Search

The kinases ATM and ATR (Tel1 and Mec1 in the yeast Saccharomyces cerevisiae) control the response to DNA damage. We report that S. cerevisiae Tel2 acts at an early step of the TEL1/ATM pathway of DNA damage signaling. We show that Tel1 and Tel2 interact, and that even when Tel1 protein levels are high, this interaction is specifically required for Tel1 localization to a DNA break and its activation of downstream targets. Computational analysis revealed structural homology between Tel2 and Ddc2 (ATRIP in vertebrates), a partner of Mec1, suggesting a common structural principle used by partners of phoshoinositide 3-kinase-like kinases.

Telomeres are an unusual component of the genome because they do not encode genes, but their structure and cellular maintenance machinery (which we define as “telotype”) are essential for chromosome stability. Cells can switch between different phenotypic states. One such example is when they switch from maintenance mediated by telomerase (TERT telotype) to one of the two alternative mechanisms of telomere preservation (ALT I and ALT II telotype). The nature of this switch is largely unknown. Reintroduction of telomerase into ALT II, but not ALT I, yeast led to the loss of their ability to survive a second round of telomerase withdrawal. Mating-based genetic analysis of ALT I and II revealed that both types of telomerase-independent telomere maintenance are inherited as a non-Mendelian trait dominant over senescence (SEN telotype). Additionally, inheritance of ALT I and ALT II did not depend on either the mitochondrial genome or a prion-based mechanism. Type I, but not type II, survivor cells exhibited impaired gene silencing, potentially connecting the switch to the ALT telotype epigenetic changes. These data provide evidence that nonprion epigenetic-like mechanisms confer flexibility on cells as a population to adjust to the life-threatening situation of telomerase loss, allowing cells to switch from TERT to ALT telotypes that can sustain viable populations.

© 2015 Elsevier B.V. All rights reserved. Background Several psychiatric disorders may be characterized by peripheral telomere shortening. However, it is unclear whether telomere shortening is associated with these psychiatric disorders per se or, rather, with underlying dimensional parameters that are often, but not necessarily, associated with them. We explored the association between dimensional psychopathological measures and telomere length (TL) in granulocytes among veterans independent of psychiatric diagnosis. Methods Seventy-six combat-exposed male veterans (41 psychiatrically healthy, 18 with Posttraumatic Stress Disorder [PTSD] and 17 with concomitant PTSD and Major Depressive Disorder [MDD]) had TL assayed. Assessments included Clinician-Administered PTSD Scale (CAPS), Beck Depression Inventory-II (BDI-II), Early Trauma Inventory (ETI), Symptom Checklist-90-R Global Severity Index (SCL-90-GSI), Perceived Stress Scale (PSS) and Positive and Negative Affect Schedule (PANAS). Correlations were corrected for age, BMI, antidepressants and ethnicity. Results Across subjects, TL was negatively correlated with early trauma (p

We studied peripheral leukocyte telomere length (LTL) as a predictor of antidepressant response to PPAR-γ agonist in patients with unremitted depression. In addition we examined correlation between LTL and the insulin resistance (IR) status in these subjects. Forty-two medically stable men and women ages 23–71 with non-remitted depression participated in double-blind placebo-controlled add-on of Pioglitazone to treatment-as-usual. Oral glucose tolerance tests were administered at baseline and at 12 weeks. Diagnostic evaluation of psychiatric disorders was performed at baseline and mood severity was followed weekly throughout the duration of the trial. At baseline, no differences in LTL were detected by depression severity, duration or chronicity. LTL was also not significantly different between insulin-resistant and insulin-sensitive subjects at baseline. Subjects with longer telomeres exhibited greater declines in depression severity in the active arm, but not in a placebo arm, P=0.005, r=−0.63, 95% confidence interval (95% CI)=(−0.84,−0.21). In addition, LTL predicted improvement in insulin sensitivity in the group overall and did not differ between intervention arms, P=0.036, r=−0.44, 95% CI=(−0.74,0.02) for the active arm, and P=0.026, r=−0.50, 95% CI=(−0.78,−0.03) for the placebo arm. LTL may emerge as a viable predictor of antidepressant response. An association between insulin sensitization and LTL regardless of the baseline IR status points to potential role of LTL as a non-specific moderator of metabolic improvement in these patients.

Introduction HIV infection is a risk factor for opportunistic pneumonias such as tuberculosis (TB) and for age-associated health complications. Short telomeres, markers of biological aging, are also associated with an increased risk of age-associated diseases and mortality. Our goals were to use a single cohort of HIV-infected and HIV-uninfected individuals hospitalized with pneumonia to assess whether shortened telomere length was associated with HIV infection, TB diagnosis, and 2-month mortality. Methods This was a sub-study of the IHOP Study, a prospective observational study. Participants consisted of 184 adults admitted to Mulago Hospital in Kampala, Uganda who underwent evaluation for suspected TB and were followed for 2 months. Standardized questionnaires were administered to collect demographic and clinical data. PBMCs were isolated and analyzed using quantitative PCR to determine telomere length. The association between HIV infection, demographic and clinical characteristics, and telomere length was assessed, as were the associations between telomere length, TB diagnosis and 2-month mortality. Variables with a P≤0.2 in bivariate analysis were included in multivariate models. Results No significant demographic or clinical differences were observed between the HIV-infected and HIV-uninfected subjects. Older age (P

We have entered a transformative period in cancer prevention (including early detection). Remarkable progress in precision medicine and immune-oncology, driven by extraordinary recent advances in genome-wide sequencing, big-data analytics, blood-based technologies, and deep understanding of the tumor immune microenvironment (TME), has provided unprecedented possibilities to study the biology of premalignancy. The pace of research and discovery in precision medicine and immunoprevention has been astonishing and includes the following clinical firsts reported in 2015: driver mutations detected in circulating cell-free DNA in patients with premalignant lesions (lung); clonal hematopoiesis shown to be a premalignant state; molecular selection in chemoprevention randomized controlled trial (RCT; oral); striking efficacy in RCT of combination chemoprevention targeting signaling pathway alterations mechanistically linked to germline mutation (duodenum); molecular markers for early detection validated for lung cancer and showing promise for pancreatic, liver, and ovarian cancer. Identification of HPV as the essential cause of a major global cancer burden, including HPV16 as the single driver of an epidemic of oropharyngeal cancer in men, provides unique opportunities for the dissemination and implementation of public health interventions. Important to immunoprevention beyond viral vaccines, genetic drivers of premalignant progression were associated with increasing immunosuppressive TME; and Kras vaccine efficacy in pancreas genetically engineered mouse (GEM) model required an inhibitory adjuvant (Treg depletion). In addition to developing new (e.g., epigenetic) TME regulators, recent mechanistic studies of repurposed drugs (aspirin, metformin, and tamoxifen) have identified potent immune activity. Just as precision medicine and immune-oncology are revolutionizing cancer therapy, these approaches are transforming cancer prevention. Here, we set out a brief agenda for the immediate future of cancer prevention research (including a “Pre-Cancer Genome Atlas” or “PCGA”), which will involve the inter-related fields of precision medicine and immunoprevention – pivotal elements of a broader domain of personalized public health. Cancer Prev Res; 9(1); 2–10. ©2016 AACR.

Author(s): Goglin, Sarah E; Farzaneh-Far, Ramin; Epel, Elissa S; Lin, Jue; Blackburn, Elizabeth H; Whooley, Mary A | Abstract: BackgroundShort telomere length independently predicts mortality in patients with coronary heart disease. Whether 5-year change in telomere length predicts subsequent mortality in patients with coronary heart disease has not been evaluated.MethodsIn a prospective cohort study of 608 individuals with stable coronary artery disease, we measured leukocyte telomere length at baseline and after five years of follow-up. We divided the sample into tertiles of telomere change: shortened, maintained or lengthened. We used Cox survival models to evaluate 5-year change in telomere length as a predictor of mortality.ResultsDuring an average of 4.2 years follow-up, there were 149 deaths. Change in telomere length was inversely predictive of all-cause mortality. Using the continuous variable of telomere length change, each standard deviation (325 base pair) greater increase in telomere length was associated with a 24% reduction in mortality (HR 0.76, 95% CI 0.61-0.94; p = 0.01), adjusted for age, sex, waist to hip ratio, exercise capacity, LV ejection fraction, serum creatinine, and year 5 telomere length. Mortality occurred in 39% (79/203) of patients who experienced telomere shortening, 22% (45/203) of patients whose telomere length was maintained, and 12% (25/202) of patients who experienced telomere lengthening (pl0.001). As compared with patients whose telomere length was maintained, those who experienced telomere lengthening were 56% less likely to die (HR 0.44, 95% CI, 0.23-0.87).ConclusionsIn patients with coronary heart disease, an increase in leukocyte telomere length over 5 years is associated with decreased mortality.

Telomeres, the protective DNA-protein complexes at the ends of linear chromosomes, are important for genome stability. Leukocyte or peripheral blood mononuclear cell (PBMC) telomere length is a potential biomarker for human aging that integrates genetic, environmental, and lifestyle factors and is associated with mortality and risks for major diseases. However, only a limited number of studies have examined longitudinal changes of telomere length and few have reported data on sorted circulating immune cells. We examined the average telomere length (TL) in CD4+, CD8+CD28+, and CD8+CD28− T cells, B cells, and PBMCs, cross-sectionally and longitudinally, in a cohort of premenopausal women. We report that TL changes over 18 months were correlated among these three T cell types within the same participant. Additionally, PBMC TL change was also correlated with those of all three T cell types, and B cells. The rate of shortening for B cells was significantly greater than for the three T cell types. CD8+CD28− cells, despite having the shortest TL, showed significantly more rapid attrition when compared to CD8+CD28+ T cells. These results suggest systematically coordinated, yet cell type-specific responses to factors and pathways contribute to telomere length regulation.

Using a modified single telomere length analysis protocol (STELA) to clone and examine the sequence composition of individual human XpYp telomeres, we discovered a distinct class of extremely short telomeres in human cancer cells with active telomerase. We name them “t-stumps”, to distinguish them from the well-regulated longer bulk telomeres. T-stumps contained arrangements of telomeric repeat variants and a minimal run of seven canonical telomeric TTAGGG repeats, but all could bind at least one TRF1 or TRF2 in vitro. The abundance of t-stumps was unaffected by ATM alteration, but could be changed by manipulating telomerase catalytic subunit (hTERT) levels in cancer cells. We propose that in the setting of active telomerase and compromised checkpoints characteristic of human cancer cells, t-stumps define the minimal telomeric unit that can still be protected by a TRF1/TRF2-capping complex and, further, that hTERT (or telomerase) may have a role in protecting t-stumps.

Several studies have reported that adults with major depressive disorder have shorter telomere length and reduced hippocampal volumes. Moreover, studies of adult populations without major depressive disorder suggest a relationship between peripheral telomere length and hippocampal volume. However, the relationship of these findings in adolescents with major depressive disorder has yet to be explored. We examined whether adolescent major depressive disorder is associated with altered peripheral telomere length and hippocampal volume, and whether these measures relate to one another. In 54 unmedicated adolescents (13–18 years) with major depressive disorder and 63 well-matched healthy controls, telomere length was assessed from saliva using quantitative polymerase chain reaction methods, and bilateral hippocampal volumes were measured with magnetic resonance imaging. After adjusting for age and sex (and total brain volume in the hippocampal analysis), adolescents with major depressive disorder exhibited significantly shorter telomere length and significantly smaller right, but not left hippocampal volume. When corrected for age, sex, diagnostic group and total brain volume, telomere length was not significantly associated with left or right hippocampal volume, suggesting that these cellular and neural processes may be mechanistically distinct during adolescence. Our findings suggest that shortening of telomere length and reduction of hippocampal volume are already present in early-onset major depressive disorder and thus unlikely to be only a result of accumulated years of exposure to major depressive disorder.

We developed an efficient, automated 2-step purification protocol for the production of milligram quantities of untagged recombinant rat lactate dehydrogenase A (rLDHA) from E. coli, using the ÄKTAxpress™ chromatography system. Cation exchange followed by size exclusion results in average final purity in excess of 93% and yields ~ 14 milligrams per 50 ml of original cell culture in EnPresso B media, in under 8 hrs, including all primary sample processing and column equilibration steps. The protein is highly active and coherent biophysically and a viable alternative to the more problematic human homolog for structural and ligand-binding studies; an apo structure of untagged rLDHA was solved to a resolution 2.29 Å (PDB ID 5ES3). Our automated methodology uses generic commercially available pre-packed columns and simple buffers, and represents a robust standard method for the production of milligram amounts of untagged rLDHA, facilitating a novel fragment screening approach for new inhibitors.

Cyclophilin 40 (Cyp40) comprises an N-terminal cyclophilin domain with peptidyl-prolyl isomerase (PPIase) activity and a C-terminal tetratricopeptide repeat (TPR) domain that binds to the C-terminal -EEVD sequence common to both heat shock protein 70 (Hsp70) and Hsp90. We show in the present study that binding of peptides containing the MEEVD motif reduces the PPIase activity by similar to 30%. CD and fluorescence assays show that the TPR domain is less stable than the cyclophilin domain and is stabilized by peptide binding. Isothermal titration calorimetry (ITC) shows that the affinity for the -MEEVD peptide is temperature sensitive in the physiological temperature range. Results from these biophysical studies fit with the MD simulations of the apo and holo (peptide-bound) structures which show a significant reduction in root mean square (RMS) fluctuation in both TPR and cyclophilin domains when -MEEVD is bound. The MD simulations of the apo-protein also highlight strong anti-correlated motions between residues around the PPIase-active site and a band of residues running across four of the seven helices in the TPR domain. Peptide binding leads to a distortion in the shape of the active site and a significant reduction in these strongly anti-correlated motions, providing an explanation for the allosteric effect of ligand binding and loss of PPIase activity. Together the experimental and MD results suggest that on heat shock, dissociation of Cyp40 from complexes mediated by the TPR domain leads to an increased pool of free Cyp40 capable of acting as an isomerase/chaperone in conditions of cellular stress., British Council UK-Turkey Partnership Programme; Wellcome Trust; Biotechnology and Biological Sciences Research Council; Wellcome-UoE ISSF award; Scottish Universities Life Science Alliance (SULSA)

Background: Telomere length (TL) is a marker of cellular aging, with the majority of lifetime attrition occurring during the first 4 y. Little is known about risk factors for telomere shortening in childhood. Objective: We evaluated the relation between early life feeding variables and preschool TL. Design: We assessed the relation between dietary, feeding, and weight-associated risk factors measured from birth and TL from blood samples taken at 4 y of age (n = 108) and 5 y of age (n = 92) in a cohort of urban, Latino children (n = 121 individual children). Feeding variables were evaluated in children with repeat measurements (n = 77). Results: Mean TL (in bp) was associated with exclusive breastfeeding at 4–6 wk of age (adjusted coefficient: 353.85; 95% CI: 72.81, 634.89; P = 0.01), maternal TL (adjusted coefficient: 0.32; 95% CI: 0.11, 0.54; P < 0.01), and older paternal age (adjusted coefficient: 33.27; 95% CI: 4.10, 62.44; P = 0.03). The introduction of other foods or drinks in addition to breast-milk or replacement-milk substitutes before 4–6 wk of age was associated with mean TL at 4 and 5 y of age (adjusted coefficient: −457.01; 95% CI: −720.50, −193.51; P < 0.01). Infant obesity at 6 mo of age and soda consumption at 4 y of age mediated the relation in part between exclusive breastfeeding at 4–6 wk of age and mean TL at 4 and 5 y of age. High soda consumption at 3 y of age was associated with an accelerated attrition from 4 to 5 y of age (adjusted coefficient: −515.14; 95% CI: −986.06, −41.22; P = 0.03). Conclusion: Exclusive breastfeeding at 4–6 wk of age may have long-term effects on child health as evidenced by longer TL at 4 and 5 y of age.

© 2015 Elsevier Inc. Introduction: Mitochondrial abnormalities may be involved in PTSD, although few studies have examined this. Mitochondrial DNA copy number (mtDNAcn) in blood cells is an emerging systemic index of mitochondrial biogenesis and function. The present study assessed mtDNAcn in male combat-exposed veterans with PTSD compared to those without PTSD as well as its correlation with clinical scales. Methods: mtDNAcn was assessed with a TaqMan multiplex assay in granulocytes of 43 male combat veterans with (n. =43) or without (n. =44) PTSD. Twenty of the PTSD subjects had co-morbid major depressive disorder (MDD). The Clinician Administered PTSD Scale (CAPS), the Positive and Negative Affect Schedule (PANAS), the Early Trauma Inventory (ETI) and the Beck Depression Inventory II (BDI-II) were used for the clinical assessments. All analyses were corrected for age and BMI. Results: mtDNAcn was significantly lower in subjects with PTSD (p.

Telomerase is commonly expressed in human cancer cells. Increased telomerase expression produces vulnerability of cancer cells, distinguishing them from normal cells in the body, although normal cells do also have some active telomerase. Recent studies also suggest that telomerase is implicated in

Numerous studies demonstrate links between chronic stress and indices of poor health, including risk factors for cardiovascular disease and poorer immune function. Nevertheless, the exact mechanisms of how stress gets “under the skin” remain elusive. We investigated the hypothesis that stress impacts health by modulating the rate of cellular aging. Here we provide evidence that psychological stress— both perceived stress and chronicity of stress—is significantly associated with higher oxidative stress, lower telomerase activity, and shorter telomere length, which are known determinants of cell senescence and longevity, in peripheral blood mononuclear cells from healthy premenopausal women. Women with the highest levels of perceived stress have telomeres shorter on average by the equivalent of at least one decade of additional aging compared to low stress women. These findings have implications for understanding how, at the cellular level, stress may promote earlier onset of age-related diseases.

The ribonucleoprotein telomerase is a specialized reverse transcriptase minimally composed of an RNA, TER, and a protein catalytic subunit, TERT. The TER and TERT subunits of telomerase associate to form a dimeric enzyme in several organisms, including human. A small portion of TER, the template domain, is used by telomerase for the synthesis of tandem repeats of telomeric DNA. We studied some of the requirements for processive template usage by human telomerase. A blunt-ended duplex DNA primer was not utilized by telomerase. With a duplex telomeric DNA primer, a single-stranded 3' overhang with a minimum length of approximately 6 bases was required for efficient priming activity. Large substitutions in the human TER templating domain did not abolish enzymatic activity, although insertion of two residues into this sequence reduced processivity, as did a template mutation that results in a mismatch between the template region used for copying DNA and the region used for alignment of the substrate primer. Finally, by using a complementary pair of catalytically inactive telomerase RNA pseudoknot mutants in combination with a marked template, we demonstrated that processive synthesis by an obligatory dimer of human telomerase does not require template switching. These results indicate that processive template usage by human telomerase, like that of Tetrahymena telomerase, is strongly dependent on the base identities in the template domain and that a dimeric human telomerase can processively utilize a single template.

Telomere length is negatively regulated by proteins of the telomeric DNA-protein complex. Rap1p in Saccharomyces cerevisiae binds the telomeric TG(1-3) repeat DNA, and the Rap1p C terminus interacts with Rif1p and Rif2p. We investigated how these three proteins negatively regulate telomere length. We show that direct tethering of each Rif protein to a telomere shortens that telomere proportionally to the number of tethered molecules, similar to previously reported counting of Rap1p. Surprisingly, Rif proteins could also regulate telomere length even when the Rap1p C terminus was absent, and tethered Rap1p counting was completely dependent on the Rif proteins. Thus, Rap1p counting is in fact Rif protein counting. In genetic settings that cause telomeres to be abnormally long, tethering even a single Rif2p molecule was sufficient for maximal effectiveness in preventing the telomere overelongation. We show that a heterologous protein oligomerization domain, the mammalian PDZ domain, when fused to Rap1p can confer telomere length control. We propose that a nucleation and spreading mechanism is involved in forming the higher-order telomere structure that regulates telomere length.

Purpose: To test ribozymes targeting mouse telomerase RNA (mTER) for suppression of the progression of B16-F10 murine melanoma metastases in vivo. Experimental Design: Hammerhead ribozymes were designed to target mTER. The ribozyme sequences were cloned into a plasmid expression vector containing EBV genomic elements that substantially prolong expression of genes delivered in vivo. The activity of various antitelomerase ribozymes or control constructs was examined after i.v. injection of cationic liposome:DNA complexes containing control or ribozyme constructs. Expression of ribozymes and mTER at various time points were evaluated by quantitative real-time PCR. Telomerase activity was examined using the telomeric repeat amplification protocol. Results: Systemic administration of cationic liposome:DNA complexes containing a plasmid-expressed ribozyme specifically targeting a cleavage site at mTER nucleotide 180 significantly reduced the metastatic progression of B16-F10 murine melanoma. The antitumor activity of the anti-TER 180 ribozyme in mice was abolished by a single inactivating base mutation in the ribozyme catalytic core. The EBV-based expression plasmid produced sustained levels of ribozyme expression for the full duration of the antitumor studies. In addition to antitumor activity, cationic liposome:DNA complex-based ribozyme treatment also produced reductions in both TER levels and telomerase enzymatic activity in tumor-bearing mice. Conclusions: Systemic, plasmid-based ribozymes specifically targeting TER can reduce both telomerase activity and metastatic progression in tumor-bearing hosts. The work reported here demonstrates the potential utility of plasmid-based anti-TER ribozymes in the therapy of melanoma metastasis.

Replication of the linear DNA of a eukaryotic chromosome imposes a problem of end replication, as originally predicted by Watson (40) and Olovnikov (31). While the synthesis of the leading strand can proceed to the very end of the template, the lagging strand is predicted to shorten upon every round of replication in each cell cycle. Most eukaryotes solve the end-replication problem by maintaining specific repetitive DNA sequences at their chromosome ends, called telomeres, by the enzyme telomerase, which elongates the 3′ end of the telomeric DNA in a sequence-specific manner. In those rarer situations in which a eukaryote does not have telomerase, other multiple repeats, such as transposable elements in the fruit fly Drosophila melanogaster, are periodically added to their chromosome ends. The yeast Saccharomyces cerevisiae has telomeres containing ∼250 to 350 bp of TG1-3 repeats and uses telomerase for their maintenance. About two-thirds of the 32 telomeres in haploid cells carry one or more copies of subtelomeric Y′ elements (see reference 32 for a review). Members of the major class of Y′ elements are 6.7 kb long, and there is a minor 5.2-kb class; they are always arranged in the same orientation, such that multiple Y′ elements form directly repeating arrays separated by short stretches of telomeric TG1-3 DNA. Replication of eukaryotic chromosomes initiates at autonomously replicating sequence (ARS) elements—origins of replication present at multiple locations on every chromosome. Every Y′ element contains an ARS (4). While many genomic ARSs are fired early in S phase, it has been reported, by using the density transfer method, that Y′ repeats replicate late in S phase (11, 28, 33, 39). Telomeric chromatin has been implicated in determining the timing of activation of subtelomeric ARSs. An ARS placed on a circular plasmid containing telomeric TG1-3 repeats initiates replication early, but if the plasmid is linearized and therefore contains telomeres, the ARS is fired late (11). Furthermore, deletion of Sir3p, one of the components of telomeric heterochromatin, causes the telomeres to replicate in early S phase (39). These results raise the question of whether all the Y′ elements initiate their ARSs synchronously in late S phase or whether the timing of replication is different for the terminal and internal elements, since only the former are positioned next to a terminal telomeric TG1-3 tract. Replication fork movement does not proceed monotonically. Programmed replication fork pausing is conserved from bacteria to higher plants and animals and can contribute to genomic stability. Polar replication fork blocks can ensure unidirectional replication at a certain region of a genome, thereby playing regulatory roles in different cellular processes. For example, in bacteria, polar replication pausing coordinates termination of bidirectional chromosome replication so that the two replication forks meet at the terminus region where newly synthesized chromosomes are to be decatenated (reviewed in reference 34). On either side of the terminus, three sites (Ter) are bound by the protein Tus, and the resultant DNA-protein complex blocks the progression of replication forks in a polar manner. In the mating type locus of the fission yeast Schizosaccharomyces pombe, replication pausing regulates the orientation of DNA replication, thereby determining whether cells undergo mating type switching (9). Many organisms have been shown to have specific replication blocks near ribosomal DNA (rDNA) genes. In the budding yeast S. cerevisiae, replication fork pausing has been found at rDNA and telomeres, and the Rrm3 helicase has been shown to alleviate it at both loci (19, 20). While the replication pausing at rDNA is caused by the DNA-binding protein Fob1 (21, 22), the key determinants that impose the replication barrier at telomeres are unknown. Toward a fuller understanding of the role of replication in telomere maintenance in vivo, here we have analyzed replication of natural yeast telomeric regions, including the subtelomeric Y′ elements, to address the question of the nature and architecture of replication fork pausing at Y′ telomeres.

Telomeres are the protective DNA–protein complexes found at the ends of eukaryotic chromosomes. Telomeric DNA consists of tandem repeats of a simple, often G–rich, sequence specified by the action of telomerase, and complete replication of telomeric DNA requires telomerase. Telomerase is a specialized cellular ribonucleoprotein reverse transcriptase. By copying a short template sequence within its intrinsic RNA moiety, telomerase synthesizes the telomeric DNA strand running 5' to 3' towards the distal end of the chromosome, thus extending it. Fusion of a telomere, either with another telomere or with a broken DNA end, generally constitutes a catastrophic event for genomic stability. Telomerase acts to prevent such fusions. The molecular consequences of telomere failure, and the molecular contributors to telomere function, with an emphasis on telomerase, are discussed here.

Telomeres protect chromosome ends from fusing to double-stranded breaks (DSBs). Using a quantitative real-time PCR assay, we show that nonhomologous end joining between a telomere and an inducible DSB was undetectable in wild-type cells, but occurred within a few hours of DSB induction in approximately 1/2000 genomes in telomerase-deficient cells and in >1/1000 genomes in telomerase-deficient cells also lacking the ATM homolog Tel1p. The fused telomeres contained very little telomeric DNA, suggesting that catastrophic telomere shortening preceded fusion. Lengthening of telomeres did not prevent such catastrophic telomere shortening and fusion events. Telomere-DSB fusion also occurred in cells containing a catalytically inactive telomerase and in tel1 mec1 cells where telomerase cannot elongate telomeres. Thus, telomerase and Tel1p function in telomere protection as well as in telomere elongation.

In Saccharomyces cerevisiae, telomeric DNA is protected by a nonnucleosomal protein complex, tethered by the protein Rap1. Rif and Sir proteins, which interact with Rap1p, are thought to have further interactions with conventional nucleosomic chromatin to create a repressive structure that protects the chromosome end. We showed by microarray analysis that Rif1p association with the chromosome ends extends to subtelomeric regions many kilobases internal to the terminal telomeric repeats and correlates strongly with the previously determined genomic footprints of Rap1p and the Sir2-4 proteins in these regions. Although the end-protection function of telomeres is essential for genomic stability, telomeric DNA must also be copied by the conventional DNA replication machinery and replenished by telomerase, suggesting that transient remodeling of the telomeric chromatin might result in distinct protein complexes at different stages of the cell cycle. Using chromatin immunoprecipitation, we monitored the association of Rap1p, Rif1p, Rif2p, and the protein component of telomerase, Est2p, with telomeric DNA through the cell cycle. We provide evidence for dynamic remodeling of these components at telomeres.

The Kaiser Permanente Research Program on Genes, Environment, and Health (RPGEH) Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort includes DNA specimens extracted from saliva samples of 110,266 individuals. Because of its relationship to aging, telomere length measurement was considered an important biomarker to develop on these subjects. To assay relative telomere length (TL) on this large cohort over a short time period, we created a novel high throughput robotic system for TL analysis and informatics. Samples were run in triplicate, along with control samples, in a randomized design. As part of quality control, we determined the within-sample variability and employed thresholds for the elimination of outlying measurements. Of 106,902 samples assayed, 105,539 (98.7%) passed all quality control (QC) measures. As expected, TL in general showed a decline with age and a sex difference. While telomeres showed a negative correlation with age up to 75 years, in those older than 75 years, age positively correlated with longer telomeres, indicative of an association of longer telomeres with more years of survival in those older than 75. Furthermore, while females in general had longer telomeres than males, this difference was significant only for those older than age 50. An additional novel finding was that the variance of TL between individuals increased with age. This study establishes reliable assay and analysis methodologies for measurement of TL in large, population-based human studies. The GERA cohort represents the largest currently available such resource, linked to comprehensive electronic health and genotype data for analysis.

Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2-associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity., La lista completa de autores que integran el documento puede consultarse en el archivo, Instituto de Física de Líquidos y Sistemas Biológicos

Background/Aims: Telomere biology plays a fundamental role in genomic integrity and cell physiology. The newborn setting of telomere length (TL) likely has important implications for telomere dynamics over the lifespan; however, its determinants are poorly understood. Folate is essential for DNA integrity. The maternal compartment is the only source of folate for the developing fetus. We, therefore, tested the hypothesis that variation in maternal folate during pregnancy is associated with newborn TL. Methods: A prospective, longitudinal study was conducted in 119 mother-newborn dyads. Eligible mothers were enrolled at 9.5 (SD ±2.1) weeks gestation and followed through birth. Concentrations of maternal serum folate were measured in the first trimester of pregnancy. Newborn telomere length was measured in cord blood mononuclear cells (CBMC). Results: After accounting for the effects of other established determinants of newborn TL, each 10 ng/ml increase in maternal total folate was associated with a 5.8% increase in median TL (p = 0.03). The median TL in newborns of mother in the lowest quartile of total folate levels was approximately 10% shorter than that of newborns of mothers in the highest folate quartile. Conclusions: Our findings suggest that fetal TL exhibits developmental plasticity, and provide evidence that maternal nutrition may exert a ‘programming' effect on this system.

Discovery and use of a chemical tool., Developing approaches to discover protein–protein interactions (PPIs) remains a fundamental challenge. A chemical biology platform is applied here to identify novel PPIs for the AAA+ superfamily oncoprotein reptin. An in silico screen coupled with chemical optimization provided Liddean, a nucleotide-mimetic which modulates reptin's oligomerization status, protein-binding activity and global conformation. Combinatorial peptide phage library screening of Liddean-bound reptin with next generation sequencing identified interaction motifs including a novel reptin docking site on the p53 tumor suppressor protein. Proximity ligation assays demonstrated that endogenous reptin forms a predominantly cytoplasmic complex with its paralog pontin in cancer cells and Liddean promotes a shift of this complex to the nucleus. An emerging view of PPIs in higher eukaryotes is that they occur through a striking diversity of linear peptide motifs. The discovery of a compound that alters reptin's protein interaction landscape potentially leads to novel avenues for therapeutic development.

© 2015 Elsevier Ireland Ltd. Accelerated cell aging, indexed in peripheral leukocytes by telomere shortness and in peripheral blood mononuclear cells (PBMCs) by telomerase activity, has been reported in several studies of major depressive disorder (MDD). However, the relevance of these peripheral measures for brain indices that are presumably more directly related to MDD pathophysiology is unknown. In this study, we explored the relationship between PBMC telomerase activity and leukocyte telomere length and magnetic resonance imaging-estimated hippocampal volume in un-medicated depressed individuals and healthy controls. We predicted that, to the extent peripheral and central telomerase activity are directly related, PBMC telomerase activity would be positively correlated with hippocampal volume, perhaps due to hippocampal telomerase-associated neurogenesis, neuroprotection or neurotrophic facilitation, and that this effect would be clearer in individuals with increased PBMC telomerase activity, as previously reported in un-medicated MDD. We did not have specific hypotheses regarding the relationship between leukocyte telomere length and hippocampal volume, due to conflicting reports in the published literature. We found, in 25 un-medicated MDD subjects, that PBMC telomerase activity was significantly positively correlated with hippocampal volume; this relationship was not observed in 18 healthy controls. Leukocyte telomere length was not significantly related to hippocampal volume in either group (19 unmedicated MDD subjects and 17 healthy controls). Although the nature of the relationship between peripheral telomerase activity and telomere length and the hippocampus is unclear, these preliminary data are consistent with the possibility that PBMC telomerase activity indexes, and may provide a novel window into, hippocampal neuroprotection and/or neurogenesis in MDD.

Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template. We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template. Each initially produced normally regulated telomeres. One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats. This mutant differs from previously studied delayed elongation mutants in a number of properties. The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity. TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions. Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres. These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover.

Telomeres play a critical role in maintaining genome integrity. Telomere shortening is associated with the risk of many aging-related diseases. Classic twin studies have shown that genetic components may contribute up to 80% of the heritability of telomere length. In the study we report here, we used a multi-stage genome-wide association study (GWAS) to identify genetic determinants of telomere length. The mean telomere length in peripheral blood leukocytes was measured by quantitative real-time polymerase chain reaction. We first analyzed 300,000 single-nucleotide polymorphisms (SNPs) in 459 healthy controls, finding 15,120 SNPs associated with telomere length at P < 0.05. We then validated these SNPs in two independent populations comprising 890 and 270 healthy controls, respectively. Four SNPs, including rs398652 on 14q21, were associated with telomere length across all three populations (pooled P-values of < 10−5). The variant alleles of these SNPs were associated with longer telomere length. We then analyzed the association of these SNPs with the risk of bladder cancer in a large case-control study. The variant allele of rs398652 was associated with a significantly reduced risk of bladder cancer (odds ratio = 0.81; 95% confidence interval, 0.67–0.97; P = 0.025), consistent with the correlation of this variant allele with longer telomeres. We then conducted a mediation analysis to examine whether the association between rs398652 and reduced bladder cancer risk is mediated by telomere length, finding that telomere length was a significant mediator of the relationship between rs398652 and bladder cancer (P = 0.013), explaining 14% of the effect. In conclusion, we found that the SNP rs398652 on 14q21 was associated with longer telomere length and a reduced risk of bladder cancer and that a portion of the effect of this SNP on bladder cancer risk was mediated by telomere length.

Background— Advanced age is the most important risk factor for atrial fibrillation (AF); however, the mechanism remains unknown. Telomeres, regions of DNA that shorten with cell division, are considered reliable markers of biological aging. We sought to examine the association between leukocyte telomere length (LTL) and incident AF in a large population-based cohort using direct LTL measurements and genetic data. To further explore our findings, we compared atrial cell telomere length and LTL in cardiac surgery patients. Methods and Results— Mean LTL and the TERT rs2736100 single nucleotide polymorphism were assessed as predictors of incident AF in the Cardiovascular Health Study (CHS). Among the surgical patients, within subject comparison of atrial cell telomere length versus LTL was assessed. Among 1639 CHS participants, we observed no relationship between mean LTL and incident AF before and after adjustment for potential confounders (adjusted hazard ratio, 1.09; 95% confidence interval: 0.92–1.29; P =0.299); chronologic age remained strongly associated with AF in the same model. No association was observed between the TERT rs2736100 single nucleotide polymorphism and incident AF (adjusted hazard ratio: 0.95; 95% confidence interval: 0.88–1.04; P =0.265). In 35 cardiac surgery patients (26 with AF), atrial cell telomere length was longer than LTL (1.19±0.20 versus 1.02±0.25 [T/S ratio], P Conclusions— Our study revealed no evidence of an association between LTL and incident AF and no evidence of relative atrial cell telomere shortening in AF. Chronological aging independent of biological markers of aging is the primary risk factor for AF.

MOTIVATION: Using molecular similarity to discover bioactive small molecules with novel chemical scaffolds can be computationally demanding. We describe Ultra-fast Shape Recognition with Atom Types (UFSRAT), an efficient algorithm that considers both the 3D distribution (shape) and electrostatics of atoms to score and retrieve molecules capable of making similar interactions to those of the supplied query.RESULTS: Computational optimization and pre-calculation of molecular descriptors enables a query molecule to be run against a database containing 3.8 million molecules and results returned in under 10 seconds on modest hardware. UFSRAT has been used in pipelines to identify bioactive molecules for two clinically relevant drug targets; FK506-Binding Protein 12 and 11β-hydroxysteroid dehydrogenase type 1. In the case of FK506-Binding Protein 12, UFSRAT was used as the first step in a structure-based virtual screening pipeline, yielding many actives, of which the most active shows a KD, app of 281 µM and contains a substructure present in the query compound. Success was also achieved running solely the UFSRAT technique to identify new actives for 11β-hydroxysteroid dehydrogenase type 1, for which the most active displays an IC50 of 67 nM in a cell based assay and contains a substructure radically different to the query. This demonstrates the valuable ability of the UFSRAT algorithm to perform scaffold hops.AVAILABILITY AND IMPLEMENTATION: A web-based implementation of the algorithm is freely available at http://opus.bch.ed.ac.uk/ufsrat/.

BackgroundAdverse childhood experiences (ACEs) are associated with poor physical and mental health outcomes in adulthood. Adverse childhood experiences are also associated with shortened leukocyte telomere length (LTL) in adults, suggesting accelerated cell aging. No studies have yet assessed the relationship of ACEs to LTL in individuals with major depressive disorder (MDD), despite the high incidence of antecedent ACEs in individuals with MDD. Further, no studies in any population have assessed the relationship of ACEs to the activity of telomerase, the major enzyme responsible for maintaining LTL, or the relationship between telomerase and LTL in individuals with ACEs.MethodsTwenty healthy, unmedicated adults with MDD and 20 healthy age-, sex- and ethnicity-matched controls had ACEs assessed and had blood drawn for LTL and peripheral blood mononuclear cell (PBMC) resting telomerase activity.ResultsIn healthy controls, greater ACE exposure was associated with shorter LTL (p

Shorter leukocyte telomere length (LTL) has been associated with a wide range of age-related disorders including cardiovascular disease (CVD) and diabetes. Obesity is an important risk factor for CVD and diabetes. The association of LTL with obesity is not well understood. This study for the first time examines the association of LTL with obesity indices including body mass index, waist circumference, percent body fat, waist-to-hip ratio, and waist-to-height ratio in 3,256 American Indians (14-93 years old, 60% women) participating in the Strong Heart Family Study. Association of LTL with each adiposity index was examined using multivariate generalized linear mixed model, adjusting for chronological age, sex, study center, education, lifestyle (smoking, alcohol consumption, and total energy intake), high-sensitivity C-reactive protein, hypertension and diabetes. Results show that obese participants had significantly shorter LTL than non-obese individuals (age-adjusted P=0.0002). Multivariate analyses demonstrate that LTL was significantly and inversely associated with all of the studied obesity parameters. Our results may shed light on the potential role of biological aging in pathogenesis of obesity and its comorbidities.

Linear motifs mediate protein–protein interactions (PPI) that allow expansion of a target protein interactome at a systems level. This study uses a proteomics approach and linear motif sub-stratifications to expand on PPIs of MDM2. MDM2 is a multi-functional protein with over one hundred known binding partners not stratified by hierarchy or function. A new linear motif based on a MDM2 interaction consensus is used to select novel MDM2 interactors based on Nutlin-3 responsiveness in a cell-based proteomics screen. MDM2 binds a subset of peptide motifs corresponding to real proteins with a range of allosteric responses to MDM2 ligands. We validate cyclophilin B as a novel protein with a consensus MDM2 binding motif that is stabilised by Nutlin-3 in vivo, thus identifying one of the few known interactors of MDM2 that is stabilised by Nutlin-3. These data invoke two modes of peptide binding at the MDM2 N-terminus that rely on a consensus core motif to control the equilibrium between MDM2 binding proteins. This approach stratifies MDM2 interacting proteins based on the linear motif feature and provides a new biomarker assay to define clinically relevant Nutlin-3 responsive MDM2 interactors.

Background: Telomerase is a ribonucleoprotein that copies a short RNA template into telomeric DNA, maintaining eukaryotic chromosome ends and preventing replicative senescence. Telomeres differentiate chromosome ends from DNA double-stranded breaks. Nevertheless, the DNA damage-responsive ATM kinases Tel1p and Mec1p are required for normal telomere maintenance in Saccharomyces cerevisiae . We tested whether the ATM kinases are required for telomerase enzyme activity or whether it is their action on the telomere that allows telomeric DNA synthesis. Results: Cells lacking Tel1p and Mec1p had wild-type levels of telomerase activity in vitro. Furthermore, altering telomere structure in three different ways showed that telomerase can function in ATM kinase-deleted cells: tel1 mec1 cells senesced more slowly than tel1 mec1 cells that also lacked TLC1 , which encodes telomerase RNA, suggesting that tel1 mec1 cells have residual telomerase function; deleting the telomere-associated proteins Rif1p and Rif2p in tel1 mec1 cells prevented senescence; we isolated a point mutation in the telomerase RNA template domain ( tlc1-476A ) that altered telomeric DNA sequences, causing uncontrolled telomeric DNA elongation and increasing single strandedness. In tel1 mec1 cells, tlc1-476A telomerase was also capable of uncontrolled synthesis, but only after telomeres had shortened for >30 generations. Conclusion: Our results show that, without Tel1p and Mec1p, telomerase is still active and can act in vivo when the telomere structure is disrupted by various means. Hence, a primary function of the ATM-family kinases in telomere maintenance is to act on the substrate of telomerase, the telomere, rather than to activate the enzymatic activity of telomerase.

Mutations in the template region of a telomerase RNA gene can lead to the corresponding sequence alterations appearing in newly synthesized telomeric repeats. We analyzed a set of mutations in the template region of the telomerase RNA gene ( TER1 ) of the budding yeast Kluyveromyces lactis that were predicted to lead to synthesis of mutant telomeric repeats with disrupted binding of the telomeric protein Rap1p. We showed previously that mutating the left side of the 12-bp consensus Rap1p binding site led to immediate and severe telomere elongation. Here, we show that, in contrast, mutating either the right side of the site or both sides together leads initially to telomere shortening. On additional passaging, certain mutants of both categories exhibit telomere–telomere fusions. Often, six new Bal-31-resistant, telomere repeat-containing bands appeared, and we infer that each of the six K. lactis chromosomes became circularized. These fusions were not stable, appearing occasionally to resolve and then reform. We demonstrate directly that a linear minichromosome introduced into one of the fusion mutant strains circularized by means of end-to-end fusions of the mutant repeat tracts. In contrast to the chromosomal circularization reported previously in Schizosaccharomyces pombe mutants defective in telomere maintenance, the K. lactis telomere fusions retained their telomeric DNA repeat sequences.

Telomerases contain an essential RNA subunit (TER), as well as an essential protein reverse transcriptase subunit (TERT). The RNA subunit includes a short template region that is copied into telomeric DNA, but otherwise it is large and divergent. However, phylogenetic studies have revealed a conserved core secondary structure for TER. Much of the divergence can be accounted for by the acquisition of different types of RNA domains that function in RNA stabilization. Some of the nontemplate portions of TER, which include regions in the conserved core, are important for aspects of telomerase enzymatic activity independent of their role in telomerase assembly. Mutational studies indicate that telomerase enzyme function results from a collaboration of both protein and RNA functional groups contributed by TERT and TER.

Telomerase is a reverse transcriptase minimally composed of a reverse transcriptase protein subunit and an internal RNA component that contains the templating region. Point mutations of template RNA bases can cause loss of enzymatic activity, reduced processivity and misincorporation in vitro. Here we report the first complete replacement of the nine base TETRAHYMENA: thermophila telomerase templating region in vivo with non-telomeric sequences. Rather than ablating telomerase activity, three such replaced telomerases (U9, AUN and AU4) were effective in polymerization in vitro. In vivo, the AU4 and AUN genes caused telomere shortening. We demonstrated the fidelity of the AUN and U9 telomerases in vitro and utilized AUN telomerase to demonstrate that 5' end primer recognition by telomerase is independent of template base pairing. However, the mutant AUN template telomerase catalyzed an abnormal DNA cleavage reaction. For these U-only and AU- substituted templates, we conclude that base-specific interactions between the telomerase template and protein (or distant parts of the RNA) are not absolutely required for the minimal core telomerase functions of nucleotide addition and base discrimination.

The telomerase ribonucleoprotein has a phylogenetically divergent RNA subunit, which contains a short template for telomeric DNA synthesis. To understand how telomerase RNA participates in mechanistic aspects of telomere synthesis, we studied a conserved secondary structure adjacent to the template. Disruption of this structure caused DNA synthesis to proceed beyond the normal template boundary, resulting in altered telomere sequences, telomere shortening, and cellular growth defects. Compensatory mutations restored normal telomerase function. Thus, the RNA structure, rather than its sequence, specifies the template boundary. This study reveals a specific function for an RNA structure in the enzymatic action of telomerase.

Until this month, women had never won more than one Nobel science prize in a single year. This year's quartet of laureates talk about what their success might mean for science and society.

The past year has seen the ectopic expression of human telomerase and the consequent increased replicative lifespan of cells, whereas mice lacking telomerase have lived and reproduced for six generations. Core telomerase activity from various organisms was reconstituted in vitro, yet how its action is regulated remains largely unknown. Telomerase activation preceded oncogenic transformation in some human cell types, yet was lacking in other transformed cells. These advances highlight the potentials of telomerase-based therapeutics and warn of their pitfalls.

We have identified telomerase activity in extracts of three evolutionarily diverse kinetoplastid species: Trypanosoma brucei, Leishmania major , and Leishmania tarentolae . Telomerase activity was initially detected in extracts from insect form cells of all three kinetoplastid species by using a modification of the one-tube telomere repeat amplification protocol [Kim, N., et al. (1994) Science 266, 2011–2015], although better results were subsequently achieved with the two-tube telomere repeat amplification protocol [Autexier, C., Pruzan, R., Funk, W. & Greider, C. (1996) EMBO J. 15, 5928–5935]. The activity in T. brucei extracts was sufficiently robust to enable its detection in a direct assay of telomerase; enzyme processivity was found to be relatively low. The in vitro properties of telomerase suggest a possible templating domain sequence for the telomerase RNA of T. brucei . Telomerase activity is likely to contribute to telomere maintenance in these parasitic organisms and provides a new target for chemotherapeutic intervention.

The telomeric d(GGGGTT).d(AACCCC) repeat tracts (G4T2 repeats) in Tetrahymena thermophila macronuclei were shown previously to be packaged in a non-nucleosomal DNA-protein complex. Here, we demonstrate that these telomeric repeats, together with a short region of the immediately adjacent non-telomeric sequence, exist in two distinct types of chromatin. The non-nucleosomal complex (type I complex) comprises approximately 90 to 97% of telomeric DNA, has no apparent underlying periodic nucleosomal substructure, and includes the whole telomeric tract as well as the immediately adjacent sequence. Type II chromatin, comprising the remaining approximately 3 to 10% of the total telomeric DNA, consists of tightly packed nucleosomes clustered at the inner border of the telomeric tracts, with a periodicity of 154(+/-3) bp. This packing is similar to that of telomeric nucleosomes in vertebrates. However, in contrast to the unstability of vertebrate telomeric mononucleosomes, the T. thermophila mononucleosomes were stable to micrococcal nuclease digestion. During the natural lengthening of the T. thermophila telomeric DNA tracts that occurs in vegetatively dividing cells, the overall ratio of type I and type II chromatin did not change. However, type I complex expanded with the length of the telomeric DNA repeat tract, and the number of telomeric nucleosomes increased from an average of one, up to three to four, per telomeric tract. This finding of telomeric nucleosomes in T. thermophila suggests that the difference between vertebrates and lower eukaryotes in telomeric chromatin structure is quantitative rather than qualitative. We propose that deposition of nucleosomes competes with non-nucleosomal complex formation on telomeric DNA, resulting in a sub-population of chimeric telomeres containing inner nucleosomes abutting a distal, variable length of type I complex.

Telomerase is a specialized reverse transcriptase with a built‐in RNA template. Base pairing between the templating domain of telomerase RNA and a telomeric DNA primer is normally a characteristic of elongation of telomeric DNA. Here we demonstrate the mechanism by which Tetrahymena telomerase bypasses a requirement for template–primer pairing in order to add telomeric DNA de novo to completely non‐telomeric DNA primers. We show that this reaction initiates by copying the template residue at the 3′ boundary of the telomerase RNA template sequence. Unexpectedly, as the RNA template moves through the telomerase catalytic center, the number of required potential Watson–Crick base pairs between RNA template and DNA primer increases from zero to five. We propose that this unprecedented position specificity of a base pairing potential requirement in a polymerase underlies the chromosome healing mechanism of telomerase, and reflects constraints inherent in an internal template.