Single-stranded Telomeric Overhang Research Articles

Protection of the Telomeric Junction by the Shelterin Complex.

Shelterin serves critical roles in suppressing superfluous DNA damage repair pathways on telomeres. The junction between double-stranded telomeric tracts (dsTEL) and single-stranded telomeric overhang (ssTEL) is the most accessible region of the telomeric DNA. The shelterin complex contains dsTEL and ssTEL binding proteins and can protect this junction by bridging the ssTEL and dsTEL tracts. To test this possibility, we monitored shelterin binding to telomeric DNA substrates with varying ssTEL and dsTEL lengths and quantified its impact on telomere accessibility using single-molecule fluorescence microscopy methods in vitro. We identified the first dsTEL repeat nearest the junction as the preferred binding site for creating the shelterin bridge. Shelterin requires at least two ssTEL repeats, while the POT1 subunit of shelterin that binds to ssTEL requires longer ssTEL tracts for stable binding to telomeres and effective protection of the junction region. The ability of POT1 to protect the junction is significantly enhanced by the 5'-phosphate at the junction. Collectively, our results show that shelterin enhances the binding stability of POT1 to ssTEL and provides more effective protection compared with POT1 alone by bridging single- and double-stranded telomeric tracts.

Slow G-Quadruplex Conformation Rearrangement and Accessibility Change Induced by Potassium in Human Telomeric Single-Stranded DNA.

The guanine-rich telomeric repeats can form G-quadruplexes (G4s) that alter the accessibility of the single-stranded telomeric overhang. In this study, we investigated the effects of Na+ and K+ on G4 folding and accessibility through cation introduction and exchange. We combined differential scanning calorimetry (DSC), circular dichroism (CD), and single molecule Förster resonance energy transfer (smFRET) to monitor the stability, conformational dynamics, and complementary strand binding accessibility of G4 formed by single-stranded telomeric DNA. Our data showed that G4 formed through heating and slow cooling in K+ solution exhibited fewer conformational dynamics than G4 formed in Na+ solution, which is consistent with the higher thermal stability of G4 in K+. Monitoring cation exchange with real time smFRET at room temperature shows that Na+ and K+ can replace each other in G4. When encountering high K+ at room or body temperature, G4 undergoes a slow conformational rearrangement process which is mostly complete by 2 h. The slow conformational rearrangement ends with a stable G4 that is unable to be unfolded by a complementary strand. This study provides new insights into the accessibility of G4 forming sequences at different time points after introduction to a high K+ environment in cells, which may affect how the nascent telomeric overhang interacts with proteins and telomerase.

POT1 mutations cause differential effects on telomere length leading to opposing disease phenotypes.

The protection of telomereprotein (POT1) is a telomere-binding protein and is an essential component of the six-memberedshelterin complex, which is associated with the telomeres. POT1 directly binds to the3' single-stranded telomeric overhang and prevents the activation of DNA damage response at telomeres thus preventing the telomere-telomere fusions and genomic instability. POT1 also plays a pivotal role in maintaining telomere length by regulating telomerase-mediated telomere elongation. Mutations in POT1 proteins resultin three different telomere phenotypes, which include long, short, or aberrant telomere length. Long telomeres predispose individuals to cancer, while short or aberrant telomere phenotypes result in pro-aging diseases referred to as telomeropathies. Here, we review the function of POT1 proteins in telomere length hemostasis and how the spectrum of mutations reported in POT1 can be segregated towarddeveloping very distinct diseasephenotypes of cancer and telomeropathies.

Full length RTEL1 is required for the elongation of the single-stranded telomeric overhang by telomerase.

Telomeres cap the ends of eukaryotic chromosomes and distinguish them from broken DNA ends to suppress DNA damage response, cell cycle arrest and genomic instability. Telomeres are elongated by telomerase to compensate for incomplete replication and nuclease degradation and to extend the proliferation potential of germ and stem cells and most cancers. However, telomeres in somatic cells gradually shorten with age, ultimately leading to cellular senescence. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and diverse symptoms including bone marrow failure, immunodeficiency, and neurodevelopmental defects. HHS is caused by germline mutations in telomerase subunits, factors essential for its biogenesis and recruitment to telomeres, and in the helicase RTEL1. While diverse phenotypes were associated with RTEL1 deficiency, the telomeric role of RTEL1 affected in HHS is yet unknown. Inducible ectopic expression of wild-type RTEL1 in patient fibroblasts rescued the cells, enabled telomerase-dependent telomere elongation and suppressed the abnormal cellular phenotypes, while silencing its expression resulted in gradual telomere shortening. Our observations reveal an essential role of the RTEL1 C-terminus in facilitating telomerase action at the telomeric 3′ overhang. Thus, the common etiology for HHS is the compromised telomerase action, resulting in telomere shortening and reduced lifespan of telomerase positive cells.

The Replisome Mediates A-NHEJ Repair of Telomeres Lacking POT1-TPP1 Independently of MRN Function

SUMMARYTelomeres use shelterin to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), repressing ataxia-telangiectasia, mutated (ATM) and ATM and Rad3-related (ATR) dependent DNA damage checkpoint responses. The MRE11 nuclease is thought to be essential for the resection of the 5′ C-strand to generate the microhomologies necessary for alternative non-homologous end joining (A-NHEJ) repair. In the present study, we uncover DNA damage signaling and repair pathways engaged by components of the replisome complex to repair dysfunctional telomeres. In cells lacking MRN, single-stranded telomeric overhangs devoid of POT1-TPP1 do not recruit replication protein A (RPA), ATR-interacting protein (ATRIP), and RAD 51. Rather, components of the replisome complex, including Claspin, Proliferating cell nuclear antigen (PCNA), and Downstream neighbor of SON (DONSON), initiate DNA-PKcs-mediated p-CHK1 activation and A-NHEJ repair. In addition, Claspin directly interacts with TRF2 and recruits EXO1 to newly replicated telomeres to promote 5′ end resection. Our data indicate that MRN is dispensable for the repair of dysfunctional telomeres lacking POT1-TPP1 and highlight the contributions of the replisome in telomere repair.

Structural biology of telomeres and telomerase.

Telomeres are protein-DNA complexes that protect chromosome ends from illicit ligation and resection. Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA to counter telomere shortening. Human telomeres are composed of complexes between telomeric DNA and a six-protein complex known as shelterin. The shelterin proteins TRF1 and TRF2 provide the binding affinity and specificity for double-stranded telomeric DNA, while the POT1-TPP1 shelterin subcomplex coats the single-stranded telomeric G-rich overhang that is characteristic of all our chromosome ends. By capping chromosome ends, shelterin protects telomeric DNA from unwanted degradation and end-to-end fusion events. Structures of the human shelterin proteins reveal a network of constitutive and context-specific interactions. The shelterin protein-DNA structures reveal the basis for both the high affinity and DNA sequence specificity of these interactions, and explain how shelterin efficiently protects chromosome ends from genome instability. Several protein-protein interactions, many provided by the shelterin component TIN2, are critical for upholding the end-protection function of shelterin. A survey of these protein-protein interfaces within shelterin reveals a series of "domain-peptide" interactions that allow for efficient binding and adaptability towards new functions. While the modular nature of shelterin has facilitated its part-by-part structural characterization, the interdependence of subunits within telomerase has made its structural solution more challenging. However, the exploitation of several homologs in combination with recent advancements in cryo-EM capabilities has led to an exponential increase in our knowledge of the structural biology underlying telomerase function. Telomerase homologs from a wide range of eukaryotes show a typical retroviral reverse transcriptase-like protein core reinforced with elements that deliver telomerase-specific functions including recruitment to telomeres and high telomere-repeat addition processivity. In addition to providing the template for reverse transcription, the RNA component of telomerase provides a scaffold for the catalytic and accessory protein subunits, defines the limits of the telomeric repeat sequence, and plays a critical role in RNP assembly, stability, and trafficking. While a high-resolution definition of the human telomerase structure is only beginning to emerge, the quick pace of technical progress forecasts imminent breakthroughs in this area. Here, we review the structural biology surrounding telomeres and telomerase to provide a molecular description of mammalian chromosome end protection and end replication.

Heterozygous RTEL1 variants in Patients with Bone Marrow Failure Associate with Telomere Dysfunction in the Absence of Telomere Shortening

The pathophysiology of bone marrow failure (BMF) can be immune, as in acquired aplastic anemia (AA), or constitutional, due to germline mutations in genes critical for DNA repair and telomere maintenance. Variability in penetrance and phenotype can complicate diagnosis, as patients with underlying genetic defects may present in adulthood and without characteristic physical anomalies. RTEL1 encodes a helicase crucial for telomere maintenance and DNA repair. The gene has two main transcripts in human cells: the 1300 amino acid isoform 3 and the 1219 amino acid isoform 1. RTEL1 isoform 3 contains a conserved C4C4-RING domain responsible for resolving the t-loop required for telomere replication. Using next-generation sequencing (NGS), RTEL1 germline variants with unknown clinical significance have been found in AA patients. Functional tests may elucidate RTEL1 variants' pathogenic role in telomere biology. Here, we describe RTEL1 heterozygous germline mutations in patients with BMF and investigate their impact in telomere maintenance.We screened 63 patients with a suggestive familial phenotype for germline mutations in peripheral blood cells using a targeted, 49 gene NGS panel. To investigate variants' impact in telomere functions, telomere length (TL) was measured by Southern blot (SB), t-circles were quantified by telomere circle assay, and single-stranded overhang was measured by non-denaturing SB.Eight patients carried novel heterozygous non-synonymous RTEL1 variants: four nucleotide changes were located in the RAD3 domain, six in the harmonin-like domain, and one in the RING domain. Clinical features and TL were heterogeneous (Table 1). The only RTEL1 variant predicted as pathogenic in silico was F1262L (c.3786 C>G) in patient 2; this mutation affects a highly conserved amino acid residue located in the RING domain, which is responsible for RTEL1 interaction with TRF2 at telomeres and t-loop unwinding. Patient 2 had very short telomeres, abnormal accumulation of t-circles, and eroded single-stranded telomeric overhangs in leukocytes, indicating a disrupted RTEL1 RING domain. To confirm observations made in clinical samples, 293T cells transfected with a plasmid carrying wild-type RTEL1-FLAG isoform 3 or its F1262L mutated version were assessed for TRF2 and FLAG co-localization in the nucleus. By confocal microscopy, wild-type RTEL1, but not mutant RTEL1 co-localized with TRF2. These findings strongly implicate RTEL1-F1262L as pathogenic, and thus the first autosomal dominant mutation in the RING domain in an AA patient. In patient 1, D743N variant in silico prediction was indeterminate, but telomeres were very short and there was a family history of typical telomeropathy (AA, liver cirrhosis, and pulmonary fibrosis) without any other suspicious germline mutations. The D743N variant is located close to the V745M variant that has been reported in a patient with dyskeratosis congenita. Increased amounts of t-circles and telomeric overhang attrition were observed in three other patients (#4, 5, and 7). While not specific for RTEL1 function, these results suggest telomere dysfunction, despite TLs in the normal range for patient 4 and 5. The RTEL1 P82L variant also appeared related to clonal evolution and leukemic progression observed in patient 5. For patients 3, 4, 6, 7, and 8, several mutations were observed in other genes concomitant to RTEL1, and a more complex genomic architecture may be the cause of patients’ phenotype. A previously reported TERC variant, and a TERT variant of undetermined in silico prediction, could be pathogenic in patients 7 and 6, respectively. In these cases, RTEL1 variants may modulate disease, or represent only coincidental abnormalities.To our knowledge, this is the first report of heterozygous RTEL1 mutations in AA. We also describe a TL-independent association between RTEL1 haploinsufficiency and telomere dysfunction in humans. Haploinsufficiency of RTEL1 may disrupt DNA repair, destabilize the genome, and promote leukemogenesis by a mechanism different than typical accelerated telomere attrition associated with very short telomeres. T-circle quantification and overhang measurement may be better measures of telomere dysfunction in patients with RTEL1 variants than simple TL assessment. The combination of different functional tests was useful to the assessment of novel variants impact in telomere maintenance and DNA repair. [Display omitted] DisclosuresFernandez Ibanez:GSK/Novartis: Research Funding. Desierto:GSK/Novartis: Research Funding. Townsley:GSK/Novartis: Research Funding. Young:GSK/Novartis: Research Funding.

Binding of TPP1 Protein to TIN2 Protein Is Required for POT1a,b Protein-mediated Telomere Protection

The single-stranded DNA binding proteins in mouse shelterin, POT1a and POT1b, accumulate at telomeres as heterodimers with TPP1, which binds TIN2 and thus links the TPP1/POT1 dimers with TRF1 and TRF2/Rap1. When TPP1 is tethered to TIN2/TRF1/TRF2, POT1a is thought to block replication protein A binding to the single-stranded telomeric DNA and prevent ataxia telangiectasia and Rad3-related kinase activation. Similarly, TPP1/POT1b tethered to TIN2 can control the formation of the correct single-stranded telomeric overhang. Consistent with this view, the telomeric phenotypes following deletion of POT1a,b or TPP1 are phenocopied in TIN2-deficient cells. However, the loading of TRF1 and TRF2/Rap1 is additionally compromised in TIN2 KO cells, leading to added phenotypes. Therefore, it could not be excluded that, in addition to TIN2, other components of shelterin contribute to the recruitment of TPP1/POT1a,b as suggested by previous reports. To test whether TIN2 is the sole link between TPP1/POT1a,b and telomeres, we defined the TPP1 interaction domain of TIN2 and generated a TIN2 allele that was unable to interact with TPP1 but retained its interaction with TRF1 and TRF2. We demonstrated that cells expressing TIN2ΔTPP1 instead of wild-type TIN2 phenocopy the POT1a,b knockout setting without showing additional phenotypes. Therefore, these results are consistent with TIN2 being the only mechanism by which TPP1/POT1 heterodimers bind to shelterin and function in telomere protection.

Abstract A131: Drugging the telomere: A rational approach for the selection quadruplex DNA targeting agents for the treatment of cancer.

Abstract The pentacyclic acridinium class of small molecule telomere targeting agents have been shown to stabilise the 3'single-stranded guanine-rich telomeric overhang to fold into a G-quadruplex structure. Stabilisation of the latter is incompatible with an attachment of telomerase and a collection of protective proteins referred to as the shelterin complex to the telomere. G-quadruplex ligands can effectively inhibit the telomere maintenance functions of telomerase while revealing the chromosomal termini to the unwanted attention of DNA repair proteins resulting in telomere specific damage. Potent antitumor properties of the G-quadruplex ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) have been reported and shown to be driven by telomere uncapping events. However, in preliminary toxological studies the original lead compound (RHPS4) revealed cardiovascular liabilities related to interactions with hERG receptor (IC50 0.2 uM) as well as additional receptor interactions including muscarinic M1, M2 and M3 and Beta2 adrenergic receptors. Pharminox have designed and synthesised second generation agents with significantly improved on and off target profiles. Here we describe the modification and optimisation of new telomere targeting agents with an emphasis on their specific effect on telomeres. Specific bio-molecular assays have been performed on the best compounds of the series, as defined by telomeric DNA binding characteristics and cancer cell activities, “normal” cell selectivities and off target affinities. It was found that all new compounds examined were more potent than RHPS4 as telomere damage inducers. Specifically, at the dose of 0,1 uM, the compounds PMX-000177-761-785 trigger a percentage of TIF-positive cells comparable to that induced by RHPS4 at a 5 fold higher drug concentration. Activation of damage response mainly occurred in replicating cells for the compounds PMX-000177 and -785, suggesting that they can work with a RHPS4-like mechanism of action. As part of a defined screening cascade and candidate target profile, these studies are being used to differentiate between compounds as they progress towards pre-clinical candidate selection status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A131.

Rapid regulation of telomere length is mediated by poly(ADP-ribose) polymerase-1

Shelterin/telosome is a multi-protein complex at mammalian telomeres, anchored to the double-stranded region by the telomeric-repeat binding factors-1 and -2. In vitro modification of these proteins by poly(ADP-ribosyl)ation through poly(ADP-ribose) polymerases-5 (tankyrases) and -1/-2, respectively, impairs binding. Thereafter, at least telomeric-repeat binding factor-1 is degraded by the proteasome. We show that pharmacological inhibition of poly(ADP-ribose) polymerase activity in cells from two different species leads to rapid decrease in median telomere length and stabilization at a lower setting. Specific knockdown of poly(ADP-ribose) polymerase-1 by RNA interference had the same effect. The length of the single-stranded telomeric overhang as well as telomerase activity were not affected. Release of inhibition led to a fast re-gain in telomere length to control levels in cells expressing active telomerase. We conclude that poly(ADP-ribose) polymerase-1 activity and probably its interplay with telomeric-repeat binding factor-2 is an important determinant in telomere regulation. Our findings reinforce the link between poly(ADP-ribosyl)ation and aging/longevity and also impact on the use of poly(ADP-ribose) polymerase inhibitors in tumor therapy.

Photosensitized damage to telomere overhang and telomerase RNA by riboflavin

By ESR spin elimination and photocleavage assay, the mechanisms of one-electron oxidation damage of oligonucleotides by excited triplet state of riboflavin (Rb) have been elucidated. The results demonstrate that Rb, an endogenous photosensitizer, is capable of cleaving single-stranded telomeric overhang and the template region of telomerase RNA under UVA irradiation, resulting in blocking of reverse transcription of telomeric DNA which leads to the apoptosis of cancer cells ultimately.

G-Quadruplex stabilization by telomestatin induces TRF2 protein dissociation from telomeres and anaphase bridge formation accompanied by loss of the 3′ telomeric overhang in cancer cells

Inhibition of telomerase activity by telomerase inhibitors induces a gradual loss of telomeres, and this in turn causes cancer cells to enter to a crisis stage. Here, we report the telomerase inhibitor telomestatin, which is known to stabilize G-quadruplex structures at 3' single-stranded telomeric overhangs (G-tails), rapidly dissociates TRF2 from telomeres in cancer cells within a week, when given at a concentration that does not cause normal cells to die. The G-tails were dramatically reduced upon short-term treatment with the drug in cancer cell lines, but not in normal fibroblasts and epithelial cells. In addition, telomestatin also induced anaphase bridge formation in cancer cell lines. These effects of telomestatin were similar to those of dominant negative TRF2, which also causes a prompt loss of the telomeric G-tails and induces an anaphase bridge. These results indicate that telomestatin exerts its anticancer effect not only through inhibiting telomere elongation, but also by rapidly disrupting the capping function at the very ends of telomeres. Unlike conventional telomerase inhibitors that require long-term treatments, the G-quadruplex stabilizer telomestatin induced prompt cell death, and it was selectively effective in cancer cells. This study also identifies the TRF2 protein as a therapeutic target for treating many types of cancer which have the TRF2 protein at caps of the telomere DNA of each chromosome.

G-tail telomere HPA: simple measurement of human single-stranded telomeric overhangs

Accurate measurement of telomeric 3'-overhang (G-tail) lengths is essential for investigation of the biological effects of telomere dysfunction. G-tail telomere hybridization protection assay (Gt-telomere HPA) has the advantages of being simple to perform, accurate and highly sensitive for G tails as short as 20 nucleotides. Furthermore, Gt-telomere HPA is specific and quantitative for human G tails, and can be used to assay cell lysates as well as genomic DNA.

Taz1 Binding to a Fission Yeast Model Telomere: FORMATION OF TELOMERIC LOOPS AND HIGHER ORDER STRUCTURES

Similar to its human homologues TRF1 and TRF2, fission yeast Taz1 protein is a component of telomeric chromatin regulating proper telomere maintenance. As mammalian TRF1 and TRF2 proteins have been shown to directly bind telomeric DNA to form protein arrays and looped structures, termed t-loops, the ability of Taz1p to act on fission yeast telomeric DNA in similar ways was examined using purified protein and model DNA templates. When incubated with Taz1p, model telomeres containing 3' single-stranded telomeric overhangs formed t-loops at a frequency approaching 13%. Termini with blunt ends and non-telomeric overhangs were deficient in t-loop formation. In addition, we observed arrays of multiple Taz1p molecules bound to the telomeric regions, resembling the pattern of TRF1 binding. The presence of t-loops larger than the telomeric tract, a high frequency of end-bound DNAs and a donut shape of the Taz1p complex suggest that Taz1p binds the 3' overhang then extrudes a loop that grows in size as the donut slides along the duplex DNA. Based on these in vitro results we discuss possible general implications for fission yeast telomere dynamics.

Telomerase Maintains Telomere Structure in Normal Human Cells

In normal human cells, telomeres shorten with successive rounds of cell division, and immortalization correlates with stabilization of telomere length. These observations suggest that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression. Here, we demonstrate that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling primary presenescent human fibroblasts, previously believed to lack hTERT expression and telomerase activity. Disruption of telomerase activity in normal human cells slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3′ single-stranded telomeric overhang without changing the rate of overall telomere shortening. Together, these observations support the view that telomerase and telomere structure are dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence.

Electron microscopic analysis supports a dual role for the mitochondrial telomere-binding protein of Candida parapsilosis

Linear mitochondrial genomes exist in several yeast species which are closely related to yeast that harbor circular mitochondrial genomes. Several lines of evidence suggest that the conversion from one form to another occurred accidentally through a relatively simple mechanism. Previously, we (L.T. & J.N.) reported the identification of the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of Candida parapsilosis linear mtDNA, and sequence analysis of the corresponding nuclear gene MTP1 revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded (ss) DNA-binding (SSB) proteins. In this study, the DNA-binding properties of mtTBP in vitro and in vivo were analyzed by electron microscopy (EM). When M13 ssDNA was used as a substrate, mtTBP exhibited similar DNA binding characteristics as human mitochondrial SSB: mtTBP formed protein globules along the DNA substrate, and the bound proteins were randomly distributed, indicating that the binding of mtTBP to M13 ssDNA is not highly cooperative. EM analysis demonstrated that mtTBP is able to recognize the 5′ single-stranded telomeric overhangs in their natural context. Using isopycnic centrifugation of mitochondrial lysates of C. papsilosis we show that mtTBP is a structural part of mitochondrial nucleoids of C. parapsilosis and is predominantly bound to the mitochondrial telomeres. These data support a dual role of mtTBP in mitochondria of C. parapsilosis, serving both as a typical mitochondrial SSB and as a specific component of the mitochondrial telomeric chromatin.