A persistent variant telomere sequence in a human pedigree.

The telomere sequence, TTAGGG, is conserved across all vertebrates and plays an essential role in suppressing the DNA damage response by binding a set of proteins termed shelterin. Changes in the telomere sequence impair shelterin binding, initiate a DNA damage response, and are toxic to cells. Here we identify a family with a variant in the telomere template sequence of telomerase, the enzyme responsible for telomere elongation, that led to a non-canonical telomere sequence. The variant is inherited across at least one generation and one family member reports no significant medical concerns despite ~9% of their telomeres converting to the novel sequence. The variant template disrupts telomerase repeat addition processivity and decreased the binding of the telomere-binding protein POT1. Despite these disruptions, the sequence is readily incorporated into cellular chromosomes. Incorporation of a variant sequence prevents POT1-mediated inhibition of telomerase suggesting that incorporation of a variant sequence may influence telomere addition. These findings demonstrate that telomeres can tolerate substantial degeneracy while remaining functional and provide insights as to how incorporation of a non-canonical telomere sequence might alter telomere length dynamics.

Fate of telomere entanglements is dictated by the timing of anaphase midregion nuclear envelope breakdown.

Persisting replication intermediates can confer mitotic catastrophe. Loss of the fission yeast telomere protein Taz1 (ortholog of mammalian TRF1/TRF2) causes telomeric replication fork (RF) stalling and consequently, telomere entanglements that stretch between segregating mitotic chromosomes. At ≤20 °C, these entanglements fail to resolve, resulting in lethality. Rif1, a conserved DNA replication/repair protein, hinders the resolution of telomere entanglements without affecting their formation. At mitosis, local nuclear envelope (NE) breakdown occurs in the cell's midregion. Here we demonstrate that entanglement resolution occurs in the cytoplasm following this NE breakdown. However, in response to taz1Δ telomeric entanglements, Rif1 delays midregion NE breakdown at ≤20 °C, in turn disfavoring entanglement resolution. Moreover, Rif1 overexpression in an otherwise wild-type setting causes cold-specific NE defects and lethality, which are rescued by membrane fluidization. Hence, NE properties confer the cold-specificity of taz1Δ lethality, which stems from postponement of NE breakdown. We propose that such postponement promotes clearance of simple stalled RFs, but resolution of complex entanglements (involving strand invasion between nonsister telomeres) requires rapid exposure to the cytoplasm.

F2RL3 Regulates Epithelial-Mesenchymal Transition and Angiogenesis in Gastric Cancer through the Rap1/MAPK Signaling Pathway.

BACKGROUND: Gastric cancer (GC) is frequently diagnosed at advanced stages, when cancer cells have already metastasized. Therefore, patients with GC have a low survival rate and poor prognosis even after treatment. METHODS: We downloaded GC-related RNA sequencing (RNA-Seq) data, copy number variation (CNV) data, and clinical data for bioinformatics analysis to screen prognostic genes of GC. Single-sample gene set enrichment analysis and survival analyses were performed on the RNA-Seq data, and differential and correlation analyses were conducted on the CNV data to obtain CNV-driven differentially expressed genes (DEGs). Prognostic genes were identified through univariate Cox analyses of the CNV-driven DEGs, combined with the clinical data. F2R like thrombin or trypsin receptor 3 (F2RL3) was finally selected for verification after functional and survival analyses of the prognostic genes. RESULTS: F2RL3 expression was lower in paracancer tissue than in GC tissue, and lower in GES-1 gastric epithelial cells than in GC cells. The cell culture supernatants from F2RL3-knockdown GC cells were collected and used to culture human umbilical vein endothelial cells (HUVECs). It was observed that F2RL3 enhanced the activity, metastasis, invasion, and angiogenesis of GC cells; promoted the epithelial-mesenchymal transition (EMT) of GC cells; and impacted the Ras-associated protein 1 (Rap1)/mitogen-activated protein kinase (MAPK) pathway. To further explore the involvement of the Rap1/MAPK pathway in GC development, a pathway activator was added to GC cells with knockdown of F2RL3 expression. This pathway activator not only enhanced the activity, invasion, and migration of GC cells but also promoted the EMT and blood vessel formation. CONCLUSIONS: F2RL3 regulates the angiogenesis and EMT of GC cells through the Rap1/MAPK pathway, thus influencing the onset and progression of GC.

A needle in a haystack? The impact of a targeted epilepsy gene panel in the identification of a treatable but rapidly progressive metabolic epilepsy: CLN2 disease.

BACKGROUND: Neuronal ceroid lipofuscinoses (NCL) are a group of autosomal recessive, inherited, lysosomal, and neurodegenerative diseases that causes progressive dementia, seizures, movement disorders, language delay/regression, progressive visual failure, and early death. Neuronal ceroid lipofuscinosis type 2 (CLN2), caused by biallelic pathogenic variants of the TPP1 gene, is the only NCL with an approved targeted therapy. The laboratory diagnosis of CLN2 is established through highly specific tests, leading to diagnostic delays and eventually hampering the provision of specific treatment for patients with CLN2. Epilepsy is a common and clinically-identifiable feature among NCLs, and seizure onset is the main driver for families to seek medical care. OBJECTIVE: To evaluate the results of the Latin America Epilepsy and Genetics Program, an epilepsy gene panel, as a comprehensive tool for the investigation of CLN2 among other genetic causes of epilepsy. METHODS: A total of 1,284 patients with epilepsy without a specific cause who had at least 1 symptom associated with CLN2 were screened for variants in 160 genes associated with epilepsy or metabolic disorders presenting with epilepsy through an epilepsy gene panel. RESULTS: Variants of the TPP1 gene were identified in 25 individuals (1.9%), 21 of them with 2 variants. The 2 most frequently reported variants were p.Arg208* and p.Asp276Val, and 2 novel variants were detected in the present study: p.Leu308Pro and c.89 + 3G > C Intron 2. CONCLUSION: The results suggest that these genetic panels can be very useful tools to confirm or exclude CLN2 diagnosis and, if confirmed, provide disease-specific treatment for the patients.

ANTECEDENTES: As lipofuscinoses ceroides neuronais (neuronal ceroid lipofuscinoses, NCLs, em inglês) são um grupo de doenças autossômicas recessivas, hereditárias, lisossomais e neurodegenerativas que causam demência progressiva, crises epiléticas, distúrbios de movimento, atraso/regressão da linguagem, deficiência visual progressiva e morte precoce. A lipofuscinose ceroide neuronal tipo 2 (neuronal ceroid lipofuscinosis type 2, CLN2, em inglês), causada por variantes patogênicas bialélicas do gene TPP1, é a única com terapia-alvo aprovada. O diagnóstico laboratorial é realizado por testes específicos, o que leva a atrasos diagnósticos e, consequentemente, prejudica a disponibilização de tratamento. A epilepsia é uma característica comum e clinicamente identificável entre as NCLs, e o início das convulsões é o principal motivo para as famílias buscarem atendimento médico. OBJETIVO: Avaliar os resultados do Programa de Epilepsia e Genética da América Latina, um painel genético, como uma ferramenta abrangente para a investigação de CLN2 entre outras causas genéticas de epilepsia. MéTODOS: Um total de 1.284 pacientes com epilepsia sem uma causa específica e que tinham pelo menos 1 sintoma associado à CLN2 foram rastreados em busca de variantes em 160 genes associados à epilepsia ou a distúrbios metabólicos que apresentam epilepsia, por meio de um painel genético. RESULTADOS: Variantes do gene TPP1 foram identificadas em 25 indivíduos (1,9%), sendo que ; 21 apresentavam duas variantes. As duas variantes mais frequentes foram p.Arg208* e p.Asp276Val, e duas variantes novas foram detectadas neste: p.Leu308Pro e c.89 + 3G > C Intron 2. CONCLUSãO: Os resultados sugerem que os painéis genéticos de epilepsia podem ser uma ferramenta útil para confirmar ou excluir o diagnóstico de CLN2 e, se confirmado, fornecer tratamento específico para os pacientes.

TPP1 is associated with risk of advanced precursors and cervical cancer survival.

It is unclear how telomere-binding protein TPP1 interacts with human telomerase reverse transcriptase (hTERT) and influences cervical cancer development and progression. This study included all eligible 156 cervical cancers diagnosed during 2003-2008 and followed up through 2014, 102 cervical intraepithelial neoplasia (CIN) patients, and 16 participants with normal cervix identified at the same period. Correlation of expression of TPP1 and hTERT in these lesions was assessed using Kappa statistics. TPP1 was knocked down by siRNA in three cervical cancer cell lines. We assessed mRNA expression using quantitative real-time polymerase chain reaction and protein expression using tissue microarray-based immunohistochemical staining. We further analyzed the impact of TPP1 expression on the overall survival of cervical cancer patients by calculating the hazard ratio (HR) with 95% confidence intervals (CIs) using the multivariable-adjusted Cox regression model. Compared to the normal cervix, high TPP1expression was significantly associated with CIN 3 and cervical cancers (P<0.001 for both). Expressions of TPP1 and hTERT were highly correlated in CIN 3 (Kappa statistics = 0.50, P = 0.005), squamous cell carcinoma (Kappa statistics = 0.22, P = 0.011), and adenocarcinoma/adenosquamous carcinoma (Kappa statistics = 0.77, P = 0.001). Mechanistically, knockdown of TPP1 inhibited the expression of hTERT in both mRNA and protein levels. High expression of TPP1 (HR = 2.61, 95% CI 1.23-5.51) and co-high expression of TPP1 and hTERT (HR = 2.38, 95% CI 1.28-4.43) were independently associated with worse survival in cervical cancer patients. TPP1 and hTERT expression was correlated and high expression of TPP1 was associated with high risk of CIN 3 and cervical cancer and could predict a worse survival in cervical cancer.

Caenorhabditis elegans telomere-binding proteins TEBP-1 and TEBP-2 adapt the Myb module to dimerize and bind telomeric DNA.

Protecting chromosome ends from misrecognition as double-stranded (ds) DNA breaks is fundamental to eukaryotic viability. The protein complex shelterin prevents a DNA damage response at mammalian telomeres. Mammalian shelterin proteins TRF1 and TRF2 and their homologs in yeast and protozoa protect telomeric dsDNA. N-terminal homodimerization and C-terminal Myb-domain-mediated dsDNA binding are two structural hallmarks of end protection by TRF homologs. Yet our understanding of how Caenorhabditis elegans protects its telomeric dsDNA is limited. Recently identified C. elegans proteins TEBP-1 (also called DTN-1) and TEBP-2 (also called DTN-2) are functional homologs of TRF proteins, but how they bind DNA and whether or how they dimerize is not known. TEBP-1 and TEBP-2 harbor three Myb-containing domains (MCDs) and no obvious dimerization domain. We demonstrate biochemically that only the third MCD binds DNA. We solve the X-ray crystal structure of TEBP-2 MCD3 with telomeric dsDNA to reveal the structural mechanism of telomeric dsDNA protection in C. elegans. Mutagenesis of the DNA-binding site of TEBP-1 and TEBP-2 compromises DNA binding in vitro, and increases DNA damage signaling, lengthens telomeres, and decreases brood size in vivo. Via an X-ray crystal structure, biochemical validation of the dimerization interface, and SEC-MALS analysis, we demonstrate that MCD1 and MCD2 form a composite dimerization module that facilitates not only TEBP-1 and TEBP-2 homodimerization but also heterodimerization. These findings provide fundamental insights into C. elegans telomeric dsDNA protection and highlight how different eukaryotes have evolved distinct strategies to solve the chromosome end protection problem.

TIN2-mediated reduction of mitophagy induces RPE senescence under high glucose.

The telomere-associated protein TIN2 localizes to both telomeres and mitochondria. Nevertheless, the impact of TIN2 on retinal pigment epithelial (RPE) cells in diabetic retinopathy (DR) remains unclear. This research aims to examine the role of TIN2 in the senescence of RPE and its potential as a therapeutic target. Western blotting and immunofluorescence staining were utilized to identify TIN2 expression and mitophagy. RT-qPCR was employed to identify senescent associated secretory phenotype (SASP) in ARPE-19 cells infected with TIN2 overexpression. To examine mitochondria and the cellular senescence of RPE, TEM, SA-ß-gal staining, and cell cycle analysis were used. The impact of TIN2 was examined using OCT and immunohistochemistry in mice. DHE staining and ZO-1 immunofluorescence were applied to detect RPE oxidative stress and tight junctions. Our research revealed that increased mitochondria-localized TIN2 aggravated the cellular senescence of RPE cells both in vivo and in vitro under hyperglycemia. TIN2 overexpression stimulated the mTOR signaling pathway in ARPE-19 cells and exacerbated the inhibition of mitophagy levels under high glucose, which can be remedied through the mTOR inhibitor, rapamycin. Knockdown of TIN2 significantly reduced senescence and mitochondrial oxidative stress in ARPE-19 cells under high glucose and restored retinal thickness and RPE cell tight junctions in DR mice. Our study indicates that increased mitochondria-localized TIN2 induced cellular senescence in RPE via compromised mitophagy and activated mTOR signaling. These results propose that targeting TIN2 could potentially serve as a therapeutic strategy in the treatment of DR.

Telomere-lengthening germline variants predispose to a syndromic papillary thyroid cancer subtype.

Papillary thyroid cancer (PTC) is the most common endocrine malignancy. 10% to 15% of individuals show familial clustering with three or more affected members, but the factors underlying this risk are unknown. In a group of recently studied individuals with POT1 pathogenic variants and ultra-long telomere length, PTC was the second most common solid tumor. We tested whether variants in POT1 and four other telomere-maintenance genes associated with familial cancer underlie PTC susceptibility. Among 470 individuals, we identified pathogenic or likely pathogenic variants in three genes encoding telomere-binding proteins: POT1, TINF2, and ACD. They were found in 4.5% and 1.5% of familial and unselected cases, respectively. Individuals harboring these variants had ultra-long telomere length, and 15 of 18 (83%) developed other cancers, of which melanoma, lymphoma, and sarcoma were most common. Among individuals with PTC and melanoma, 22% carried a deleterious germline variant, suggesting that a long telomere syndrome might be clinically recognizable. Successive generations had longer telomere length than their parents and, at times, developed more cancers at younger ages. Tumor sequencing identified a single oncogenic driver, BRAF p.Val600Glu, in 10 of 10 tumors studied, but no telomere-maintenance mechanism, including at the TERT promoter. These data identify a syndromic subset of PTCs with locus heterogeneity and telomere lengthening as a convergent mechanism. They suggest these germline variants lower the threshold to cancer by obviating the need for an acquired telomere-maintenance mechanism in addition to sustaining the longevity of oncogenic mutations.

Decreased telomere length in a subgroup of young individuals with bipolar disorders: replication in the FACE-BD cohort and association with the shelterin component POT1.

Bipolar disorder (BD) has been associated with premature cellular aging with shortened telomere length (TL) as compared to the general population. We recently identified a subgroup of young individuals with prematurely shortened TL. The aims of the present study were to replicate this observation in a larger sample and analyze the expression levels of genes associated with age or TL in a subsample of these individuals. TL was measured on peripheral blood DNA using quantitative polymerase chain reaction in a sample of 542 individuals with BD and clustering analyses were performed. Gene expression level of 29 genes, associated with aging or with telomere maintenance, was analyzed in RNA samples from a subsample of 129 individuals. Clustering analyses identified a group of young individuals (mean age 29.64 years), with shorter TL. None of the tested clinical variables were significantly associated with this subgroup. Gene expression level analyses showed significant downregulation of MYC, POT1, and CD27 in the prematurely aged young individuals compared to the young individuals with longer TL. After adjustment only POT1 remained significantly differentially expressed between the two groups of young individuals. This study confirms the existence of a subgroup of young individuals with BD with shortened TL. The observed decrease of POT1 expression level suggests a newly described cellular mechanism in individuals with BD, that may contribute to telomere shortening.

Upregulation of shelterin and CST genes and longer telomeres are associated with unfavorable prognostic characteristics in prostate cancer.

INTRODUCTION: Search for new clinical biomarkers targets in prostate cancer (PC) is urgent. Telomeres might be one of these targets. Telomeres are the extremities of linear chromosomes, essential for genome stability and control of cell divisions. Telomere homeostasis relies on the proper functioning of shelterin and CST complexes. Telomeric dysfunction and abnormal expression of its components are reported in most cancers and are associated with PC. Despite this, there are only a few studies about the expression of the main telomere complexes and their relationship with PC progression. We aimed to evaluate the role of shelterin (POT1, TRF2, TPP1, TIN2, and RAP1) and CST (CTC1, STN1, and TEN1) genes and telomere length in the progression of PC. METHODS: We evaluated genetic alterations of shelterin and CST by bioinformatics in samples of localized (n = 499) and metastatic castration-resistant PC (n = 444). We also analyzed the expression of the genes using TCGA (localized PC n = 497 and control n = 152) and experimental approaches, with surgical specimens (localized PC n = 81 and BPH n = 10) and metastatic cell lines (LNCaP, DU145, PC3 and PNT2 as control) by real-time PCR. Real-time PCR also determined the telomere length in the same experimental samples. All acquired data were associated with clinical parameters. RESULTS: Genetic alterations are uncommon in PC, but POT1, TIN2, and TEN1 showed significantly more amplifications in the metastatic cancer. Except for CTC1 and TEN1, which are differentially expressed in localized PC samples, we did not detect an expression pattern relative to control and cell lines. Nevertheless, except for TEN1, the upregulation of all genes is associated with a worse prognosis in localized PC. We also found that increased telomere length is associated with disease aggressiveness in localized PC. CONCLUSION: The upregulation of shelterin and CST genes creates an environment that favors telomere elongation, giving selective advantages for localized PC cells to progress to more aggressive stages of the disease.

Guardians of the Genome: How the Single-Stranded DNA-Binding Proteins RPA and CST Facilitate Telomere Replication.

Telomeres act as the protective caps of eukaryotic linear chromosomes; thus, proper telomere maintenance is crucial for genome stability. Successful telomere replication is a cornerstone of telomere length regulation, but this process can be fraught due to the many intrinsic challenges telomeres pose to the replication machinery. In addition to the famous "end replication" problem due to the discontinuous nature of lagging strand synthesis, telomeres require various telomere-specific steps for maintaining the proper 3' overhang length. Bulk telomere replication also encounters its own difficulties as telomeres are prone to various forms of replication roadblocks. These roadblocks can result in an increase in replication stress that can cause replication forks to slow, stall, or become reversed. Ultimately, this leads to excess single-stranded DNA (ssDNA) that needs to be managed and protected for replication to continue and to prevent DNA damage and genome instability. RPA and CST are single-stranded DNA-binding protein complexes that play key roles in performing this task and help stabilize stalled forks for continued replication. The interplay between RPA and CST, their functions at telomeres during replication, and their specialized features for helping overcome replication stress at telomeres are the focus of this review.

A POT1 Founder Variant Associated with Early Onset Recurrent Melanoma and Various Solid Malignancies.

POT1 (Protection of Telomeres 1) is a key component of the six-membered shelterin complex that plays a critical role in telomere protection and length regulation. Germline variants in the POT1 gene have been implicated in predisposition to cancer, primarily to melanoma and chronic lymphocytic leukemia (CLL). We report the identification of POT1 p.(I78T), previously ranked with conflicting interpretations of pathogenicity, as a founder pathogenic variant among Ashkenazi Jews (AJs) and describe its unique clinical landscape. A directed database search was conducted for individuals referred for genetic counselling from 2018 to 2023. Demographic, clinical, genetic, and pathological data were collected and analyzed. Eleven carriers, 25 to 67 years old, from ten apparently unrelated families were identified. Carriers had a total of 30 primary malignancies (range 1-6); nine carriers (82%) had recurrent melanoma between the ages of 25 and 63 years, three carriers (27%) had desmoid tumors, three (27%) had papillary thyroid cancer (PTC), and five women (63% of female carriers) had breast cancer between the ages of 44 and 67 years. Additional tumors included CLL; sarcomas; endocrine tumors; prostate, urinary, and colorectal cancers; and colonic polyps. A review of a local exome database yielded an allelic frequency of the variant of 0.06% among all ethnicities and of 0.25% in AJs. A shared haplotype was found in all carriers tested. POT1 p.(I78T) is a founder disease-causing variant associated with early-onset melanoma and additional various solid malignancies with a high tumor burden. We advocate testing for this variant in high-risk patients of AJ descent. The inclusion of POT1 in germline panels for various types of cancer is warranted.

Human CST complex restricts excessive PrimPol repriming upon UV induced replication stress by suppressing p21.

DNA replication stress, caused by various endogenous and exogenous agents, halt or stall DNA replication progression. Cells have developed diverse mechanisms to tolerate and overcome replication stress, enabling them to continue replication. One effective strategy to overcome stalled replication involves skipping the DNA lesion using a specialized polymerase known as PrimPol, which reinitiates DNA synthesis downstream of the damage. However, the mechanism regulating PrimPol repriming is largely unclear. In this study, we observe that knockdown of STN1 or CTC1, components of the CTC1/STN1/TEN1 complex, leads to enhanced replication progression following UV exposure. We find that such increased replication is dependent on PrimPol, and PrimPol recruitment to stalled forks increases upon CST depletion. Moreover, we find that p21 is upregulated in STN1-depleted cells in a p53-independent manner, and p21 depletion restores normal replication rates caused by STN1 deficiency. We identify that p21 interacts with PrimPol, and STN1 depletion stimulates p21-PrimPol interaction and facilitates PrimPol recruitment to stalled forks. Our findings reveal a previously undescribed interplay between CST, PrimPol and p21 in promoting repriming in response to stalled replication, and shed light on the regulation of PrimPol repriming at stalled forks.

Exploring TRF2-Dependent DNA Distortion Through Single-DNA Manipulation Studies.

TRF2 is a component of shelterin, a telomere-specific protein complex that protects the ends of mammalian chromosomes from DNA damage signaling and improper repair. TRF2 functions as a homodimer and its interaction with telomeric DNA has been studied, but its full-length DNA-binding properties are unknown. This study examines TRF2's interaction with single-DNA strands and focuses on the conformation of the TRF2-DNA complex and TRF2's preference for DNA chirality. The results show that TRF2-DNA can switch between extended and compact conformations, indicating multiple DNA-binding modes, and TRF2's binding does not have a strong preference for DNA supercoiling chirality when DNA is under low tension. Instead, TRF2 induces DNA bending under tension. Furthermore, both the N-terminal domain of TRF2 and the Myb domain enhance its affinity for the telomere sequence, highlighting the crucial role of multivalent DNA binding in enhancing its affinity and specificity for telomere sequence. These discoveries offer unique insights into TRF2's interaction with telomeric DNA.

TERT-independent telomere elongation and shelterin dysregulation after pulmonary exposure to stainless-steel welding fume in-vivo.

Telomeres are inert DNA sequences (TTAGGG) at the end of chromosomes that protect genetic information and maintain DNA integrity. Emerging evidence has demonstrated that telomere alteration can be closely related to occupational exposure and the development of various disease conditions, including cancer. However, the functions and underlying molecular mechanisms of telomere alteration and shelterin dysregulation after welding fume exposures have not been broadly defined. In this study, we analyzed telomere length and shelterin complex proteins in peripheral blood mononuclear cells (PBMCs) and in lung tissue recovered from male Sprague-Dawley rats following exposure by intratracheal instillation (ITI) to 2 mg/rat of manual metal arc-stainless steel (MMA-SS) welding fume particulate or saline (vehicle control). PBMCs and lung tissue were harvested at 30 d after instillation. Our study identified telomere elongation and shelterin dysregulation in PBMCs and lung tissue after welding fume exposure. Mechanistically, telomere elongation was independent of telomerase reverse transcriptase (TERT) activation. Collectively, our findings demonstrated that welding fume-induced telomere elongation was (a) TERT-independent and (b) associated with shelterin complex dysregulation. It is possible that an alteration of telomere length and its regulatory proteins may be utilized as predictive biomarkers for various disease conditions after welding fume exposure. This needs further investigation.

CST-polymerase α-primase solves a second telomere end-replication problem.

Telomerase adds G-rich telomeric repeats to the 3' ends of telomeres1, counteracting telomere shortening caused by loss of telomeric 3' overhangs during leading-strand DNA synthesis ('the end-replication problem'2). Here we report a second end-replication problem that originates from the incomplete duplication of the C-rich telomeric repeat strand (C-strand) by lagging-strand DNA synthesis. This problem is resolved by fill-in synthesis mediated by polymerase α-primase bound to Ctc1-Stn1-Ten1 (CST-Polα-primase). In vitro, priming for lagging-strand DNA replication does not occur on the 3' overhang and lagging-strand synthesis stops in a zone of approximately 150 nucleotides (nt) more than 26 nt from the end of the template. Consistent with the in vitro data, lagging-end telomeres of cells lacking CST-Polα-primase lost 50-60 nt of telomeric CCCTAA repeats per population doubling. The C-strands of leading-end telomeres shortened by around 100 nt per population doubling, reflecting the generation of 3' overhangs through resection. The measured overall C-strand shortening in the absence of CST-Polα-primase fill-in is consistent with the combined effects of incomplete lagging-strand synthesis and 5' resection at the leading ends. We conclude that canonical DNA replication creates two telomere end-replication problems that require telomerase to maintain the G-rich strand and CST-Polα-primase to maintain the C-strand.

Ccq1 restrains Mre11-mediated degradation to distinguish short telomeres from double-strand breaks.

Telomeres protect chromosome ends and are distinguished from DNA double-strand breaks (DSBs) by means of a specialized chromatin composed of DNA repeats bound by a multiprotein complex called shelterin. We investigated the role of telomere-associated proteins in establishing end-protection by studying viable mutants lacking these proteins. Mutants were studied using a Schizosaccharomyces pombe model system that induces cutting of a 'proto-telomere' bearing telomere repeats to rapidly form a new stable chromosomal end, in contrast to the rapid degradation of a control DSB. Cells lacking the telomere-associated proteins Taz1, Rap1, Poz1 or Rif1 formed a chromosome end that was stable. Surprisingly, cells lacking Ccq1, or impaired for recruiting Ccq1 to the telomere, converted the cleaved proto-telomere to a rapidly degraded DSB. Ccq1 recruits telomerase, establishes heterochromatin and affects DNA damage checkpoint activation; however, these functions were separable from protection of the new telomere by Ccq1. In cells lacking Ccq1, telomere degradation was greatly reduced by eliminating the nuclease activity of Mre11 (part of the Mre11-Rad50-Nbs1/Xrs2 DSB processing complex), and higher amounts of nuclease-deficient Mre11 associated with the new telomere. These results demonstrate a novel function for S. pombe Ccq1 to effect end-protection by restraining Mre11-dependent degradation of the DNA end.

Rif2 interaction with Rad50 counteracts Tel1 functions in checkpoint signalling and DNA tethering by releasing Tel1 from MRX binding.

The yeast Rif2 protein is known to inhibit Mre11 nuclease and the activation of Tel1 kinase through a short motif termed MIN, which binds the Rad50 subunit and simulates its ATPase activity in vitro. The mechanism by which Rif2 restrains Tel1 activation and the consequences of this inhibition at DNA double-strand breaks (DSBs) are poorly understood. In this study, we employed AlphaFold-Multimer modelling to pinpoint and validate the interaction surface between Rif2 MIN and Rad50. We also engineered the rif2-S6E mutation that enhances the inhibitory effect of Rif2 by increasing Rif2-Rad50 interaction. Unlike rif2Δ, the rif2-S6E mutation impairs hairpin cleavage. Furthermore, it diminishes Tel1 activation by inhibiting Tel1 binding to DSBs while leaving MRX association unchanged, indicating that Rif2 can directly inhibit Tel1 recruitment to DSBs. Additionally, Rif2S6E reduces Tel1-MRX interaction and increases stimulation of ATPase by Rad50, indicating that Rif2 binding to Rad50 induces an ADP-bound MRX conformation that is not suitable for Tel1 binding. The decreased Tel1 recruitment to DSBs in rif2-S6E cells impairs DSB end-tethering and this bridging defect is suppressed by expressing a Tel1 mutant variant that increases Tel1 persistence at DSBs, suggesting a direct role for Tel1 in the bridging of DSB ends.

Characterization of novel mutations in the TEL-patch domain of the telomeric factor TPP1 associated with telomere biology disorders.

Telomeres are nucleoprotein structures that protect the chromosome ends from degradation and fusion. Telomerase is a ribonucleoprotein complex essential to maintain the length of telomeres. Germline defects that lead to short and/or dysfunctional telomeres cause telomere biology disorders (TBDs), a group of rare and heterogeneous Mendelian diseases including pulmonary fibrosis, dyskeratosis congenita, and Høyeraal-Hreidarsson syndrome. TPP1, a telomeric factor encoded by the gene ACD, recruits telomerase at telomere and stimulates its activity via its TEL-patch domain that directly interacts with TERT, the catalytic subunit of telomerase. TBDs due to TPP1 deficiency have been reported only in 11 individuals. We here report four unrelated individuals with a wide spectrum of TBD manifestations carrying either heterozygous or homozygous ACD variants consisting in the recurrent and previously described in-frame deletion of K170 (K170∆) and three novel missense mutations G179D, L184R, and E215V. Structural and functional analyses demonstrated that the four variants affect the TEL-patch domain of TPP1 and impair telomerase activity. In addition, we identified in the ACD gene several motifs associated with small deletion hotspots that could explain the recurrence of the K170∆ mutation. Finally, we detected in a subset of blood cells from one patient, a somatic TERT promoter-activating mutation that likely provides a selective advantage over non-modified cells, a phenomenon known as indirect somatic genetic rescue. Together, our results broaden the genetic and clinical spectrum of TPP1 deficiency and specify new residues in the TEL-patch domain that are crucial for length maintenance and stability of human telomeres in vivo.