustness and yielded a 100% similarity between the Y' elements of the 2 independent
12 assemblies of our control strain. The similarity measure was derived from BLAST alignment results, using
13 the best alignment between each pair of sequences. The formula used was sim = %identity x
14 alignment_length/alignement_span where alignment_span is the length of the alignment plus the length
15 of the unaligned flanking regions, and %identity was obtained from BLAST.

16 To assess the precision of our method, another set of reads was split and assembled into 2 independent
17 assemblies, which yielded similarity values between homologous Y' between 99.985% and 100%. Labels
18 were then assigned to Y' elements of our control strain. Two or more Y' elements were given the same
19 label if their similarity score was higher than 99.9%.

20 Hierarchical clustering of the Y' elements of our control strain was performed on their similarity matrix
21 using Seaborn's clustermap function with default parameters. The optimal number of 11 clusters was
22 determined by determining the elbow point of the silhouette score.
1

2 **Subtelomeric alteration detection**

3 To detect Y' element structure alterations, we compared the labels assigned to each element to the labels
4 of the elements in the corresponding control strain. Any label that was present at a position in the control
5 and not in a survivor was considered a loss. Any label present at a position in a survivor and not in the
6 control was considered a gain.

7 To detect subtelomeric events not including Y' elements or telomeres, sequences upstream of the X
element at each extremity were aligned over 15 kb using BLAST against their control counterparts, and
9 any deviation from perfect alignment was flagged for manual investigation.

10 To check whether a new Y' sequence not present in the control genome could be explained by a mosaic
11 of elements in the control genome, we used a custom python script to look for perfect alignments of size
12 >50 between a new Y' element and all known Y' elements. A Y' element was considered mosaic if it could
13 be covered by the union of such alignments.

14

15 **Data availability**

16 All new sequencing data (fast5, fastq and fasta files) were deposited in the European Nucleotide Archive
17 (https://www.ebi.ac.uk/ena/ browser/home) with the project accession PRJEB93986.

18

19 **Acknowledgement**

20 We thank Stéphane Delmas and Teresa Teixeira for their critical reviews of the paper. We thank Nicolas
21 Agier for his help and advice with Nanopore sequencing and subsequent analyses. Work in ZX's lab was
1 supported by Ville de Paris (Programme Émergence(s)), the Emergence grant of Sorbonne Université,

2 Ligue Contre le Cancer (Subvention Recherche Scientifique 2022), Fondation ARC pour la recherche sur le

3 cancer (ARCPJA202160003865 and ARCPGA2023110007341_7967), and by Agence Nationale de la

4 Recherche (ANR-24-CE12-7740-01 and ANR-24-CE12-0083-03).

5

6 Author contributions

7 LD: Investigation, Methodology, Formal Analysis, Writing – Original Draft. CG: Methodology, Formal

8 Analysis. OI: Investigation. JSB: Conceptualization, Supervision. ZX: Conceptualization, Supervision,

9 Writing – Original Draft. All authors – Writing – Review & Editing.

10

11 Conflict of interest

12 The authors declare that they have no conflict of interest.

13

14 References

15 Aguilera, P., Dubarry, M., Hardy, J., Lisby, M., Simon, M., & Géli, V. (2022). Telomeric C-circles localize at
16 nuclear pore complexes in *Saccharomyces cerevisiae*. *The EMBO Journal*, *41*(6), e108736.

17 https://doi.org/10.15252/embj.2021108736

18 Anand, R., Memisoglu, G., & Haber, J. (2017). Cas9-mediated gene editing in Saccharomyces cerevisiae.

19 *Protocol Exchange*. https://doi.org/10.1038/protex.2017.021a

20 Cesare, A. J., & Reddel, R. R. (2010). Alternative lengthening of telomeres: Models, mechanisms and

21 implications. *Nature Reviews Genetics*, *11*(5), 319–330. https://doi.org/10.1038/nrg2763
1 Chen, Q., Ijpma, A., & Greider, C. W. (2001). Two Survivor Pathways That Allow Growth in the Absence

2 of Telomerase Are Generated by Distinct Telomere Recombination Events. *Molecular and*

3 *Cellular Biology, 21*(5), 1819–1827. https://doi.org/10.1128/MCB.21.5.1819-1827.2001

4 Churikov, D., Charifi, F., Simon, M.-N., & Géli, V. (2014). Rad59-Facilitated Acquisition of Y' Elements by

5 Short Telomeres Delays the Onset of Senescence. *PLoS Genetics, 10*(11), e1004736.

6 https://doi.org/10.1371/journal.pgen.1004736

7 Churikov, D., Corda, Y., Luciano, P., & Géli, V. (2013). Cdc13 at a crossroads of telomerase action.

8 *Frontiers in Oncology, 3*. https://doi.org/10.3389/fonc.2013.00039

9 Coutelier, H., Xu, Z., Morisse, M. C., Lhuillier-Akakpo, M., Pelet, S., Charvin, G., Dubrana, K., & Teixeira,

10 M. T. (2018). Adaptation to DNA damage checkpoint in senescent telomerase-negative cells

11 promotes genome instability. *Genes & Development, 32*(23–24), 1499–1513.

12 https://doi.org/10.1101/gad.318485.118

13 de Lange, T. (2018). Shelterin-Mediated Telomere Protection. *Annual Review of Genetics, 52*(1), 223–

14 247. https://doi.org/10.1146/annurev-genet-032918-021921

15 Ebert, P., Audano, P. A., Zhu, Q., Rodriguez-Martin, B., Porubsky, D., Bonder, M. J., Sulovari, A., Ebler, J.,

16 Zhou, W., Serra Mari, R., Yilmaz, F., Zhao, X., Hsieh, P., Lee, J., Kumar, S., Lin, J., Rausch, T., Chen,

17 Y., Ren, J., ... Eichler, E. E. (2021). Haplotype-resolved diverse human genomes and integrated

18 analysis of structural variation. *Science, 372*(6537), eabf7117.

19 https://doi.org/10.1126/science.abf7117

20 Enomoto, S., Glowczewski, L., & Berman, J. (2002). *MEC3*, *MEC1*, and *DDC2* Are Essential Components

21 of a Telomere Checkpoint Pathway Required for Cell Cycle Arrest during Senescence in

22 *Saccharomyces cerevisiae*. *Molecular Biology of the Cell, 13*(8), 2626–2638.

23 https://doi.org/10.1091/mbc.02-02-0012
1 Fellerhoff, B., Eckardt-Schupp, F., & Friedl, A. A. (2000). Subtelomeric Repeat Amplification Is Associated

2 With Growth at Elevated Temperature in yku70 Mutants of Saccharomyces cerevisiae. *Genetics*,

3 154(3), 1039-1051. https://doi.org/10.1093/genetics/154.3.1039

4 Forstemann, K. (2000). Telomerase-dependent repeat divergence at the 3' ends of yeast telomeres.

5 *Nucleic Acids Research*, 28(14), 2690-2694. https://doi.org/10.1093/nar/28.14.2690

6 Galgoczy, D. J., & Toczyski, D. P. (2001). Checkpoint Adaptation Precedes Spontaneous and Damage-

7 Induced Genomic Instability in Yeast. *Molecular and Cellular Biology*, 21(5), 1710-1718.

8 https://doi.org/10.1128/MCB.21.5.1710-1718.2001

9 Garrido, C., Kornobis, E., Agier, N., Ilioaia, O., Fischer, G., & Xu, Z. (2025). Natural diversity of telomere

10 length distributions across 100 yeast strains. *bioRxiv*, 2025.05.13.653712.

11 https://doi.org/10.1101/2025.05.13.653712

12 Garvik, B., Carson, M., & Hartwell, L. (1995). Single-stranded DNA arising at telomeres in cdc13 mutants

13 may constitute a specific signal for the RAD9 checkpoint. *Molecular and Cellular Biology*, 15(11),

14 6128-6138. https://doi.org/10.1128/MCB.15.11.6128

15 Gatbonton, T., Imbesi, M., Nelson, M., Akey, J. M., Ruderfer, D. M., Kruglyak, L., Simon, J. A., & Bedalov,

16 A. (2006). Telomere Length as a Quantitative Trait: Genome-Wide Survey and Genetic Mapping

17 of Telomere Length-Control Genes in Yeast. *PLoS Genetics*, 2(3), e35.

18 https://doi.org/10.1371/journal.pgen.0020035

19 Grandin, N. (2001). Cdc13 prevents telomere uncapping and Rad50-dependent homologous

20 recombination. *The EMBO Journal*, 20(21), 6127-6139.

21 https://doi.org/10.1093/emboj/20.21.6127

22 Grandin, N., & Charbonneau, M. (2003). The Rad51 Pathway of Telomerase-Independent Maintenance

23 of Telomeres Can Amplify TG 1-3 Sequences in yku and cdc13 Mutants of *Saccharomyces*
1 *cerevisiae*. *Molecular and Cellular Biology*, 23(11), 3721–3734.

2 https://doi.org/10.1128/MCB.23.11.3721-3734.2003

3 Grandin, N., & Charbonneau, M. (2013). RPA provides checkpoint-independent cell cycle arrest and

4 prevents recombination at uncapped telomeres of Saccharomyces cerevisiae. *DNA Repair*, 12(3),

5 212–226. https://doi.org/10.1016/j.dnarep.2012.12.002

6 Hackett, J. A., Feldser, D. M., & Greider, C. W. (2001). Telomere Dysfunction Increases Mutation Rate

7 and Genomic Instability. *Cell*, 106(3), 275–286. https://doi.org/10.1016/S0092-8674(01)00457-3

8 Hackett, J. A., & Greider, C. W. (2003). End Resection Initiates Genomic Instability in the Absence of

9 Telomerase. *Molecular and Cellular Biology*, 23(23), 8450–8461.

10 https://doi.org/10.1128/MCB.23.23.8450-8461.2003

11 Hartwell, L. H., & Smith, D. (1985). ALTERED FIDELITY OF MITOTIC CHROMOSOME TRANSMISSION IN

12 CELL CYCLE MUTANTS OF S. CEREVISIAE. *Genetics*, 110(3), 381–395.

13 https://doi.org/10.1093/genetics/110.3.381

14 IJpma, A. S., & Greider, C. W. (2003). Short Telomeres Induce a DNA Damage Response in

15 *Saccharomyces cerevisiae*. *Molecular Biology of the Cell*, 14(3), 987–1001.

16 https://doi.org/10.1091/mbc.02-04-0057

17 Ira, G., & Haber, J. E. (2002). Characterization of RAD51 -Independent Break-Induced Replication That

18 Acts Preferentially with Short Homologous Sequences. *Molecular and Cellular Biology*, 22(18),

19 6384–6392. https://doi.org/10.1128/MCB.22.18.6384-6392.2002

20 Jain, D., & Cooper, J. P. (2010). Telomeric Strategies: Means to an End. *Annual Review of Genetics*, 44(1),

21 243–269. https://doi.org/10.1146/annurev-genet-102108-134841

22 Klar, A. J., Strathern, J. N., Hicks, J. B., & Prudente, D. (1983). Efficient Production of a Ring Derivative of

23 Chromosome III by the Mating-Type Switching Mechanism in *Saccharomyces cerevisiae*.

24 *Molecular and Cellular Biology*, 3(5), 803–810. https://doi.org/10.1128/mcb.3.5.803-810.1983
1 Kockler, Z. W., Comeron, J. M., & Malkova, A. (2021). A unified alternative telomere-lengthening

2 pathway in yeast survivor cells. *Molecular Cell*, **81**(8), 1816-1829.e5.

3 https://doi.org/10.1016/j.molcel.2021.02.004

4 Larrivée, M., LeBel, C., & Wellinger, R. J. (2004). The generation of proper constitutive G-tails on yeast

5 telomeres is dependent on the MRX complex. *Genes & Development*, **18**(12), 1391-1396.

6 https://doi.org/10.1101/gad.1199404

7 Larrivée, M., & Wellinger, R. J. (2006). Telomerase- and capping-independent yeast survivors with

8 alternate telomere states. *Nature Cell Biology*, **8**(7), 741-747. https://doi.org/10.1038/ncb1429

9 Le, S., Moore, J. K., Haber, J. E., & Greider, C. W. (1999). RAD50 and RAD51 Define Two Pathways That

10 Collaborate to Maintain Telomeres in the Absence of Telomerase. *Genetics*, **152**(1), 143-152.

11 https://doi.org/10.1093/genetics/152.1.143

12 Lee, S. E., Moore, J. K., Holmes, A., Umezu, K., Kolodner, R. D., & Haber, J. E. (1998). Saccharomyces

13 Ku70, Mre11/Rad50, and RPA Proteins Regulate Adaptation to G2/M Arrest after DNA Damage.

14 *Cell*, **94**(3), 399-409. https://doi.org/10.1016/S0092-8674(00)81482-8

15 Lin, C.-Y., Chang, H.-H., Wu, K.-J., Tseng, S.-F., Lin, C.-C., Lin, C.-P., & Teng, S.-C. (2005).

16 Extrachromosomal Telomeric Circles Contribute to Rad52-, Rad50-, and Polymerase ζ-Mediated

17 Telomere-Telomere Recombination in. *EUKARYOT. CELL*, **4**.

18 Longtine, M. S., Mckenzie III, A., Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., &

19 Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion

20 and modification in Saccharomyces cerevisiae. *Yeast*, **14**(10), 953-961.

21 https://doi.org/10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U

22 Lundblad, V., & Blackburn, E. H. (1993). An alternative pathway for yeast telomere maintenance rescues

23 est1- senescence. *Cell*, **73**(2), 347-360. https://doi.org/10.1016/0092-8674(93)90234-H
1 Lundblad, V., & Szostak, J. W. (1989). A mutant with a defect in telomere elongation leads to senescence

2 in yeast. *Cell*, 57(4), 633-643. https://doi.org/10.1016/0092-8674(89)90132-3

3 Lydall, D., & Weinert, T. (1995). *Yeast Checkpoint Genes in DNA Damage Processing: Implications for*

4 Repair and Arrest. *Science*, 270(5241), 1488-1491.

5 https://doi.org/10.1126/science.270.5241.1488

6 Lydeard, J. R., Jain, S., Yamaguchi, M., & Haber, J. E. (2007). Break-induced replication and telomerase-

7 independent telomere maintenance require Pol32. *Nature*, 448(7155), 820-823.

8 https://doi.org/10.1038/nature06047

9 Marcand, S., Pardo, B., Gratias, A., Cahun, S., & Callebaut, I. (2008). Multiple pathways inhibit NHEJ at

10 telomeres. *Genes & Development*, 22(9), 1153-1158. https://doi.org/10.1101/gad.455108

11 Mersaoui, S. Y., & Wellinger, R. J. (2019). Fine tuning the level of the Cdc13 telomere-capping protein for

12 maximal chromosome stability performance. *Current Genetics*, 65(1), 109-118.

13 https://doi.org/10.1007/s00294-018-0871-3

14 Mieczkowski, P. A., Mieczkowska, J. O., Dominska, M., & Petes, T. D. (2003). Genetic regulation of

15 telomere-telomere fusions in the yeast *Saccharomyces cerevisae*. *Proceedings of the National*

16 *Academy of Sciences*, 100(19), 10854-10859. https://doi.org/10.1073/pnas.1934561100

17 Natarajan, S., & McEachern, M. J. (2002). Recombinational Telomere Elongation Promoted by DNA

18 Circles. *Molecular and Cellular Biology*, 22(13), 4512-4521.

19 https://doi.org/10.1128/MCB.22.13.4512-4521.2002

20 Sandell, L. L., & Zakian, V. A. (1993). Loss of a yeast telomere: Arrest, recovery, and chromosome loss.

21 *Cell*, 75(4), 729-739. https://doi.org/10.1016/0092-8674(93)90493-A

22 Smolka, M., Paulin, L. F., Grochowski, C. M., Horner, D. W., Mahmoud, M., Behera, S., Kalef-Ezra, E.,

23 Gandhi, M., Hong, K., Pehlivan, D., Scholz, S. W., Carvalho, C. M. B., Proukakis, C., & Sedlazeck, F.
1 J. (2024). Detection of mosaic and population-level structural variants with Sniffles2. *Nature*
2 *Biotechnology*, **42**(10), 1571–1580. https://doi.org/10.1038/s41587-023-02024-y
3 Soudet, J., Jolivet, P., & Teixeira, M. T. (2014). Elucidation of the DNA End-Replication Problem in
4 Saccharomyces cerevisiae. *Molecular Cell*, **53**(6), 954–964.
5 https://doi.org/10.1016/j.molcel.2014.02.030
6 Strathern, J. N., Newlon, C. S., Herskowitz, I., & Hicks, J. B. (1979). Isolation of a circular derivative of
7 yeast chromosome III: Implications for the mechanism of mating type interconversion. *Cell*,
8 **18**(2), 309–319. https://doi.org/10.1016/0092-8674(79)90050-3
9 Teng, S.-C., Chang, J., McCowan, B., & Zakian, V. A. (2000). Telomerase-Independent Lengthening of
10 Yeast Telomeres Occurs by an Abrupt Rad50p-Dependent, Rif-Inhibited Recombinational
11 Process. *Molecular Cell*, **6**(4), 947–952. https://doi.org/10.1016/S1097-2765(05)00094-8
12 Teng, S.-C., & Zakian, V. A. (1999). Telomere-Telomere Recombination Is an Efficient Bypass Pathway for
13 Telomere Maintenance in *Saccharomyces cerevisiae*. *Molecular and Cellular Biology*, **19**(12),
14 8083–8093. https://doi.org/10.1128/MCB.19.12.8083
15 Toczyski, D. P., Galgoczy, D. J., & Hartwell, L. H. (1997). CDC5 and CKII Control Adaptation to the Yeast
16 DNA Damage Checkpoint. *Cell*, **90**(6), 1097–1106. https://doi.org/10.1016/S0092-8674(00)80375-X
17
18 Török, T., Rockhold, D., & King, A. D. (1993). Use of electrophoretic karyotyping and DNA-DNA
19 hybridization in yeast identification. *International Journal of Food Microbiology*, **19**(1), 63–80.
20 https://doi.org/10.1016/0168-1605(93)90124-Y
21 Wellinger, R. J., & Zakian, V. A. (2012). Everything You Ever Wanted to Know About *Saccharomyces*
22 *cerevisiae* Telomeres: Beginning to End. *Genetics*, **191**(4), 1073–1105.
23 https://doi.org/10.1534/genetics.111.137851
1 Yue, J.-X., & Liti, G. (2018). Long-read sequencing data analysis for yeasts. *Nature Protocols*, *13*(6), 1213–

2 1231. https://doi.org/10.1038/nprot.2018.025

3 Zubko, M. K., & Lydall, D. (2006). Linear chromosome maintenance in the absence of essential telomere-

4 capping proteins. *Nature Cell Biology*, *8*(7), 734–740. https://doi.org/10.1038/ncb1428

5

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