# Stn1-Ten1 and Taz1 independently promote replication of subtelomeric fragile sequences in fission yeast

Mélina Vaurs, Karel Naiman, Chaïnez Bouabboune, Sudhir Rai, Katarzyna Ptasińska, Marion Rives, Samah Matmati, Antony Carr, Vincent Géli, Stéphane Coulon

► To cite this version:

Mélina Vaurs, Karel Naiman, Chaïnez Bouabboune, Sudhir Rai, Katarzyna Ptasińska, et al. Stn1-Ten1 and Taz1 independently promote replication of subtelomeric fragile sequences in fission yeast. Cell Reports, 2023, 42 (6), pp.112537. 10.1016/j.celrep.2023.112537. hal-05393000

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**Cell Reports**

# Stn1-Ten1 and Taz1 independently promote replication of subtelomeric fragile sequences in fission yeast

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Authors

Mélina Vaurs, Karel Naiman, Chaïnez Bouabboune, ..., Antony M. Carr, Vincent Géli, Stéphane Coulon

## Correspondence

vincent.geli@inserm.fr (V.G.), stephane.coulon@inserm.fr (S.C.)

## In brief

Vauris et al. show that the Stn1-Ten1 complex is crucial for efficient replication of the subtelomeric STE3-2 region. While both Taz1 and Stn1-Ten1 bind STE3-2, Stn1-Ten1 function is independent of Taz1 but relies on Pot1-Tpz1-Poz1. With dysfunctional Stn1, an HR-based fork restart mechanism becomes necessary for STE3-2 replication.

## Highlights

* Stn1-Ten1 is crucial for replication of a hard-to-replicate subtelomeric region

* Taz1 and Stn1-Ten1 bind to this hard-to-replicate subtelomeric region

* Stn1-Ten1 function relies on its association with Pot1-Tpz1-Poz1 but not Taz1

* An HR-based fork restart mechanism occurs when Stn1 is dysfunctional

<figure><img src="image_3.png" /></figure>
Article

# Stn1-Ten1 and Taz1 independently promote replication of subtelomeric fragile sequences in fission yeast

Mélina Vauris,<fnref n="1" />,<fnref n="3" /> Karel Naiman,<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> Chaínez Bouabboune,<fnref n="1" /> Sudhir Rai,<fnref n="1" /> Katarzyna Ptasińska,<fnref n="2" /> Marion Rives,<fnref n="1" /> Samah Matmati,<fnref n="1" /> Antony M. Carr,<fnref n="2" /> Vincent Géli,<fnref n="1" />,* and Stéphane Coulon<fnref n="1" />,<fnref n="4" />,*

## SUMMARY

Efficient replication of terminal DNA is crucial to maintain telomere stability. In fission yeast, Taz1 and the Stn1-Ten1 (ST) complex play prominent roles in DNA-ends replication. However, their function remains elusive. Here, we have analyzed genome-wide replication and show that ST does not affect genome-wide replication but is crucial for the efficient replication of a subtelomeric region called STE3-2. We further show that, when ST function is compromised, a homologous recombination (HR)-based fork restart mechanism becomes necessary for STE3-2 stability. While both Taz1 and Stn1 bind to STE3-2, we find that the STE3-2 replication function of ST is independent of Taz1 but relies on its association with the shelterin proteins Pot1-Tpz1-Poz1. Finally, we demonstrate that the firing of an origin normally inhibited by Rif1 can circumvent the replication defect of subtelomeres when ST function is compromised. Our results help illuminate why fission yeast telomeres are terminal fragile sites.

## INTRODUCTION

During each cell cycle, accurate replication of the genome ensures its integrity. In eukaryotes, DNA replication is stochastically initiated at replication origins that constitute binding sites for the origin recognition complex (ORC) that subsequently load the minichromosome maintenance protein complex (MCM) (reviewed in Hyrien,<fnref n="1" /> Urban et al.,<fnref n="2" /> and Fragkos et al.<fnref n="3" />). Replication initiates bidirectionally, and individual forks continue along a chromosome until they meet a fork initiated by an adjacent origin. At the end of the chromosome, replication is unidirectional because initiation mostly originates within subtelomeric regions, with the replication fork moving toward the telomeric repeats.<fnref n="4" />⁻⁶ However, if such terminal forks arrest, replication can eventually initiate within the telomeric repeats to complete replication.⁵⁻⁷

Telomeric sequences themselves are fragile sites that are hard to replicate, and replication forks frequently stall when entering telomeric tracks.⁵,⁸ Indeed, these G-rich regions are prone to forming secondary structures, such as G-quadruplex (G4), T-loop super-structures, and R-loops or other RNA:DNA hybrids associated with telomere repeat-containing RNA (TERRA) transcription. These are potential sources of endogenous replication blocks that can impede the progression of a fork replicating telomeric repeat sequences.⁹,¹⁰

The fission yeast *Schizosaccharomyces pombe* contains three chromosomes and six telomeres. Terminal sequences are

composed of degenerate telomere repeats ($G_2$-$_6$TTAC[A]) and are ~300 bp in length. The Taz1 protein specifically binds to duplex telomeric repeat DNA, and Pot1 recognizes the telomeric 3'-overhang, or G-tail. Four additional telomeric proteins, Tpz1, Rap1, Poz1, and Ccq1, bridge Taz1 to Pot1 through a network of protein-protein interactions¹¹,¹² (Figure 1A). In contrast, subtelomeric regions contain a mosaic of multiple segments of homologous sequences that span ~50 kb of the telomere-proximal DNA regions of chromosome I and II. These repeat sequences include the subtelomeric elements 1 (STE1), 2 (STE2), and 3 (STE3), which are enriched in poly[dA:dT] tracts, and centromere-homologous sequences (*cenH*), which are embedded in *th* genes that encode putative telomere-linked helicases (TIhs) of the RecQ family¹³⁻¹⁶ (Figure 1B). Chromosome III subtelomeric regions generally contain rDNA repeats, although in some strains, the telomere-proximal left arm of chromosome III mirrors the structure of chromosomes I and II.¹⁶ Adjacent to these subtelomeric regions, an additional ~50 kb region forms a highly condensed subtelomeric chromatin body (Knob) that separates euchromatin from subtelomeres.¹⁷,¹⁸

In fission yeast,⁸ as in mammalian cells, replication forks slow down when in the proximity of telomeric repeats, and telomere maintenance requires efficient replication.¹⁹,²⁰ Fission yeast Taz1 plays a prominent role in telomere replication, although its precise function is not yet established.⁸ In addition to Taz1, further components including the RPA heterotrimer, the Pfh1

<figure><img src="image_4.png" /></figure>

<footnote n="1">¹CNRS, INSERM, Aix Marseille Univ, Institut Paoli-Calmettes, CRCM, Ligue Nationale Contre le Cancer (équipe labellisée), Marseille, France</footnote>

<footnote n="2">²Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer BN1 9RQ, UK</footnote>

<footnote n="3">³These authors contributed equally</footnote>

<footnote n="4">⁴Lead contact</footnote>

<footnote>*Correspondence: vincent.geli@inserm.fr (V.G.), stephane.coulon@inserm.fr (S.C.)</footnote>

<footnote>https://doi.org/10.1016/j.celrep.2023.112537</footnote>
<figure><img src="image_5.png" /></figure>

A

Shelterin

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>

C

<figure><img src="image_8.png" /></figure>

D

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E

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F

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(legend on next page)
helicase, and telomerase itself contribute to promoting efficient replication of telomeres.²¹⁻²⁴ Recently, we and others have shown that the Stn1-Ten1 complex fulfills an important function during telomere replication.²⁵,²⁶ stn1 and ten1 are both essential genes, deletion mutants of which eventually survive by circularizing their chromosomes. This suggests that both proteins fulfill crucial functions during telomere maintenance.²⁷ Stn1-Ten1 (ST) is known to negatively regulate telomerase through interaction with the SUMOylated form of Tpz1 and to promote C-strand synthesis through Primase-Polα recruitment.²⁵,²⁸⁻³⁰ The stn1-226 allele, which results in amino acid changes in the SIM (SUMO-interacting motif) of Stn1 confers a thermo-sensitive (ts) phenotype.²⁵ Intriguingly, deletion of rif1⁺ rescues the inviability and the telomere loss associated with the stn1-226 mutant at the restrictive temperature.²⁵,²⁶ Rif1 is a highly conserved and multifaceted protein that was initially identified as a negative regulator of telomere length in budding and fission yeasts.³¹⁻³³ In addition, Rif1 controls the timing of replication by regulating the firing of late origins.³⁴⁻³⁷

In humans, the Stn1 and Ten1 homologs STN1 and TEN1 are components of the CST (CTC1-ST) complex.³⁸,³⁹ As in yeast, the human CST complex acts as an inhibitor of telomerase by promoting the synthesis of the complementary strand through Primase-Polα recruitment.⁴⁰ Independently of this C-strand fill-in reaction, CST has been shown to be essential for the replication of duplex telomeric DNA⁴¹⁻⁴³ and also helps to resolve replication problems throughout the genome, likely at GC-rich repetitive sequences⁴⁴ or when replication is challenged.⁴⁵,⁴⁶ These activities may also function via promoting Polα chromatin association. In addition, it has been reported that CST can suppress origin licensing by preventing MCM helicase loading.⁴⁷ Thus, the CST complex has crucial functions at telomeres and during genome maintenance that explain why mutations in either CTC1 and STN1 can cause the Coats plus syndrome, a severe genetic disease.⁴⁸,⁴⁹ Human RIF1 also plays key roles in replication initiation and the regulation of replication timing by counteracting DDK-dependent phosphorylation through PP1 activity.⁵⁰,⁵¹ RIF1 also functions in double-strand break (DSB) repair, acting with 53BP1 to promote non-homologous end joining by antagonizing resection.⁵²,⁵³ More recently, RIF1 and 53BP1, in association with Shieldin, have been shown to promote fill-in synthesis by promoting the association of a CST-Polα complex at DNA breaks and at dysfunctional telomeres.⁵⁴ Thus, in several respects, the CST complex and RIF1 operate in common pathways.

To gain insight into the fission yeast ST complex function and its connection with Rif1, we investigated genome-wide replication dynamics in stn1-226 cells. For this purpose, we used the polymerase usage sequencing (PuSeq) approach that relies on

mutant replicative polymerases that insert an increased frequency of ribonucleotides during DNA synthesis.⁵⁵,⁵⁶ By mapping the strand-specific location of ribonucleotides across the genome by high-throughput sequencing, PuSeq allows the tracking of polymerase usage across the genome. This permits the examination of replication timing and the assignment of origin efficiencies. We show that the ST complex does not affect genome-wide replication but is crucial for the replication of a subtelomeric region called STE3-2. Strikingly, the ST complex is recruited to the STE3-2 region in a manner that requires the shelterin components Pot1-Tpz1-Poz1 but that does not require Taz1. Replication forks stall at STE3-2 when ST function is compromised. These stalled forks can either be restarted by homologous recombination (HR) or rescued by the firing of an origin downstream of STE3-2. Furthermore, we show that while Taz1 is also crucial for the STE3-2 replication, it acts independently of the ST complex. Our results reveal a specific role for shelterin factors and the ST complex in promoting replication of a difficult to replicate region that has characteristics reminiscent of fragile sites.

## RESULTS

### Genome-wide replication dynamics in stn1-226

To investigate ST functions during replication, we monitored replication dynamics by tracking polymerase usage in the stn1-226 mutant grown at either the permissive temperature of 25°C or for three population doublings (9 h) at the semi-permissive temperature of 34°C. stn1-226 cells grown at 34°C exhibit a mild ts phenotype but sustain sufficient growth to perform PuSeq. Indeed, 9 h following the temperature shift, telomeric and subtelomeric DNA is still maintained in stn1-226 cells (Figure S1), contrasting with its loss at 36°C.²⁵ From the polymerase usage data we first calculated, as described previously,⁵⁵,⁵⁶ T-rep (the time at which 50% of the locus of interest is replicated) to examine the relative replication timing across all three chromosomes of *S. pombe*. The global profile of replication dynamics did not change in stn1-226 when compared with wild type (WT) at either 25°C or 34°C (Figure S2A). Because of the known genetic interaction between stn1-226 and rif1Δ, we next focused our analysis on the 35 high-affinity Rif1-binding sites that have been identified on the fission yeast chromosomes.⁵⁷ At these sites, we specifically monitored the average origin activity, reflecting the relative probability of each locus to be used as an origin (see Katarzyna Ptasińska's PhD).⁵⁸ Around these Rif1-binding sites, the origin activity in stn1-226 and WT was similar at both temperatures (Figure S2B).

Because human CST has been shown to resolve replication problems both at telomeres and also throughout the genome,

<caption>**Figure 1. Replication dynamics of subtelomeres analyzed by PuSeq at the permissive temperature of 25°C**</caption>

* (A) Schematic representation of the fission yeast shelterin.
* (B) Schematic illustration of the structures of subtelomeres (~100 kb) of chromosomes 1 and 2 in strain 972.
* (C) Detailed map of *tlh2* and STE regions of the right arm of chromosome 2 (C2-R).
* (D) Top panel: PuSeq traces for the Watson and Crick strands of C2-R at 25°C: in wild type (WT), the usage of Polδ (blue) and Polε (red), and in the stn1-226 strain, the usage of Polδ (black) and Polε (green). Positions of *CenH*, *tlh2*, and STE1-3 are indicated.
* (E and F) Schematic representation of the dynamic of replication at C2-R in WT and the stn1-226 mutant at 25°C. Polδ-bias represents the total contribution of Polδ to the synthesis of both DNA strands at each individual locus.
A

C2-R 34°C
Watson strand

Crick strand

Replication direction

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

B

C2-R 34°C
WT
Replication direction

δ-bias on
both strands

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C

C2-R 34°C
stn1-226
Replication direction

δ-bias on
both strands

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D

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E

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(legend on next page)
likely at GC-rich repetitive sequences that are prone to form G4s, we next examined the influence of *stn1-226* on replication dynamics at 446 GC-rich sequences⁵⁹ that are predicted to fold into G4s (named here as G4 sites). Around these G4 sites, the Stn1 SIM mutation did not significantly modify the origin activity when compared with WT at either 25°C or 34°C (Figure S2C). To further analyze the role of ST at GC-rich sequences, we monitored the stability of the minisatellite CEB (CEB25-L1T) in the *stn1-226* mutant (Figure S3A). CEB25-L1T was previously introduced into the genome of *S. pombe* at the *leu1* locus⁶⁰ and contains 14 repeats of a 44 nt sequence (0.62 kb) that can form stable G4s.⁶¹,⁶² In striking contrast to what has been observed for an RPA mutant (*rpa1-D223Y*),²² no increased instability of CEB was detected in the *stn1-226* mutant at either 25°C or at 36°C when compared with WT (Figures S3B–S3D).

We concluded that the *stn1-226* mutant does not influence origin activity at either Rif1-binding sites or G4 sites and does not impact the maintenance of GC-rich sequences in the fission yeast genome. However, it is notable that we have previously shown *stn1-226* cells to be sensitive to genotoxic agents at semi-permissive temperatures.²⁵ This may suggest a role for the ST complex when replication is challenged by the addition of drugs, similar to the activity of CST in human cells.⁴⁵,⁴⁶,⁶³

## Replication dynamics at subtelomeres

ST has been shown to be important for the replication of subtelomeric regions. We therefore specifically assessed the replication profile of a telomere-proximal ~20 kb on the right arm of chromosome II (C2-R). We extended the sequence available in the database¹³ up to the STE1 regions by flanking the subtelomeric region with the sequence from telomeric plasmid pNSU70 (Figure 1C). In WT at 25°C, two origins were evident: the first and most active maps to approximately 4,520 kb (ori-4520), while the second is less active and maps to approximately 4,525 (ori-4525). In the majority of cells, rightward-moving forks from these two origins are responsible for the replication of the entire subtelomeric region of C2-R: Pol ε usage (leading strand synthesis) is >50% on the Watson strand and does not change dramatically, and this is mirrored by Pol δ (lagging strand synthesis) on the Crick strand (Figure 1D). In the graph, the contribution of each polymerase to the synthesis of both the Watson and Crick strands is plotted. The dashed line represents a 50% contribution to strand synthesis. A schematic representation of the replication dynamic of C2-R in WT based on the PuSeq data is shown in Figure 1E. We have added a plot of the cumulative ratio of Pol δ usage on both strands (δ-bias). δ-bias above 50% (dotted line) reflects usage of Pol δ on both strands, while 50% corresponds to the equal division of labor between the two polymerases. It should be noted that we observed a slight increase above 50% in the δ-bias close to the telomeric sequences at STE1.

However, this elevated δ-bias was not observed in *stn1-226* (cf. Figure 1F) or at 34°C in the WT (cf. Figure 2B).

In *stn1-226* at the permissive temperature of 25°C, the overall replication profile was equivalent to WT, and the division of labor between Pol ε and Pol δ was maintained throughout the C2-R (Figure 1F): ori-4520 was fired with an efficiency similar to that seen in WT, and the less-active ori-4525 was also evident. Thus, we have confirmed that, at the permissive temperature of 25°C, the ST complex mutation does not influence C2-R replication. Next, we monitored replication at 34°C (Figure 2). In WT, in addition to ori-4520 and ori-4525, an origin was active within the STE2 element (ori-4537). Despite this, the replication of the Watson (top) and Crick (bottom) strands across C2-R was again mainly performed by Pol ε and Pol δ, respectively (Figures 2A and 2B). This suggests that ori-4537 was fired late, likely in response to replication forks slowing down or stalling. In the *stn1-226* mutant, the same origins were active: ori-4520, ori-4525, and ori-4539 located within STE2 element (Figures 2A and 2C). Strikingly, although the ori-4539 was active, Pol δ synthetized a significant proportion of both the Watson and Crick strands over the STE3 and STE2 regions, represented by a high δ-bias (Figure 2C). This elevated Pol δ-bias likely reflects the occurrence of HR-mediated replication fork restart between *tlh2* and the STE3-2 region.⁶⁴⁻⁶⁶ This observation is consistent with genetic analysis that demonstrated Rad51 is crucial to maintain cellular growth of *stn1-226* cells.²⁵ Taken together, these data show that STE3 and STE2 likely represent “hard-to-replicate” regions, which require an HR-based replication fork restart mechanism in the absence of a functional ST complex. This, in turn, indicates that ST is an important accessory factor for replication of the STE3-2 region.

## The absence of subtelomeric regions restores the viability of *stn1-226* cells

Since the main defect identified by PuSeq in *stn1-226* cells is the replication of the subtelomeric STE3-2 region, we hypothesized that the removal of subtelomeric regions would suppress the growth defect of *stn1-226* cells at 36°C. We took advantage of the *SD5* strain (subtelomere deletion), constructed by Junko Kanoh's group, in which the subtelomeric sequences (including the whole STE1-3 region) of all five chromosome ends have been removed.¹⁶ We introduced the *stn1-226* allele in the *SD5* strain and monitored its growth. Removing the five subtelomeric sequences partially suppressed the growth defect of *stn1-226* cells at 36°C (Figure 2D). Consistent with this result, when we introduced the *stn1-226* allele into *trt1Δ* cells (in which the three chromosomes are circular and lack telomeres and subtelomeres⁶⁷), the viability of *stn1-226* cells was completely restored (Figure 2E). This is consistent with the fact that *stn1Δ* and *ten1Δ* cells survive

<caption>**Figure 2. Replication of the STE3-2 region is impaired in *stn1-226* at the restrictive temperature of 34°C**</caption>

(A) Top panel: PuSeq traces for the Watson and Crick strands of C2-R at 34°C: in WT, the usage of Pol δ (blue) and Pol ε (red), and in the *stn1-226* strain, the usage of Pol δ (black) and Pol ε (green). Positions of *CenH*, *tlh2*, and STE1-3 are indicated. Pol δ-bias represents the total contribution of Pol δ to the synthesis of both DNA strands at each individual locus.
(B and C) Schematic representation of the dynamic of replication at C2-R in WT and *stn1-226* mutant at 34°C.
(D and E) 5-fold serial dilution spot assay with the subtelomeric deleted strain (*SD5*) and *trt1Δ* strain with circular chromosomes combined with the *stn1-226* allele at 25°C and 36°C on YES plates grown for 2 days at 36°C and 3 days at 25°C.
<figure><img src="image_17.png" /></figure>

A

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B

ChIP Stn1-myc

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C

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D

Tpz1-I501R

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E

ChIP Stn1-myc

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F

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G

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(legend on next page)
by circularizing their chromosomes.²⁷ These results show that the ST complex becomes dispensable when hard-to-replicate telomeres and subtelomeres are removed.

## Stn1 binds to subtelomeric regions and promotes their replication

The ST complex ensures conventional replication of the STE3-2 region, suggesting that ST is recruited to this specific locus. We therefore assayed for the presence of ST at subtelomeres by chromatin immunoprecipitation (ChIP) using a myc-tagged Stn1. We designed primer sets that spread across ~20 kb from the telomere tip to the *th2* region (Figure 3A). As previously described,²⁵ we observed that Stn1 was enriched at telomeres. We also observed Stn1 to be associated with the STE regions (STE1-STE3) and that association decreased toward the *th2* region (Figure 3B). Interestingly, we observed a maximum of Stn1 binding to non-telomeric repeat sequences with the -3.7 kb primer set, which corresponds to the STE2 region.

In fission yeast, Stn1 recruitment to telomeres relies on the interaction of its SIM domain with sumoylated Tpz1.²⁸,²⁹ To assess if an equivalent Tpz1-Stn1 interaction was also necessary to ensure the replication of subtelomeres and sustain the viability of *stn1-226* cells at the restrictive temperature, we expressed a Tpz1-Stn1-226 fusion protein (adapted from Mennie et al.⁶⁸) in either *stn1-226* or *stn1Δ* cells. The Tpz1-Stn1-226 fusion restored the growth defect of both *stn1-226* at 36°C and of *stn1Δ*. This indicates that the replicative function of ST at subtelomeres is dependent on its interaction with Tpz1. In support of this, when we combined the *stn1-226* allele with mutants of Tpz1 that do not interact with either Poz1 or Pot1 (*tpz1-l501R* and *tpz1-l200R*, respectively),⁶⁹ we observed a synthetic lethal interaction in both cases (Figure 3D). This supports the suggestion that ST function requires a functional shelterin.

We next analyzed Stn1 recruitment at both the telomere (0.2 kb: cf. Figure 3A) or STE3-2 by ChIP. Stn1 recruitment was impaired in the *tpz1-l501R* and *tpz1-l200R* mutants when compared with *tpz1*⁺ cells (Figure 3E), indicating that Stn1 recruitment relies on Pot1-Tpz1-Poz1. We further combined the *stn1-226* allele with *poz1Δ* (Figure 3F) and observed that *stn1-226 poz1Δ* double mutants were viable but exhibit a synthetic sickness at 36°C (Figure 3G). This further indicates that Poz1 function is important when Stn1 is dysfunctional. This synthetic sickness contrasts with the fitness of the *stn1-226 rap1Δ* mutant, where the deletion of *rap1*⁺ partially restored the growth defect of *stn1-226*.²⁵ Taken together, these observations indicate that the interactions between Pot1, Tpz1, and Poz1 partic-

ipate in Stn1 recruitment to STE3-2, which in turn promotes the efficient replication of subtelomeric regions. Importantly, these results are corroborated by the recent observation showing that Pot1 is crucial for Stn1 recruitment in fission yeast and promotes telomere replication.⁷⁰ Thus, we conclude that Pot1-Tpz1-Poz1 are essential proteins and function in the recruitment of the ST complex to ensure the replication of chromosomes ends.

## Taz1 binds to subtelomeric regions and promotes their replication

Because Taz1 is involved in telomere replication, we assayed the presence of GFP-tagged Taz1 at subtelomeres by ChIP. We observed that Taz1 was significantly recruited at subtelomeres (Figure 4A), confirming previous observations.¹⁴ The ChIP signal for Taz1 was stronger at the STE3-2 region and was reduced as primer sets approached *th2*, mimicking the Stn1 ChIP profile. Tandem repeats of the telomere consensus sequence GGTTAC have been shown to be binding sites for Taz1 on chromosome arms and to be responsible for heterochromatin establishment and influence replication timing.⁷¹⁻⁷³ Consistent with this, we identified three Taz1-binding sites (Taz1 BSs) in the vicinity of the STE3 region, located -7.3, -7, and -6.3 kb from telomeric repeats (Figure S4). Taken together, these results demonstrated that, as with the ST complex, Taz1 is recruited at subtelomeres specifically at the hard-to-replicate STE3-2 region.

Next, we attempted to assess replication dynamics of the C2-R region in *taz1Δ* cells. While the PuSeq analysis confirmed the division of labor between Polε and Polδ through the genome in WT and in *taz1Δ* cells at 30°C (Figure S5), we were unable to establish a distinct polymerase usage pattern for the C2-R region in *taz1Δ* cells. This is reminiscent of previous observations from J. Cooper's laboratory showing that *taz1Δ* cells displayed instability of STE regions and that Taz1 is essential for replication of telomeres.⁸

Then, we asked whether the recruitment of Stn1 at subtelomeres relies on Taz1 by performing ChIP analysis of Stn1 recruitment in *taz1Δ* cells. Strikingly, Stn1 recruitment was not reduced neither at the STE3-2 region, nor at telomeres, in the absence of Taz1 (Figure 4B). This shows that Taz1 promotes the replication of the STE3-2 region independently of Stn1. Thus, Taz1 functions in subtelomeric replication independently of ST recruitment.

Because both Stn1 and Taz1 are recruited to STE3-2, we wondered if they could bind to an ectopic subtelomeric region. We therefore inserted the STE3-2 region into a strain with circular

### Figure 3. Stn1 is recruited to subtelomeres

(A) Detailed map of *th2* and STE regions of the chromosome 2 right end (C2-R) with regions used to monitor the binding of Stn1-myc, Taz1-GFP, Rad51, and Rpa1-myc by ChIP/qPCR marked.

(B) ChIP of Stn1-myc at the C2-R subtelomere analyzed by qPCR. Error bars show the standard error of the mean (SEM), n ≥ 3. p values are calculated with Mann-Whitney for 0.2 and 3.7 kb, with an unpaired two-tailed t test for 2, 7, 10.8, 14.2, and 19.7 kb.

(C) 5-fold serial dilution spot assay of indicated strains expressing the *tpz1-stn1-226* fusion on YES plates grown for 2 days at 36°C or 3 days at 25°C.

(D) Tetrad analysis of a mating between *stn1-226* and *tpz1-l200R* or *tpz1-l501R* mutants on YES plates at 25°C. Double mutants are synthetically lethal. Unmarked colonies are WT strains.

(E) ChIP analysis of Stn1-myc at telomeres and the STE3-2 region analyzed by qPCR in *tpz1-l200R* and *tpz1-l501R* mutants. Error bars show the SEM, n ≥ 3. p values are calculated with the Mann-Whitney test.

(F) Tetrad analysis of a mating between *stn1-226* and *poz1Δ* mutants on YES plates at 25°C. Unmarked colonies are WT strains.

(G) 5-fold serial dilution spot assay of the *stn1-226* allele combined with *poz1Δ* on YES plates grown at 36°C for 3 days.
<figure><img src="image_25.png" /><figcaption>Figure 4. Taz1 is recruited to subtelomeres</figcaption></figure>

(A) ChIP of Taz1-GFP at the subtelomere C2-R analyzed by qPCR. Error bars show the SEM, n = 3. p value are calculated with an unpaired two-tailed t test.

(B) ChIP of Stn1-myc at telomeres and STE3-2 region analyzed by qPCR in a taz1Δ strain. Error bars show the SEM, n ≥ 3. p values are calculated with Mann-Whitney between stn1+ and stn1-myc and an unpaired two-tailed t test between stn1-myc and stn1-myc taz1Δ.

<figure><img src="image_26.png" /></figure>

chromosomes (*trt1Δ* strain) lacking the subtelomeric and telomeric regions<sup>74</sup> (Figure S6A). Recruitment of Taz1-GFP and Stn1-myc to this internal STE3-2 region (intSTE3-2 circ) was analyzed by ChIP (Figures S6B and S6C). In this genetic context, we could not detect binding of Taz1 or Stn1 to the internal STE3-2 (intSTE3-2) region. We concluded that the recruitment of Taz1 and Stn1 to STE3-2 requires the presence of a telomere that could modify for example the chromatin structure and/or the low abundance of nucleosomes at the STE3-2 region.<sup>75</sup>

### HR-dependent fork restart occurs in stn1-226 allele

Our PuSeq analysis suggests that HR-dependent fork restart may occur within subtelomeric regions when ST function is impaired. Thus, we assessed the recruitment of the HR factors Rad51 and RPA by ChIP in *stn1+* and *stn1-226* backgrounds at both the permissive and semi-restrictive temperatures. No Rad51 binding was detected in *stn1+* cells at either temperature or in *stn1-226* cells at 25°C. However, a strong enrichment of Rad51 was detected at telomeres and subtelomeres in the *stn1-226* mutant cells at 34°C (Figure 5A). This is consistent with previous results obtained using the *stn1-1* allele.<sup>26</sup> Rad51 binding was consistently high at ~6/7 kb, encompassing the telomeres and STE1-3, and decreased near and within *tlh2*. A similar binding profile was observed for RPA: recruitment was evident in the *stn1-226* mutant at 34°C, but not at 25°C, and was not seen in *stn1+* cells at either temperature (Figure 5B). We conclude that the HR pathway is required when Stn1 function is compromised, likely to restart collapsed or broken forks that occur within the STE3-2 region.

To further characterize the mechanism of HR-based fork restart at the STE3-2 region, we combined *stn1-226* with mutants of the HR pathway.<sup>76-81</sup> Inactivation of Mre11-Rad50-Nbs1 (MRN) complex components Mre11 or Rad50 exacerbated the growth defect of *stn1-226* at 36°C, indicating that the MRN complex is required when ST function is impaired (Figures 5C and 5D). When *stn1-226* was combined with *mre11-D65N*, whose product is deficient in nuclease activity,<sup>82</sup> the growth defect was also enhanced. This suggests that the nuclease activity of the MRN complex is required to sustain *stn1-226* viability

at restrictive temperatures (Figure 5E). In line with this result, the absence of the MRN cofactor Ctp1 was also deleterious when combined with the *stn1-226* mutant at 36°C (Figure 5F).

We also observed that the Mus81 structure-specific endonuclease was required when Stn1 functions were altered but that another structure-specific nuclease, Slx1, was not (Figures 5G and 5H). Of note, we observed no genetic interaction at the restrictive temperature when *stn1-226* was combined with *pku70Δ* (Figure 5I). Pfh1 is a helicase that has previously been shown to facilitate telomere replication.<sup>23</sup> When we combined *stn1-226* with a mutant of Pfh1 (*pfh1-mt*<sup>*</sup>), we observed a synergistic effect on growth at 36°C (Figure 5J). This indicates that Pfh1 is important to sustain viability when Stn1 is deficient. Collectively, these results indicate that MRN-Ctp1 and Mus81 play a major function when the ST complex is deficient, likely to process DNA replication intermediates that arise upstream or downstream of HR-based fork restart. Pfh1 likely promotes the efficient replication of telomeres,<sup>23</sup> thus we suggest that both Pfh1 and the ST complex act in distinct pathways to facilitate replication of chromosome ends.

### rDNA replication dynamics are impaired in stn1-226 mutant

The rDNA loci contain well-characterized tandem repeats of replication/transcription units in which replication forks are stalled at a defied replication fork barriers (RFBs) to promote uni-directional replication of these units (Figure S7A).<sup>83,84</sup> It was recently reported by the group of F. Ishikawa that Stn1 is important to maintain rDNA stability.<sup>85</sup> Thus, we assessed the dynamic of rDNA replication in the *stn1-226* mutant by PuSeq. We observed a strong bias toward Pol δ in *stn1-226* at both 25°C and 34°C (Figures S6B and S7C), indicating that Stn1 is required to promote efficient replication of rDNA repeats (Figure S7D). This suggests that an HR-based fork restart mechanism may occur at RFB sites in the absence of ST function. Consistent with these observations, we identified multiple potential Taz1 BSs within the rDNA, located between the RFB and the rDNA replication origin (Figure S8). However, we could not show by ChIP that Taz1 was recruited to these sites (data not shown). It
<figure><img src="image_27.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_33.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>J</figcaption></figure>

<figure><img src="image_36.png" /></figure>

(legend on next page)
is therefore possible that Taz1 and Stn1 contribute to maintain the rDNA by regulating fork restart, although we have previously not observed a clear replication fork arrest defect at the RFB site by two-dimensional gel electrophoresis in stn1-226 cells.²⁵

## Global replication dynamics in stn1-226 rif1Δ double mutant

Because the deletion of rif1⁺ was reported to fully rescue the ts phenotype of the stn1-226 mutant at the restrictive temperature,²⁵ we used PuSeq to analyze replication dynamics in rif1Δ single and stn1-226 rif1Δ double mutants at 25°C and at 34°C. In agreement with previous work,³⁴⁻³⁷ the loss of Rif1 resulted in deregulation of T-rep (Figure S9A). We observed that the stn1-226 mutation did not modify the T-rep of rif1Δ, either at the permissive or restrictive temperatures (Figure S9B). As previously discussed, origin activity was upregulated around Rif1 BSs in rif1Δ cells. However, the Stn1 SIM mutation did not significantly impact the behavior of the rif1Δ mutant (Figures S2B and S2C). We hence conclude that deletion of rif1⁺ does not affect the global replication dynamic of stn1-226 and, reciprocally, that the Stn1 SIM mutation does not affect the global replication dynamics of rif1Δ.

## Deletion of rif1 abrogates δ/δ configuration observed in stn1-226 cells

We next monitored the replication profile of the C2-R subtelomeric region in rif1Δ single and rif1Δ stn1-226 double mutants. In the absence of Rif1 at 25°C, three origins were active (ori-4520, ori-4525, and ori-4537) in both stn1⁺ and stn1-226 backgrounds (Figure 6A). These observations confirm previous studies showing that Rif1 inhibits the firing of late origins at subtelomeres.⁷¹,⁸⁶ Interestingly, ori-4537, which showed increased activity at 34°C in WT cells and even more pronounced activity in stn1-226 at this semi-restrictive temperature (Figure 2A), was significantly active in all rif1Δ backgrounds at both 25°C and 34°C (Figure 6B). Indeed, the increased firing of ori-4537 allowed the progression of a leftward (centromeric moving) fork up to *th2* gene (4,530 kb), where a termination zone was detected. Consequently, the increased activity of an origin between STE3 and STE2 regions in the absence of Rif1 modifies the replication profile.

Importantly, for both rif1Δ and the rif1Δ stn1-226 double mutant at 34°C, the replication pattern was similar to that seen at 25°C (Figure 6B), and the δ-bias observed in the stn1-226 mutant at 34°C (cf. Figure 2B; interpreted as HR-restarted forks with a δ/δ configuration) was lost. This shows that the increased activity of ori-4537 reestablished a conventional replication profile across the STE3-2 region, indicating that a converging fork likely overcomes the replication constraints imposed by dysfunctional ST and facilitates the maintenance of terminal sequences. Thus, the firing of an origin usually repressed by Rif1 within subtelomeres allows the efficient replication of the

STE3-2 region and so restores the viability to stn1-226 cells at the restrictive temperature.

## DISCUSSION

In humans, the CST complex is involved in the synthesis of the complementary strand at telomeres,⁴⁰ in the replication of GC-rich sequences at chromosome ends and arms,⁴³ and in the suppression of origin licensing.⁴⁷ We explored the genome-wide function of the ST complex in fission yeast, taking advantage of the ts allele stn1-226. We did not identify any major genome-wide changes in replication dynamics or timing, and no replication changes were evident at either Rif1 BSs or G4 sequences. However, our study did reveal that the ST complex is required to efficiently replicate a specific subtelomeric region (STE3-2) (Figure 7A).

In WT cells, replication fork progression is largely unidirectional through the STE3-2 region, with forks moving ~25 kb toward the telomere through *th2*, the STE regions, and the telomeric repeats. However, when the ST complex function was compromised, the STE3-2 region was frequently replicated via an HR-based fork restart mechanism (Figure 7B). This suggests that STE3-2 exhibits features of a “hard-to-replicate” region (see below), requiring the activity of accessory factors, such as the ST complex, for its replication. Consistent with this, eliminating the subtelomeric regions from the *S. pombe* genome partially restored viability to stn1-226 cells at the restrictive temperature. In line with ST participating directly in subtelomeric replication, we demonstrated that Stn1 binds to the CR-2 subtelomere and is highly enriched at the STE3-2 region when compared with the telomere distal *th2* locus. Strikingly, Stn1 recruitment is impaired when the interaction between Tpz1 and Pot1 or Tpz1 and Poz1 is affected or when Pot1 is deficient.⁷⁰ Consistent with this, stn1 mutants (stn1Δ and stn1-226) became perfectly viable when a Tpz1-Stn1-226 chimeric protein was expressed. This clearly indicates that Stn1 recruitment at STE3-2 depends on the presence of, and the proper assembly of, the shelterin factors Pot1-Tpz1-Poz1 (Figure 7A). We also demonstrated that Taz1 is enriched at the STE3-2 region, likely through Taz1 BSs that are present nearby and within STE3. Taz1 is also required for efficient replication of subtelomeric regions but acts independently of the ST complex (Figure 7A).

We conclude that a function of the telomeric proteins Pot1-Tpz1-Poz1 is to recruit the ST complex at hard-to-replicate regions, likely to protect stalled forks that could be subjected to extensive resection. ST could promote fill-in reactions that prevent the accumulation of single-stranded DNA (ssDNA), thus avoiding the initiation of inappropriate recombination. We further suggest that Taz1 ensures the replication of telomeres and subtelomeres independently of ST complex, likely through the recruitment of additional accessory factors. Consistent with this, we previously showed that the *taz1Δ stn1-226* double mutant undergoes

Figure 5. Rad51 and Rpa1 are recruited to subtelomeres in stn1-226 cells at the restrictive temperature

(A and B) ChIP of Rad51 and Rpa1-myc at the subtelomere C2-R analyzed by qPCR in the indicated strains. p value are calculated with an unpaired two-tailed t test. Error bars show the SEM, n = 3.

(C–J) 5-fold serial dilution spotting assay of stn1-226 allele combined with mre11Δ, rad50Δ, mre11-D65N, ctp1Δ, mus81Δ, slx1Δ, pku70Δ, and pfh1-mt* strains, as indicated, on YES plates grown at indicated temperatures for 3 to 4 days.
A

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

B

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

(legend on next page)
severe chromosome rearrangements to allow cells to survive.²⁵
Our study reveals unanticipated mechanisms involving different
shelterin proteins during subtelomere replication.

The ST complex has been shown to be recruited to telomeres,
consequent on Tpz1 sumoylation, to promote C-strand synthesis
through Primase-Polα recruitment.²⁵,²⁸⁻³⁰ The results from this
study lead us to propose that ST is also recruited at stalled or
collapsed forks at specific regions, where its function is to recruit
Primase-Polα to counteract extensive Exo1-dependent resection
(Figure 7A). This is supported by the previous demonstration that
overexpression of the catalytic subunit of Polα (Pol1) was able to
restore, at least partially, the viability and telomere loss of the stn1-
226 mutant at the restrictive temperature and that exo1⁺ deletion
restores the growth defect of stn1-226 cells at the restrictive tem-
perature.²⁵ It is also possible that the ST complex protects stalled
forks that reverse by blocking Mre11-dependent degradation, as
has been proposed for human cells.³⁹ However, we have no such
evidence in S. pombe. Indeed, the mre11Δ stn1-226 double
mutant exhibits synthetic sickness, which is likely due to the
involvement of the MRN complex in processing recombination in-
termediates during fork restart (Figure 7B).

With the exception of the rDNA, we did not find any other
genomic sites (other than the STE3-2 region) that require the
ST complex for efficient replication. Nevertheless, it is likely
that the ST complex is required when replication is impaired,
as stn1-226 cells show sensitivity to several DNA-damaging
agents.²⁵ Taken together, we propose that the ST complex pro-
tects replication forks from resection (and from HR-based
restart) to facilitate the resumption of the replication.

Our observations suggest that STE3-2 exhibits specific, but
undetermined, features of a hard-to-replicate region. It was
recently demonstrated that the subtelomeric region (from
tlh2 to the telomeric repeats of C2-R) comprises a unique
chromatin structure displaying very low nucleosome occu-
pancy.⁷⁵ Interestingly, low nucleosome occupancy strongly
correlates with the presence of exclusive poly [dA:dT] tracts.
Factors such as shelterin components and the ORC complex
(see below) may bind to these nucleosome-depleted DNA re-
gions. At tlh2 and telomere-distal regions, poly [dA:dT] tracts
are absent, and nucleosome levels are high.⁷⁵ This may
explain why Taz1 binding is not observed at these regions.
To summarize, the STE3-2 region is located at the boundary
between a high- and a low-density nucleosome region that
is prone to binding other factors (Figure 7). When a replication
fork progresses unidirectionally from subtelomere toward the
telomeric repeats, it is likely that it slows down and/or pauses
when approaching the STE3-2 region. We hypothesize that
the distinct features of STE3-2 thus require the association
of accessory factors with STE3-2, such as the ST complex,
to it be efficiently replicated.

Deletion of rif1⁺ restores the viability and telomere loss of the stn1-226 mutant at the restrictive temperature.²⁵,²⁶ PuSeq analysis allowed us to determine that, in the rif1Δ background, the increased activity of a telomere-proximal origin within the STE region compensates for the replication defect observed in stn1-226 cells at 34°C, thereby bypassing the need for HR-based replication restart when ST is deficient (Figure 7C). In a rif1Δ strain, the replication of the subtelomeric regions at chromosome ends is no longer unidirectional, and a fork that moves from the STE3-2 region toward tlh2 appears to overcome constraints that normally block the unidirectional incoming fork. This shows that cryptic origins can be fired within subtelomeres, likely at AT-rich regions.⁸⁷ Our results are in agreement with the observation that Rif1 suppresses the T-rep by regulating origin the firing.³⁴⁻³⁷

Our analysis of replication dynamics also indicated that Taz1
and Stn1 contribute to maintaining the rDNA repeats by regu-
lating fork restart, corroborating the observation made by the
group of F. Ishikawa that Stn1 is important to maintain rDNA sta-
bility.⁸⁵ The group of F. Ishikawa also reported a direct interac-
tion between Taz1 and Reb1, a DNA-binding factor that partici-
pates in replication fork pausing at RFB.⁸⁵ It was proposed that
Reb1 localizes to subtelomeres in a Taz1-dependent manner,
but our data may also suggest that Taz1 and Reb1 may physi-
cally interact at the rDNA locus rather than at telomeres. More
extensive work will be necessary understand how Stn1 and
Taz1 act at rDNA.

In conclusion, our study unveiled unanticipated replication dy-
namics of a subtelomeric region in fission yeast and the involve-
ment of telomeric proteins in this process. The presence of this
hard-to-replicate region helps to explain why fission yeast telo-
meres are fragile sites and potential sources of genome insta-
bility. Indeed, cells can use an HR-based fork restart mechanism
to circumvent the absence of converging fork at terminal DNA.
However, the firing of cryptic origins within subtelomeres can
rescue stalled forks and relieve the resulting replication stress.
Although the role of fission yeast shelterin and ST complexes
at the telomeric repeats (~300 bp in length) is well characterized,
we show here that shelterin and ST functions extend to subtelo-
meres over approximately 10 kb. ORC proteins likely coexist
within this low-nucleosome-abundance region. The features of
terminal sequences in fission yeast are reminiscent of mamma-
lian chromosomes extremities, in which nucleosomes, shelterin,
and ORC proteins are also present at telomeres.⁸⁸ Thus, further
exploration of replication dynamics in yeast may reveal
conserved mechanisms in mammals.

Limitations of the study

Even though we have covered some limitations of our study in
the results and discussion, we acknowledge some caveats

<caption>Figure 6. Deletion of rif1 restores a normal distribution of Pol δ and ε labor in the stn1-226 mutant</caption>

(A) Top panel: PuSeq traces for the Watson and Crick strands for C2-R at 25°C: in *rif1Δ*, the usage of Polδ (blue) and Polε (red), and in the *rif1Δ stn1-226* strain, the usage of Polδ (black) and Polε (green). Positions of *CenH*, *tlh2*, and STE1-3 are indicated. Bottom panel, schematic representation of the dynamics of replication at C2-R in *rif1Δ* and *rif1Δ stn1-226* strains.

(B) Top panel: PuSeq traces for the Watson and Crick strands of C2-R at 34°C: in *rif1Δ*, the usage of Polδ (blue) and Polε (red), and in the *rif1Δ stn1-226* strain, the usage of Polδ (black) and Polε (green). Positions of *CenH*, *tlh2*, and STE1-3 are indicated. Bottom panel, schematic representation of the dynamics of replication at C2-R in *rif1Δ* and *rif1Δ stn1-226* strains.
<figure><img src="image_41.png" /><figcaption>Figure 7. Replication dynamic of subtelomeric regions in fission yeast</figcaption></figure>

that still remain. We show here that ST complex is crucial for the efficient replication of a subtelomeric region, called STE3-2, on C2-R of the fission yeast. One limitation is that we could not investigate the replication dynamic at the other subtelomeric regions by PuSeq. We also identified the STE3-2 of the C2-R as a

hard-to-replicate region, but we still have to determine which features of this region are at the origin of the replication stress observed at this specific site. Further investigation will be needed for a better understanding of the mechanisms involved in these processes.
## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* RESOURCE AVAILABILITY

* Lead contact

* Materials availability

* Data and code availability

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* Strains and growth conditions

* METHOD DETAILS

* Polymerase usage sequencing (PuSeq)

* Chromatin immunoprecipitation

* QUANTIFICATION AND STATISTICAL ANALYSIS

* qPCR and statistical analysis for ChIP

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2023.112537.

## ACKNOWLEDGMENTS

We thank the pombe community for sharing strains, in particular Junko Kanoh, Toru Nakamura, Feng Qiao, Paul Russell, Pascal Bernard, and Julie Cooper. We also thank the members of the V.G. team, especially Christelle Cayrou, for helpful discussions and Nagham Ghaddar for advice on ChIP experiments. K.N., K.P., and A.M.C. are supported by the Wellcome Trust, grant number 110047/Z/15/Z, and Marie Sklodowska-Curie Fellowship 749631-Rep_Fork Restart. The V.G. laboratory is supported by the "Ligue Nationale Contre le Cancer" (LNCC) (Equipe labélisée). M.V. and C.B. are supported by "Bourse du Ministère de l'Enseignement Supérieur, et de la Recherche (MESR)" and by LNCC. S.C. is supported by the Agence Nationale de la Recherche (ANR-16-CE12 TeloMito and ANR-20-CE12 TeloRPA).

## AUTHOR CONTRIBUTIONS

M.V. and K.N. performed most of experiments presented in this study. C.B. performed the experiments presented in Figures 3C, 3E, and 4C and S.R. and M.R. the experiments of Figures S3 and 5C-5I, respectively. K.P. performed the experiment of Figures 4B and S5. S.M. generated several fission yeast strains used in this study. M.V., K.N., V.G., and S.C. designed experiments and analyzed the data. M.V., V.G., and S.C. wrote the paper with input from K.N. and A.M.C.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: September 15, 2022

Revised: March 1, 2023

Accepted: May 3, 2023

Published: May 25, 2023

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Anti-Rad51</td><td>abcam</td><td>Cat# ab63799, RRID:AB_1142602</td></tr><tr><td>Anti-GFP</td><td>Invitrogen</td><td>Cat# A-11122, RRID:AB_221569</td></tr><tr><td>Anti-Myc (c-Myc antibody; 9E10)</td><td>Santa cruz</td><td>Cat# sc-40; RRID: AB_2892598</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>Proteinase K</td><td>Euromedex</td><td>EU0090</td></tr><tr><td>RNase A DNase-free</td><td>Roche</td><td>11119915001</td></tr><tr><td>Dynabeads protein G</td><td>Invitrogen</td><td>10003D</td></tr><tr><td>CutSmart buffer</td><td>New England Biolabs</td><td>B7204S</td></tr><tr><td>Apal, recombinant</td><td>New England Biolabs</td><td>R0114S</td></tr><tr><td>Nourseothricin</td><td>Jena Bioscience</td><td>AB102L</td></tr><tr><td>Geneticin</td><td>Gibco</td><td>10131-026</td></tr><tr><td>Hygromycin</td><td>Invitrogen</td><td>10687010</td></tr><tr><td>Hybond-XL membrane</td><td>Dutsher</td><td>RPN2035</td></tr><tr><td>Deoxycytidine 5'-triphosphate (α-32P)</td><td>Perkin Elmer</td><td>BLU013Z250UC</td></tr><tr><td>Formaldehyde</td><td>Sigma</td><td>F-8775</td></tr><tr><td>Glycerol</td><td>Euromedex</td><td>EU3550</td></tr><tr><td>MOPS</td><td>Euromedex</td><td>EU0034</td></tr><tr><td>Potassium acetate</td><td>Sigma-Aldrich</td><td>P1190</td></tr><tr><td>EDTA</td><td>Euromedex</td><td>EU0007</td></tr><tr><td>SDS</td><td>Euromedex</td><td>EU0360-A</td></tr><tr><td>Buffer G2</td><td>Qiagen</td><td>1014636</td></tr><tr><td>Buffer QC</td><td>Qiagen</td><td>19055</td></tr><tr><td>Buffer QBT</td><td>Qiagen</td><td>19054</td></tr><tr><td>Buffer QF</td><td>Qiagen</td><td>19056</td></tr><tr><td>N-lauroyl sarcosine</td><td>Sigma-Aldrich</td><td>61747</td></tr><tr><td>RnaseH2</td><td>New England Biolabs</td><td>M0288</td></tr><tr><td>USER</td><td>New England Biolabs</td><td>M550</td></tr><tr><td>T4 polymerase</td><td>New England Biolabs</td><td>M0203</td></tr><tr><td>Ampure XP beads</td><td>Beckman Coulter</td><td>A63880</td></tr><tr><td>NEB Next Ultra II DNA library Prep kit</td><td>New England Biolabs</td><td>E7645</td></tr><tr><td>Zymolyase</td><td>Amsbio</td><td>120493-1</td></tr><tr><td colspan="3"><strong>Critical commercial assays</strong></td></tr><tr><td>TB Green premix Ex Taq II (Tli RNase H Plus)</td><td>TAKARA</td><td>RR820W</td></tr><tr><td>InnuPrep PCR pure Kit</td><td>AnalytikJena</td><td>845-KS-5010250</td></tr><tr><td>2% agarose gel cassette</td><td>Sage Science</td><td>BEF2010</td></tr><tr><td>Genomic tips G-100</td><td>Quiagen</td><td>10223</td></tr><tr><td>High Sensitivity DNA Chips</td><td>Agilent Technology</td><td>5067-4626</td></tr><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>NCBI GEO repository (GSE207082)</td><td>NCBI</td><td>N/A</td></tr><tr><td colspan="3"><strong>Experimental models: Organisms/strains</strong></td></tr><tr><td>See Table S1 for a list of yeast strains used in this study</td><td>N/A</td><td>N/A</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>See Table S2 for a list of primers used in this study</td><td>N/A</td><td>N/A</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>Quantity One 1-D analysis software</td><td>Biorad</td><td>N/A</td></tr><tr><td>Bowtie 2</td><td>Johns Hopkins University</td><td>N/A</td></tr><tr><td>GitHub repository (https://doi.org/10.5281/zenodo.7890318)</td><td>GitHub</td><td>N/A</td></tr><tr><td>Samtools</td><td>MIT</td><td>N/A</td></tr><tr><td>R studio</td><td>GNU</td><td>N/A</td></tr><tr><td>Prism 6</td><td>GraphPad</td><td>N/A</td></tr><tr><td>Papers 3</td><td>Digital Science Research & Solutions Inc.</td><td>N/A</td></tr></tbody></table>

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Stéphane Coulon (stephane.coulon@inserm.fr).

Materials availability

Strains generated for this manuscript are listed in Table S1 and the key resources table. Please request strains by contacting the lead contact.

Data and code availability

* The datasets generated during the current study are available in the NCBI GEO repository under accession number GSE207082.

* The used script is available at: https://github.com/KarelNaiman/shelterin-dependent-recruitment-of-Stn1-Ten1.git (https://doi.org/10.5281/zenodo.7890318)

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Strains and growth conditions

S. pombe strains used in this work are listed in Table S1. Stn1-226-Myc PCR fragment was amplified from plasmid pTopo-Stn1-226-myc:KanMx with primers ³⁸¹FwStn1 and ²⁹⁴RvStn1 (see Table S2) and integrated to the genome of S. pombe strains by one-step homologous insertion. Diploid strains were sporulated or haploid strains were mated and sporulated on ME plates prior to dissection, then the genotype was verified following the genetic markers or by PCR (see Table S2). Cells are grown in YES media at indicated temperatures.

METHOD DETAILS

Polymerase usage sequencing (PuSeq)

Cells were grown at 25°C or collected 9 h after the shift at 34°C. This incubation time at the restrictive temperature corresponds to three population doublings and is necessary for cells to incorporate ribonucleotides into ~80% of the DNA during the restrictive temperature. The published protocol was used with minor modifications: size selection was performed using a Blue Pippin (Sage Science).⁵⁶ We calculated the relative replication timing (T-rep as determined in ref. ⁵⁵,⁵⁶) to examine replication dynamics across all three chromosomes of S. pombe. Briefly, T-rep was determined as a time when the population-averaged fraction of the genome replicated was equal to 50%. The telomeric plasmid pNSU70 was aligned to the right end of chromosome 2 using the current version of the S. pombe genome available on PomBase.¹³,⁸⁹ Nucleotides 4137 to 7223 of pNSU70 were added to chromosome 2 right end to create the C2-R extended version available under the accession number GSE196149.

Chromatin immunoprecipitation

Cells were grown at 25°C or collected 6 h after the shift at 34°C. 100mL of culture at OD₆₀₀nm = 0.8 was crosslinked with 1% of Formaldehyde (Sigma Aldrich) at room temperature for 15min. Crosslinking was quenched with 125mM Glycine for 5 min at room temperature and cells were washed twice with ice-cold TBS. Cell pellets were then resuspended in Lysis Buffer Low Sodium (50mM Hepes-KOH pH7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate, 2mM of PMSF, protease inhibitors) and mechanically lysed with glass beads using 4 cycles of 20 s at a speed of 6.5 in a fast-prep machine. Whole cell extracts were
sonicated for 10 min (30s ON, 30s OFF) using the Diagenode Bioruptor. Sonicated lysates were cleared by centrifugation and standardized. 3µL of anti-Rhp51 (Abcam, ab63799) mouse monoclonal antibodies, 3µL of anti-myc (9E10 sc-40, Santa Cruz) or 3µL of anti-GFP (A11122, Invitrogen), were added to solubilized chromatin and incubated overnight at 4°C. 25µL of Protein G Dynabeads (Thermofisher) were added for 3 h at 4°C. Beads were then washed twice with ice-cold Lysis Buffer Low Sodium, twice with ice-cold Lysis Buffer High Sodium (50mM Hepes-KOH pH7.5, 500mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate), twice with ice-cold Sodium Deoxycolate buffer (10mM Tri-HCl pH 8, 250mM LiCl, 0.5% NP-40, 0.5% Na-Deoxycholate, 1mM EDTA) and once with ice-cold TE buffer (10mM Tri-HCl pH 8, 1mM EDTA). Beads were then eluted in TES (TE with 1% of SDS) by incubation for 15 min at 65°C. Samples were incubated 12 h at 65°C and then treated with Proteinase K for 4 h at 37°C. DNA was purified using innuPREP PCRpure purification column (Analytikjena) and samples were analyzed by qPCR.

## QUANTIFICATION AND STATISTICAL ANALYSIS

### qPCR and statistical analysis for ChIP

Input samples were diluted to 1/40. 2µL of template DNA was added to 5µL of TB Green Premix Ex Taq II (Takara), and primers were added to a final concentration of 0.4µM. Each sample was run in triplicate on the same 96-well PCR plate in a C1000 Touch Thermal Cycler (CFX96 Biorad). The PCR program was as follows: 95°C for 30 s followed by 55 cycles at 95°C for 10 s, 54°C for 6.1kb, 56°C for 0.2kb, 2kb, 3.7kb, 10.8kb and 14.2kb, 58°C for 7kb and 19.7kb for 10 s, 72°C for 10 s and a final extension step at 72°C for 5min. Primers used for qPCR are listed in Table S2. %IP was calculated as follows: $%IP = (1/((EcrIP - EcrT)/xDF)) \times 100$ with E the efficiency of qPCR, CTIP and CTIT the mean of triplicate for IP and Input (IT) respectively, DF the dilution factor. Statistical comparisons were performed using an unpaired two-tail t test.