Telomeres cooperate with the nuclear envelope to maintain genome stability.

Mammalian telomeres have evolved safeguards to prevent their recognition as DNA double-stranded breaks by suppressing the activation of various DNA sensing and repair proteins. We have shown that the telomere-binding proteins TRF2 and RAP1 cooperate to prevent telomeres from undergoing aberrant homology-directed recombination by mediating t-loop protection. Our recent findings also suggest that mammalian telomere-binding proteins interact with the nuclear envelope to maintain chromosome stability. RAP1 interacts with nuclear lamins through KU70/KU80, and disruption of RAP1 and TRF2 function result in nuclear envelope rupture, promoting telomere-telomere recombination to form structures termed ultrabright telomeres. In this review, we discuss the importance of the interactions between shelterin components and the nuclear envelope to maintain telomere homeostasis and genome stability.

Human POT1 protects the telomeric ds-ss DNA junction by capping the 5' end of the chromosome.

Protection of telomeres 1 (POT1) is the 3' single-stranded overhang-binding telomeric protein that prevents an ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) at chromosome ends. What precludes the DDR machinery from accessing the telomeric double-stranded-single-stranded junction is unknown. We demonstrate that human POT1 binds this junction by recognizing the phosphorylated 5' end of the chromosome. High-resolution crystallographic structures reveal that the junction is capped by POT1 through a "POT-hole" surface, the mutation of which compromises junction protection in vitro and telomeric 5'-end definition and DDR suppression in human cells. Whereas both mouse POT1 paralogs bind the single-stranded overhang, POT1a, not POT1b, contains a POT-hole and binds the junction, which explains POT1a's sufficiency for end protection. Our study shifts the paradigm for DDR suppression at telomeres by highlighting the importance of protecting the double-stranded-single-stranded junction.

The shelterin protein expansion of telomere dynamics: Linking early life adversity, life history, and the hallmarks of aging.

Aging is characterized by functional decline occurring alongside changes to several hallmarks of aging. One of the hallmarks includes attrition of repeated DNA sequences found at the ends of chromosomes called telomeres. While telomere attrition is linked to morbidity and mortality, whether and how it causally contributes to lifelong rates of functional decline is unclear. In this review, we propose the shelterin-telomere hypothesis of life history, in which telomere-binding shelterin proteins translate telomere attrition into a range of physiological outcomes, the extent of which may be modulated by currently understudied variation in shelterin protein levels. Shelterin proteins may expand the breadth and timing of consequences of telomere attrition, e.g., by translating early life adversity into acceleration of the aging process. We consider how the pleiotropic roles of shelterin proteins provide novel insights into natural variation in physiology, life history, and lifespan. We highlight key open questions that encourage the integrative, organismal study of shelterin proteins that enhances our understanding of the contribution of the telomere system to aging.

RPA-like single-stranded DNA-binding protein complexes including CST serve as specialized processivity factors for polymerases.

Telomeres and other single-stranded regions of the genome require specialized management to maintain stability and for proper progression of DNA metabolism pathways. Human Replication Protein A and CTC1-STN1-TEN1 are structurally similar heterotrimeric protein complexes that have essential ssDNA-binding roles in DNA replication, repair, and telomeres. Yeast and ciliates have related ssDNA-binding proteins with strikingly conserved structural features to these human heterotrimeric protein complexes. Recent breakthrough structures have extended our understanding of these commonalities by illuminating a common mechanism used by these proteins to act as processivity factors for their partner polymerases through their ability to manage ssDNA.

Complex interaction network revealed by mutation of human telomerase 'insertion in fingers' and essential N-terminal domains and the telomere protein TPP1.

In the majority of human cancer cells, cellular immortalization depends on the maintenance of telomere length by telomerase. An essential step required for telomerase function is its recruitment to telomeres, which is regulated by the interaction of the telomere protein, TPP1, with the telomerase essential N-terminal (TEN) domain of the human telomerase reverse transcriptase, hTERT. We previously reported that the hTERT 'insertion in fingers domain' (IFD) recruits telomerase to telomeres in a TPP1-dependent manner. Here, we use hTERT truncations and the IFD domain containing mutations in conserved residues or premature aging disease-associated mutations to map the interactions between the IFD and TPP1. We find that the hTERT-IFD domain can interact with TPP1. However, deletion of the IFD motif in hTERT lacking the N-terminus and the C-terminal extension does not abolish interaction with TPP1, suggesting the IFD is not essential for hTERT interaction with TPP1. Several conserved residues in the central IFD-TRAP region that we reported regulate telomerase recruitment to telomeres, and cell immortalization compromise interaction of the hTERT-IFD domain with TPP1 when mutated. Using a similar approach, we find that the IFD domain interacts with the TEN domain but is not essential for intramolecular hTERT interactions with the TEN domain. IFD-TEN interactions are not disrupted by multiple amino acid changes in the IFD or TEN, thus highlighting a complex regulation of IFD-TEN interactions as suggested by recent cryo-EM structures of human telomerase.

Helicase mediated vectorial folding of telomere G-quadruplex.

The G-rich single-stranded telomere overhang can self-fold into G-quadruplex (G4) structure both in vivo and in vitro. In somatic cells, telomeres shorten progressively due to the end-replication. In stem cells, however, telomeres are replenished by a special enzyme, telomerase which synthesizes single-stranded telomere overhang. The active extension by the telomerase releases G-rich overhang segmentally in 5' to 3' direction as the overhang folds into G4 structure after successive elongation. To replicate such vectorial G4 folding process, we employed a superhelicase, Rep-X to release the G-rich sequence gradually. Using single-molecule assay we demonstrated that the folded conformation achieved by the vectorial folding is inherently different from the post-folding where the entire overhang is allowed to fold at once. In addition, the vectorially folded overhangs are less stable and more accessible to a complementary C-rich strand and the telomere binding protein, POT1 compared to the post-folded state. The higher accessibility may have implications for the facile loading of shelterin proteins after DNA replication.

Shelterin Components Modulate Nucleic Acids Condensation and Phase Separation in the Context of Telomeric DNA.

Telomeres are nucleoprotein complexes that protect the ends of chromosomes and are essential for chromosome stability in Eukaryotes. In cells, individual telomeres form distinct globules of finite size that appear to be smaller than expected for bare DNA. Moreover, telomeres can cluster together, form telomere-induced-foci or co-localize with promyelocytic leukemia (PML) nuclear bodies. The physical basis for collapse of individual telomeres and coalescence of multiple ones remains unclear, as does the relationship between these two phenomena. By combining single-molecule force spectroscopy measurements, optical microscopy, turbidity assays, and simulations, we show that the telomere scaffolding protein TRF2 can condense individual DNA chains and drives coalescence of multiple DNA molecules, leading to phase separation and the formation of liquid-like droplets. Addition of the TRF2 binding protein hRap1 modulates phase boundaries and tunes the specificity of solution demixing while simultaneously altering the degree of DNA compaction. Our results suggest that the condensation of single telomeres and formation of biomolecular condensates containing multiple telomeres are two different outcomes driven by the same set of molecular interactions. Moreover, binding partners, such as other telomere components, can alter those interactions to promote single-chain DNA compaction over multiple-chain phase separation.

Insights into POT1 structural dynamics revealed by cryo-EM.

Telomeres are protein-DNA complexes that protect the ends of linear eukaryotic chromosomes. Mammalian telomeric DNA consists of 5'-(TTAGGG)n-3' double-stranded repeats, followed by up to several hundred bases of a 3' single-stranded G-rich overhang. The G-rich overhang is bound by the shelterin component POT1 which interacts with TPP1, the component involved in telomerase recruitment. A previously published crystal structure of the POT1 N-terminal half bound to the high affinity telomeric ligand 5'-TTAGGGTTAG-3' showed that the first six nucleotides, TTAGGG, are bound by the OB1 fold, while the adjacent OB2 binds the last four, TTAG. Here, we report two cryo-EM structures of full-length POT1 bound by the POT1-binding domain of TPP1. The structures differ in the relative orientation of the POT1 OB1 and OB2, suggesting that these two DNA-binding OB folds take up alternative conformations. Supporting DNA binding studies using telomeric ligands in which the OB1 and OB2 binding sites were spaced apart, show that POT1 binds with similar affinities to spaced or contiguous binding sites, suggesting plasticity in DNA binding and a role for the alternative conformations observed. A likely explanation is that the structural flexibility of POT1 enhances binding to the tandemly arranged telomeric repeats and hence increases telomere protection.

Structural basis of human telomerase recruitment by TPP1-POT1.

Telomerase maintains genome stability by extending the 3' telomeric repeats at eukaryotic chromosome ends, thereby counterbalancing progressive loss caused by incomplete genome replication. In mammals, telomerase recruitment to telomeres is mediated by TPP1, which assembles as a heterodimer with POT1. We report structures of DNA-bound telomerase in complex with TPP1 and with TPP1-POT1 at 3.2- and 3.9-angstrom resolution, respectively. Our structures define interactions between telomerase and TPP1-POT1 that are crucial for telomerase recruitment to telomeres. The presence of TPP1-POT1 stabilizes the DNA, revealing an unexpected path by which DNA exits the telomerase active site and a DNA anchor site on telomerase that is important for telomerase processivity. Our findings rationalize extensive prior genetic and biochemical findings and provide a framework for future mechanistic work on telomerase regulation.

The TERB1 MYB domain suppresses telomere erosion in meiotic prophase I.

The meiosis-specific telomere-binding protein TERB1 anchors telomeres to the nuclear envelope and drives chromosome movements for the pairing of homologous chromosomes. TERB1 has an MYB-like DNA-binding (MYB) domain, which is a hallmark of telomeric DNA-binding proteins. Here, we demonstrate that the TERB1 MYB domain has lost its canonical DNA-binding activity. The analysis of Terb1 point mutant mice expressing TERB1 lacking its MYB domain showed that the MYB domain is dispensable for telomere localization of TERB1 and the downstream TERB2-MAJIN complex, the promotion of homologous pairing, and even fertility. Instead, the TERB1 MYB domain regulates the enrichment of cohesin and promotes the remodeling of axial elements in the early-to-late pachytene transition, which suppresses telomere erosion. Considering its conservation across metazoan phyla, the TERB1 MYB domain is likely to be important for the maintenance of telomeric DNA and thus for genomic integrity by suppressing meiotic telomere erosion over long evolutionary timescales.

Structure of the Human TELO2-TTI1-TTI2 Complex.

Phosphatidylinositol 3-kinase-related protein kinases (PIKKs) play critical roles in various metabolic pathways related to cell proliferation and survival. The TELO2-TTI1-TTI2 (TTT) complex has been proposed to recognize newly synthesized PIKKs and to deliver them to the R2TP complex (RUVBL1-RUVBL2-RPAP3-PIH1D1) and the heat shock protein 90 chaperone, thereby supporting their folding and assembly. Here, we determined the cryo-EM structure of the TTT complex at an average resolution of 4.2 Å. We describe the full-length structures of TTI1 and TELO2, and a partial structure of TTI2. All three proteins form elongated helical repeat structures. TTI1 provides a platform on which TELO2 and TTI2 bind to its central region and C-terminal end, respectively. The TELO2 C-terminal domain (CTD) is required for the interaction with TTI1 and recruitment of Ataxia-telangiectasia mutated (ATM). The N- and C-terminal segments of TTI1 recognize the FRAP-ATM-TRRAP (FAT) domain and the N-terminal HEAT repeats of ATM, respectively. The TELO2 CTD and TTI1 N- and C-terminal segments are required for cell survival in response to ionizing radiation.

Evolutionary mode for the functional preservation of fast-evolving Drosophila telomere capping proteins.

DNA end protection is fundamental for the long-term preservation of the genome. In vertebrates the Shelterin protein complex protects telomeric DNA ends, thereby contributing to the maintenance of genome integrity. In the Drosophila genus, this function is thought to be performed by the Terminin complex, an assembly of fast-evolving subunits. Considering that DNA end protection is fundamental for successful genome replication, the accelerated evolution of Terminin subunits is counterintuitive, as conservation is supposed to maintain the assembly and concerted function of the interacting partners. This problem extends over Drosophila telomere biology and provides insight into the evolution of protein assemblies. In order to learn more about the mechanistic details of this phenomenon we have investigated the intra- and interspecies assemblies of Verrocchio and Modigliani, two Terminin subunits using in vitro assays. Based on our results and on homology-based three-dimensional models for Ver and Moi, we conclude that both proteins contain Ob-fold and contribute to the ssDNA binding of the Terminin complex. We propose that the preservation of Ver function is achieved by conservation of specific amino acids responsible for folding or localized in interacting surfaces. We also provide here the first evidence on Moi DNA binding.

Multifunctionality of the Telomere-Capping Shelterin Complex Explained by Variations in Its Protein Composition.

Protecting telomere from the DNA damage response is essential to avoid the entry into cellular senescence and organismal aging. The progressive telomere DNA shortening in dividing somatic cells, programmed during development, leads to critically short telomeres that trigger replicative senescence and thereby contribute to aging. In several organisms, including mammals, telomeres are protected by a protein complex named Shelterin that counteract at various levels the DNA damage response at chromosome ends through the specific function of each of its subunits. The changes in Shelterin structure and function during development and aging is thus an intense area of research. Here, we review our knowledge on the existence of several Shelterin subcomplexes and the functional independence between them. This leads us to discuss the possibility that the multifunctionality of the Shelterin complex is determined by the formation of different subcomplexes whose composition may change during aging.

Novel compound heterozygous STN1 variants are associated with Coats Plus syndrome.

AIM: Coats plus syndrome (CP) is a rare autosomal recessive disorder, characterised by retinal telangiectasia exudates (Coats disease), leukodystrophy, distinctive intracranial calcification and cysts, as well as extra-neurological features including abnormal vasculature of the gastrointestinal tract, portal hypertension and osteopenia with a tendency to fractures. CP most frequently occurs due to loss-of-function mutations in CTC1. The encoded protein CTC1 constitutes part of the CST (CTC1-STN1-TEN1) complex, and three patients have been described with CP due to biallelic mutations in STN1. Together with the identification of homozygosity for a specific loss-of-function mutation in POT1 in a sibling pair, these observations highlight a defect in the maintenance of telomere integrity as the cause of CP, although the precise mechanism leading to the micro-vasculopathy seen at a pathological level remains unclear. Here, we present the investigation of a fourth child who presented to us with retinal exudates, intracranial calcifications and developmental delay, in keeping with a diagnosis of CP, and later went on to develop pancytopenia and gastrointestinal bleeding. Genome sequencing revealed compound heterozygous variants in STN1 as the likely genetic cause of CP in this present case. METHODS: We assessed the phenotype to be CP and undertook targeted sequencing. RESULTS: Whilst sequencing of CTC1 and POT1 was normal, we identified novel compound heterozygous variants in STN1 (previous gene symbol OBFC1): one loss-of-function--c.894dup (p.(Asp299Argfs*58)); and one missense--c.707T>C (p.(Leu236Pro)). CONCLUSION: Given the clinical phenotype and identified variants we suggest that this is only the fourth patient reported to date with CP due to mutations in STN1.

Mechanism of MRX inhibition by Rif2 at telomeres.

Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5' end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5' resection.

Structures of telomerase at several steps of telomere repeat synthesis.

Telomerase is unique among the reverse transcriptases in containing a noncoding RNA (known as telomerase RNA (TER)) that includes a short template that is used for the processive synthesis of G-rich telomeric DNA repeats at the 3' ends of most eukaryotic chromosomes1. Telomerase maintains genomic integrity, and its activity or dysregulation are critical determinants of human longevity, stem cell renewal and cancer progression2,3. Previous cryo-electron microscopy structures have established the general architecture, protein components and stoichiometries of Tetrahymena and human telomerase, but our understandings of the details of DNA-protein and RNA-protein interactions and of the mechanisms and recruitment involved remain limited4-6. Here we report cryo-electron microscopy structures of active Tetrahymena telomerase with telomeric DNA at different steps of nucleotide addition. Interactions between telomerase reverse transcriptase (TERT), TER and DNA reveal the structural basis of the determination of the 5' and 3' template boundaries, handling of the template-DNA duplex and separation of the product strand during nucleotide addition. The structure and binding interface between TERT and telomerase protein p50 (a homologue of human TPP17,8) define conserved interactions that are required for telomerase activation and recruitment to telomeres. Telomerase La-related protein p65 remodels several regions of TER, bridging the 5' and 3' ends and the conserved pseudoknot to facilitate assembly of the TERT-TER catalytic core.

Dynamic DNA Shortening by Telomere-Binding Protein Cdc13.

Telomeres are essential for chromosome maintenance. Cdc13 is a single-stranded telomeric DNA binding protein that caps telomeres and regulates telomerase function in yeast. Although specific binding of Cdc13 to telomeric DNA is critical for telomere protection, the detail mechanism how Cdc13-DNA complex protects telomere is unclear. Using two single-molecule methods, tethered particle motion and atomic force microscopy, we demonstrate that specific binding of Cdc13 on single-stranded telomeric DNA shortens duplex DNA into distinct states differed by ∼70-80 base pairs. DNA shortening by Cdc13 is dynamic and independent of duplex DNA sequences or length. Significantly, we found that Pif1 helicase is incapable of removing Cdc13 from the shortened DNA-Cdc13 complex, suggesting that Cdc13 forms structurally stable complex by shortening of the bound DNA. Together our data identified shortening of DNA by Cdc13 and provided an indication for efficient protection of telomere ends by the shortened DNA-Cdc13 complex.

POT1 stability and binding measured by fluorescence thermal shift assays.

The protein POT1 (Protection of Telomeres 1) is an integral part of the shelterin complex that protects the ends of human chromosomes from degradation or end fusions. It is the only component of shelterin that binds single-stranded DNA. We describe here the application of two separate fluorescent thermal shift assays (FTSA) that provide quantitative biophysical characterization of POT1 stability and its interactions. The first assay uses Sypro Orange™ and monitors the thermal stability of POT1 and its binding under a variety of conditions. This assay is useful for the quality control of POT1 preparations, for biophysical characterization of its DNA binding and, potentially, as an efficient screening tool for binding of small molecule drug candidates. The second assay uses a FRET-labeled human telomeric G-quadruplex structure that reveals the effects of POT1 binding on thermal stability from the DNA frame of reference. These complementary assays provide efficient biophysical approaches for the quantitative characterization of multiple aspects of POT1 structure and function. The results from these assays provide thermodynamics details of POT1 folding, the sequence selectivity of its DNA binding and the thermodynamic profile for its binding to its preferred DNA binding sequence. Most significantly, results from these assays elucidate two mechanisms for the inhibition of POT1 -DNA interactions. The first is by competitive inhibition at the POT1 DNA binding site. The second is indirect and is by stabilization of G-quadruplex formation within the normal POT1 single-stranded DNA sequence to prevent POT1 binding.

Non-canonical roles of canonical telomere binding proteins in cancers.

Reactivation of telomerase is a major hallmark observed in 90% of all cancers. Yet paradoxically, enhanced telomerase activity does not correlate with telomere length and cancers often possess short telomeres; suggestive of supplementary non-canonical roles that telomerase might play in the development of cancer. Moreover, studies have shown that aberrant expression of shelterin proteins coupled with their release from shortening telomeres can further promote cancer by mechanisms independent of their telomeric role. While targeting telomerase activity appears to be an attractive therapeutic option, this approach has failed in clinical trials due to undesirable cytotoxic effects on stem cells. To circumvent this concern, an alternative strategy could be to target the molecules involved in the non-canonical functions of telomeric proteins. In this review, we will focus on emerging evidence that has demonstrated the non-canonical roles of telomeric proteins and their impact on tumorigenesis. Furthermore, we aim to address current knowledge gaps in telomeric protein functions and propose future research approaches that can be undertaken to achieve this.