Pot1b deletion and telomerase haploinsufficiency in mice initiate an ATR-dependent DNA damage response and elicit phenotypes resembling dyskeratosis congenita

The Protection of telomeres 1 (POT1) protein is a single-stranded telomere binding protein that is essential for proper maintenance of telomere length. Disruption of POT1 function leads to chromosome instability and loss of cellular viability. Here, we show that targeted deletion of the mouse Pot1b gene results in increased apoptosis in highly proliferative tissues. In the setting of telomerase haploinsufficiency, loss of Pot1b results in depletion of germ cells and complete bone marrow failure due to increased apoptosis, culminating in premature death. Pot1b^−/− mTR^+/− hematopoietic progenitor and stem cells display markedly reduced survival potential in vitro. Accelerated telomere shortening, increased G overhang and elevated number of chromosome end-to-end fusions that initiate an ATR-dependent DNA damage response were also observed. These results indicate an essential role for Pot1b in the maintenance of genome integrity and the long-term viability of proliferative tissues in the setting of telomerase deficiency. Interestingly, these phenotypes closely resemble those found in the human disease dyskeratosis congenita (DC), an inherited syndrome characterized by bone marrow failure, hyperpigmentation, and nail dystrophy. We anticipate that this mouse will serve as a useful model to further understand the pathophysiology of DC.     

Tetrahymena Pot2 Is a Developmentally Regulated Paralog of Pot1 That Localizes to Chromosome Breakage Sites but Not to Telomeres

Tetrahymena telomeres are protected by a protein complex composed of Pot1, Tpt1, Pat1, and Pat2. Pot1 binds the 3′ overhang and serves multiple roles in telomere maintenance. Here we describe Pot2, a paralog of Pot1 which has evolved a novel function during Tetrahymena sexual reproduction. Pot2 is unnecessary for telomere maintenance during vegetative growth, as the telomere structure is unaffected by POT2 macronuclear gene disruption. Pot2 is expressed only in mated cells, where it accumulates in developing macronuclei around the time of two chromosome processing events: internal eliminated sequence (IES) excision and chromosome breakage. Chromatin immunoprecipitation (ChIP) demonstrated Pot2 localization to regions of chromosome breakage but not to telomeres or IESs. Pot2 association with chromosome breakage sites (CBSs) occurs slightly before chromosome breakage. Pot2 did not bind CBSs or telomeric DNA in vitro, suggesting that it is recruited to CBSs by another factor. The telomere proteins Pot1, Pat1, and Tpt1 and the IES binding factor Pdd1 fail to colocalize with Pot2. Thus, Pot2 is the first protein found to associate specifically with CBSs. The selective association of Pot2 versus Pdd1 with CBSs or IESs indicates a mechanistic difference between the chromosome processing events at these two sites. Moreover, ChIP revealed that histone marks characteristic of IES processing, H3K9me3 and H3K27me3, are absent from CBSs. Thus, the mechanisms of chromosome breakage and IES excision must be fundamentally different. Our results lead to a model where Pot2 directs chromosome breakage by recruiting telomerase and/or the endonuclease responsible for DNA cleavage to CBSs.     

The 3′ Overhangs at Tetrahymena thermophila Telomeres Are Packaged by Four Proteins,Pot1a,Tpt1, Pat1, and Pat2

Although studies with the ciliate Tetrahymena thermophila have played a central role in advancing our understanding of telomere biology and telomerase mechanisms and composition, the full complement of Tetrahymena telomere proteins has not yet been identified. Previously, we demonstrated that in Tetrahymena, the telomeric 3′ overhang is protected by a three-protein complex composed of Pot1a, Tpt1, and Pat1. Here we show that Tpt1 and Pat1 associate with a fourth protein, Pat2 (Pot1 associated Tetrahymena 2). Mass spectrometry of proteins copurifying with Pat1 or Tpt1 identified peptides from Pat2, Pot1a, Tpt1, and Pat1. The lack of other proteins copurifying with Pat1 or Tpt1 implies that the overhang is protected by a four-protein Pot1a-Tpt1-Pat1-Pat2 complex. We verified that Pat2 localizes to telomeres, but we were unable to detect direct binding to telomeric DNA. Cells depleted of Pat2 continue to divide, but the telomeres exhibit gradual shortening. The lack of growth arrest indicates that, in contrast to Pot1a and Tpt1, Pat2 is not required for the sequestration of the telomere from the DNA repair machinery. Instead, Pat2 is needed to regulate telomere length, most likely by acting in conjunction with Pat1 to allow telomerase access to the telomere.     

The effect of Pot1 binding on the repair of thymine analogs in a telomeric DNA sequence

Telomeric DNA can form duplex regions or single-stranded loops that bind multiple proteins, preventing it from being processed as a DNA repair intermediate. The bases within these regions are susceptible to damage; however, mechanisms for the repair of telomere damage are as yet poorly understood. We have examined the effect of three thymine (T) analogs including uracil (U), 5-fluorouracil (5FU) and 5-hydroxymethyluracil (5hmU) on DNA–protein interactions and DNA repair within the GGTTAC telomeric sequence. The replacement of T with U or 5FU interferes with Pot1 (Pot1pN protein of Schizosaccharomyces pombe) binding. Surprisingly, 5hmU substitution only modestly diminishes Pot1 binding suggesting that hydrophobicity of the T-methyl group likely plays a minor role in protein binding. In the GGTTAC sequence, all three analogs can be cleaved by DNA glycosylases; however, glycosylase activity is blocked if Pot1 binds. An abasic site at the G or T positions is cleaved by the endonuclease APE1 when in a duplex but not when single-stranded. Abasic site formation thermally destabilizes the duplex that could push a damaged DNA segment into a single-stranded loop. The inability to enzymatically cleave abasic sites in single-stranded telomere regions would block completion of the base excision repair cycle potentially causing telomere attrition.     

Dissection of Rad9 BRCT domain function in the mitotic checkpoint response to telomere uncapping

In Saccharomyces cerevisiae, destabilizing telomeres, via inactivation of telomeric repeat binding factor Cdc13, induces a cell cycle checkpoint that arrests cells at the metaphase to anaphase transition—much like the response to an unrepaired DNA double strand break (DSB). Throughout the cell cycle, the multi-domain adaptor protein Rad9 is required for the activation of checkpoint effector kinase Rad53 in response to DSBs and is similarly necessary for checkpoint signaling in response to telomere uncapping. Rad53 activation in G1 and S phase depends on Rad9 association with modified chromatin adjacent to DSBs, which is mediated by Tudor domains binding histone H3 di-methylated at K79 and BRCT domains to histone H2A phosphorylated at S129. Nonetheless, Rad9 Tudor or BRCT mutants can initiate a checkpoint response to DNA damage in nocodazole-treated cells. Mutations affecting di-methylation of H3 K79, or its recognition by Rad9 enhance 5′ strand resection upon telomere uncapping, and potentially implicate Rad9 chromatin binding in the checkpoint response to telomere uncapping. Indeed, we report that Rad9 binds to sub-telomeric chromatin, upon telomere uncapping, up to 10 kb from the telomere. Rad9 binding occurred within 30 min after inactivating Cdc13, preceding Rad53 phosphorylation. In turn, Rad9 Tudor and BRCT domain mutations blocked chromatin binding and led to attenuated checkpoint signaling as evidenced by decreased Rad53 phosphorylation and impaired cell cycle arrest. Our work identifies a role for Rad9 chromatin association, during mitosis, in the DNA damage checkpoint response to telomere uncapping, suggesting that chromatin binding may be an initiating event for checkpoints throughout the cell cycle.     

Abnormal micronuclear telomeres lead to an unusual cell cycle checkpoint and defects in Tetrahymena oral morphogenesis

Telomere mutants have been well studied with respect to telomerase and the role of telomere binding proteins, but they have not been used to explore how a downstream morphogenic event is related to the mutated telomeric DNA. We report that alterations at the telomeres can have profound consequences on organellar morphogenesis. Specifically, a telomerase RNA mutation termed ter1-43AA results in the loss of germ line micronuclear telomeres in the binucleate protozoan Tetrahymena thermophila. These cells also display a micronuclear mitotic arrest, characterized by an extreme delay in anaphase with an elongated, condensed chromatin and a mitotic spindle apparatus. This anaphase defect suggests telomere fusions and consequently a spindle rather than a DNA damage checkpoint. Most surprisingly, these mutants exhibit unique, dramatic defects in the formation of the cell's oral apparatus. We suggest that micronuclear telomere loss leads to a “dynamic pause” in the program of cortical development, which may reveal an unusual cell cycle checkpoint.     

Structural insights into Pot1-ssDNA,Pot1-Tpz1 and Tpz1-Ccq1 Interactions within fission yeast shelterin complex

The conserved shelterin complex caps chromosome ends to protect telomeres and regulate telomere replication. In fission yeast Schizosaccharomyces pombe, shelterin consists of telomeric single- and double-stranded DNA-binding modules Pot1-Tpz1 and Taz1-Rap1 connected by Poz1, and a specific component Ccq1. While individual structures of the two DNA-binding OB folds of Pot1 (Pot1_OB1-GGTTAC and Pot1_OB2-GGTTACGGT) are available, structural insight into recognition of telomeric repeats with spacers by the complete DNA-binding domain (Pot1_DBD) remains an open question. Moreover, structural information about the Tpz1-Ccq1 interaction requires to be revealed for understanding how the specific component Ccq1 of S. pombe shelterin is recruited to telomeres to function as an interacting hub. Here, we report the crystal structures of Pot1_DBD-single-stranded-DNA, Pot1_372-555-Tpz1_185-212 and Tpz1_425-470-Ccq1_123-439 complexes and propose an integrated model depicting the assembly mechanism of the shelterin complex at telomeres. The structure of Pot1_DBD-DNA unveils how Pot1 recognizes S. pombe degenerate telomeric sequences. Our analyses of Tpz1-Ccq1 reveal structural basis for the essential role of the Tpz1-Ccq1 interaction in telomere recruitment of Ccq1 that is required for telomere maintenance and telomeric heterochromatin formation. Overall, our findings provide valuable structural information regarding interactions within fission yeast shelterin complex at 3’ ss telomeric overhang.     

Roles of the checkpoint sensor clamp Rad9-Rad1-Hus1 (911)-complex and the clamp loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe telomere maintenance

While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1 cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1 cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes.     

The Mec1p and Tel1p checkpoint kinases allow humanized yeast to tolerate chronic telomere dysfunctions by suppressing telomere fusions

In this work we report that budding yeasts carrying human-type telomeric repeats at their chromosome termini show a chronic activation of the Rad53-dependent DNA damage checkpoint pathway and a G2/M cell cycle delay. Furthermore, in the absence of either TEL1/ATM or MEC1/ATR genes, which encodes phosphatidylinositol 3-kinase-related kinases (PIKKs), we detected telomere fusions, whose appearance correlates with a reduced cell viability and a high rate of genome instability. Based on sequence analysis, telomere fusions occurred primarily between ultrashort telomeres. Microcolony formation assays argue against the possibility that fusion-containing cells are eliminated by PIKK-dependent signalling.These findings reveal that humanized telomeres in yeast cells are sensed as a chronically damaged DNA but do not greatly impair cell viability as long as the cells have a functional DNA damage checkpoint.     

Telomere shortening impairs regeneration of the olfactory epithelium in response to injury but not under homeostatic conditions

Atrophy of the olfactory epithelium (OE) associated with impaired olfaction and dry nose represents one of the most common phenotypes of human aging. Impairment in regeneration of a functional olfactory epithelium can also occur in response to injury due to infection or nasal surgery. These complications occur more frequently in aged patients. Although age is the most unifying risk factor for atrophic changes and functional decline of the olfactory epithelium, little is known about molecular mechanisms that could influence maintenance and repair of the olfactory epithelium. Here, we analyzed the influence of telomere shortening (a basic mechanism of cellular aging) on homeostasis and regenerative reserve in response to chemical induced injury of the OE in late generation telomere knockout mice (G3 mTerc^−/−) with short telomeres compared to wild type mice (mTerc^+/+) with long telomeres. The study revealed no significant influence of telomere shortening on homeostatic maintenance of the OE during mouse aging. In contrast, the regenerative response to chemical induced injury of the OE was significantly impaired in G3 mTerc^−/− mice compared to mTerc^+/+ mice. Seven days after chemical induced damage, G3 mTerc^−/− mice exhibited significantly enlarged areas of persisting atrophy compared to mTerc^+/+ mice (p = 0.031). Telomere dysfunction was associated with impairments in cell proliferation in the regenerating epithelium. Deletion of the cell cycle inhibitor, Cdkn1a (p21) rescued defects in OE regeneration in telomere dysfunctional mice. Together, these data indicate that telomere shortening impairs the regenerative capacity of the OE by impairing cell cycle progression in a p21-dependent manner. These findings could be relevant for the impairment in OE function in elderly people.     

Fission Yeast Exo1 and Rqh1-Dna2 Redundantly Contribute to Resection of Uncapped Telomeres

The uncapping of telomeres induces a DNA damage response. In Schizosaccharomyces pombe, deletion of pot1 ⁺ causes telomere uncapping and rapid telomere resection, resulting in chromosome fusion. Using the nmt-pot1-aid strain, we previously reported that Pot1 shut-off causes telomere loss and chromosome fusion in S. pombe. However, the factors responsible for the resection of uncapped telomeres remain unknown. In this study, we investigated these factors and found that concomitant deletion of rqh1 ⁺ and exo1 ⁺ alleviated the loss of telomeres following Pot1 shut-off, suggesting that Rqh1 and Exo1 are redundantly involved in the resection of uncapped telomeres. We also investigated the role of Rqh1 helicase activity and found it to be essential for the resection of uncapped telomeres. Moreover, we found that Dna2 and Exo1 function redundantly in the resection of uncapped telomeres. Taken together, these results suggest that Exo1 and Rqh1-Dna2 redundantly contribute to the resection of uncapped telomeres. Therefore, our results demonstrate that nmt-pot1-aid is an important model strain to study the role of helicases and nucleases in the resection of uncapped telomeres and to improve our understanding of DNA double-strand break repair.     

Distinct requirements for Pot1 in limiting telomere length and maintaining chromosome stability

The fission yeast Pot1 (protection of telomeres) protein binds to the single-stranded extensions at the ends of telomeres, where its presence is critical for the maintenance of linear chromosomes. Homologs of Pot1 have been identified in a wide variety of eukaryotes, including plants, animals, and humans. We now show that Pot1 plays dual roles in telomere length regulation and chromosome end protection. Using a series of Pot1 truncation mutants, we have defined distinct areas of the protein required for chromosome stability and for limiting access to telomere ends by telomerase. We provide evidence that a large portion of Pot1, including the N-terminal DNA binding domain and amino acids close to the C terminus, is essential for its protective function. C-terminal Pot1 fragments were found to exert a dominant-negative effect by displacing endogenous Pot1 from telomeres. Reducing telomere-bound Pot1 in this manner resulted in dramatic lengthening of the telomere tract. Upon further reduction of Pot1 at telomeres, the opposite phenotype was observed: loss of telomeric DNA and chromosome end fusions. Our results demonstrate that cells must carefully regulate the amount of telomere-bound Pot1 to differentiate between allowing access to telomerase and catastrophic loss of telomeres.     

G2/M-checkpoint activation in fasciata1 rescues an aberrant S-phase checkpoint but causes genome instability

The WEE1 and ATM AND RAD3-RELATED (ATR) kinases are important regulators of the plant intra-S-phase checkpoint; consequently, WEE1^KO and ATR^KO roots are hypersensitive to replication-inhibitory drugs. Here, we report on a loss-of-function mutant allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex that suppresses the phenotype of WEE1- or ATR-deficient Arabidopsis (Arabidopsis thaliana) plants. We demonstrate that lack of FAS1 activity results in the activation of an ATAXIA TELANGIECTASIA MUTATED (ATM)- and SUPPRESSOR OF GAMMA-RESPONSE 1 (SOG1)-mediated G2/M-arrest that renders the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for the telomere erosion and loss of ribosomal DNA that are described for fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 is an important secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint.     

Dominant TEL1-hy mutations compensate for Mec1 lack of functions in the DNA damage response

Eukaryotic genome integrity is safeguarded by two highly conserved protein kinases that are called ATR and ATM for humans and Mec1 and Tel1 for Saccharomyces cerevisiae. Although they share sequence similarities and substrates, these protein kinases perform different specialized functions. In particular, Mec1 plays a key role in the DNA damage checkpoint response, whereas Tel1 primarily is involved in telomere homeostasis, and its checkpoint function is masked by the prevailing activity of Mec1. In order to understand how this specificity is achieved, we searched for TEL1 mutations able to compensate for the lack of Mec1 functions. Here, we describe seven independent dominant TEL1-hy alleles that are able to suppress, to different extents, both the hypersensitivity to genotoxic agents and the checkpoint defects of Mec1-deficient cells. Most of these alleles also cause telomere overelongation. In vitro kinase activity was increased compared to that of wild-type Tel1 in the Tel1-hy385, Tel1-hy394, Tel1-hy680, and Tel1-hy909 variants, but its activity was not affected by the TEL1-hy184 and TEL1-hy628 mutations and was slightly reduced by the TEL1-hy544 mutation. Thus, the phenotypes caused by at least some Tel1-hy variants are not simply the consequence of improved catalytic activity. Further characterization shows that Tel1-hy909 not only can sense and signal a single double-stranded DNA break, unlike wild-type Tel1, but also contributes more efficiently than Tel1 to single-stranded DNA accumulation at double-strand ends, thus enhancing Mec1 signaling activity. Moreover, it causes unscheduled checkpoint activation in unperturbed conditions and upregulates the checkpoint response to small amounts of DNA lesions. Finally, Tel1-hy544 can activate the checkpoint more efficiently than wild-type Tel1, while it causes telomere shortening, indicating that the checkpoint and telomeric functions of Tel1 can be separable.     

Regulation of Telomere Length by Checkpoint Genes in Schizosaccharomyces pombe

We have studied telomere length in Schizosaccharomyces pombe strains carrying mutations affecting cell cycle checkpoints, DNA repair, and regulation of the Cdc2 protein kinase. Telomere shortening was found in rad1, rad3, rad17, and rad26 mutants. Telomere lengths in previously characterized rad1 mutants paralleled the replication checkpoint proficiency of those mutants. In contrast, rad9, chk1, hus1, and cds1 mutants had intact telomeres. No difference in telomere length was seen in mutants affected in the regulation of Cdc2, whereas some of the DNA repair mutants examined had slightly longer telomeres than did the wild type. Overexpression of the rad1⁺ gene caused telomeres to elongate slightly. The kinetics of telomere shortening was monitored by following telomere length after disruption of the rad1⁺ gene; the rate was ~1 nucleotide per generation. Wild-type telomere length could be restored by reintroduction of the wild-type rad1⁺ gene. Expression of the Saccharomyces cerevisiae RCK1 protein kinase gene, which suppresses the radiation and hydroxyurea sensitivity of Sz. pombe checkpoint mutants, was able to attenuate telomere shortening in rad1 mutant cells and to increase telomere length in a wild-type background. The functional effects of telomere shortening in rad1 mutants were assayed by measuring loss of a linear and a circular minichromosome. A minor increase in loss rate was seen with the linear minichromosome, and an even smaller difference compared with wild-type was detected with the circular plasmid.     

Fission Yeast RecQ Helicase Rqh1 Is Required for the Maintenance of Circular Chromosomes

Protection of telomeres protein 1 (Pot1) binds to single-stranded telomere overhangs and protects chromosome ends. RecQ helicases regulate homologous recombination at multiple stages, including resection, strand displacement, and resolution. Fission yeast pot1 and RecQ helicase rqh1 double mutants are synthetically lethal, but the mechanism is not fully understood. Here, we show that the synthetic lethality of pot1Δ rqh1Δ double mutants is due to inappropriate homologous recombination, as it is suppressed by the deletion of rad51⁺. The expression of Rad51 in the pot1Δ rqh1Δ rad51Δ triple mutant, which has circular chromosomes, is lethal. Reduction of the expression of Rqh1 in a pot1 disruptant with circular chromosomes caused chromosome missegregation, and this defect was partially suppressed by the deletion of rad51⁺. Taken together, our results suggest that Rqh1 is required for the maintenance of circular chromosomes when homologous recombination is active. Crossovers between circular monomeric chromosomes generate dimers that cannot segregate properly in Escherichia coli. We propose that Rqh1 inhibits crossovers between circular monomeric chromosomes to suppress the generation of circular dimers.     

MEC3, MEC1, and DDC2 are essential components of a telomere checkpoint pathway required for cell cycle arrest during senescence in Saccharomyces cerevisiae

When telomerase is absent and/or telomeres become critically short, cells undergo a progressive decline in viability termed senescence. The telomere checkpoint model predicts that cells will respond to a damaged or critically short telomere by transiently arresting and activating repair of the telomere. We examined the senescence of telomerase-deficient Saccharomyces cerevisiae at the cellular level to ask if the loss of telomerase activity triggers a checkpoint response. As telomerase-deficient mutants were serially subcultured, cells exhibited a progressive decline in average growth rate and an increase in the number of cells delayed in the G2/M stage of the cell cycle. MEC3, MEC1, and DDC2, genes important for the DNA damage checkpoint response, were required for the cell cycle delay in telomerase-deficient cells. In contrast, TEL1, RAD9, and RAD53, genes also required for the DNA damage checkpoint response, were not required for the G2/M delay in telomerase-deficient cells. We propose that the telomere checkpoint is distinct from the DNA damage checkpoint and requires a specific set of gene products to delay the cell cycle and presumably to activate telomerase and/or other telomere repair activities.     

Fission yeast telomeric DNA binding protein Pot1 has the ability to unfold tetraplex structure of telomeric DNA

To understand the regulation mechanism of fission yeast telomeric DNA, we analyzed the structural properties of 4Gn: d(G(n)TTAC)(4) (n = 3, 4) and their interaction with the single-stranded telomeric DNA binding domain of telomere-binding protein Pot1 (Pot1DBD). 4G4 adopted only an antiparallel tetraplex in spite of a mixture of parallel and antiparallel tetraplexes of 4G3. The antiparallel tetraplex of 4G4 became unfolded upon the interaction with Pot1DBD. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of Pot1 to unfold the antiparallel tetraplex is required for telomerase-mediated telomere regulation.     

Pot1 deficiency initiates DNA damage checkpoint activation and aberrant homologous recombination at telomeres

The terminal t-loop structure adopted by mammalian telomeres is thought to prevent telomeres from being recognized as double-stranded DNA breaks by sequestering the 3' single-stranded G-rich overhang from exposure to the DNA damage machinery. The POT1 (protection of telomeres) protein binds the single-stranded overhang and is required for both chromosomal end protection and telomere length regulation. The mouse genome contains two POT1 orthologs, Pot1a and Pot1b. Here we show that conditional deletion of Pot1a elicits a DNA damage response at telomeres, resulting in p53-dependent replicative senescence. Pot1a-deficient cells exhibit overall telomere length and 3' overhang elongation as well as aberrant homologous recombination (HR) at telomeres, manifested as increased telomere sister chromatid exchanges and formation of telomere circles. Telomeric HR following Pot1a loss requires NBS1. Pot1a deletion also results in chromosomal instability. Our results suggest that POT1a is crucial for the maintenance of both telomere integrity and overall genomic stability.