Maintaining Telomere Integrity Research Articles

Telomere dynamics during hibernation in a tropical primate.

Hibernation is a widespread metabolic strategy among mammals for surviving periods of food scarcity. During hibernation, animals naturally alternate between metabolically depressed torpor bouts and energetically expensive arousals without ill effects. As a result, hibernators are promising models for investigating mechanisms that buffer against cellular stress, including telomere protection and restoration. In non-hibernators, telomeres, the protective structural ends of chromosomes, shorten with age and metabolic stress. In temperate hibernators, however, telomere shortening and elongation can occur in response to changing environmental conditions and associated metabolic state. We investigate telomere dynamics in a tropical hibernating primate, the fat-tailed dwarf lemur (Cheirogaleus medius). In captivity, these lemurs can hibernate when maintained under cold temperatures (11-15°C) with limited food provisioning. We study telomere dynamics in eight fat-tailed dwarf lemurs at the Duke Lemur Center, USA, from samples collected before, during, and after the hibernation season and assayed via qPCR. Contrary to our predictions, we found that telomeres were maintained or even lengthened during hibernation, but shortened immediately thereafter. During hibernation, telomere lengthening was negatively correlated with time in euthermia. Although preliminary in scope, our findings suggest that there may be a preemptive, compensatory mechanism to maintain telomere integrity in dwarf lemurs during hibernation. Nevertheless, telomere shortening immediately afterward may broadly result in similar outcomes across seasons. Future studies could profitably investigate the mechanisms that offset telomere shortening within and outside of the hibernation season and whether those mechanisms are modulated by energy surplus or crises.

Ginsenoside F1-Mediated Telomere Preservation Delays Cellular Senescence.

Telomeres play pivotal roles in processes closely related to somatic senescence and aging, making them a compelling target for interventions aimed at combating aging and age-related pathologies. Ginsenoside, a natural compound, has emerged as a potential remedy for promoting healthy aging, yet how it protects telomeres remains incompletely understood. Here, we show that treatment of F1 can effectively restore the level of TRF2, thereby preserving telomere integrity. This restoration leads to inhibition of the DNA damage response and improvements in mitochondrial function and, ultimately, delays in cellular senescence. Conversely, depletion of TRF2 causes mitochondrial dysfunction, accompanied by increased oxidative stress, autophagy inhibition, insufficient energy metabolism, and the onset of cellular senescence. These observations underscore the critical role of TRF2 in maintaining telomere integrity and direct association with the initiation of cellular senescence. We conduct a further analysis, suggesting F1 could bind in proximity to the TRF2 heterodimer interface, potentially enhancing dimerization stability. These findings suggest that F1 may be a promising natural remedy for anti-aging, and restoring TRF2 could potentially prevent telomere-dependent diseases commonly associated with the aging process.

Interactions between the DNA Damage Response and the Telomere Complex in Carcinogenesis: A Hypothesis.

Contrary to what was once thought, direct cancer originating from normal stem cells seems to be extremely rare. This is consistent with a preneoplastic period of telomere length reduction/damage in committed cells that becomes stabilized in transformation. Multiple observations suggest that telomere damage is an obligatory step preceding its stabilization. During tissue turnover, the telomeres of cells undergoing differentiation can be damaged as a consequence of defective DNA repair caused by endogenous or exogenous agents. This may result in the emergence of new mechanism of telomere maintenance which is the final outcome of DNA damage and the initial signal that triggers malignant transformation. Instead, transformation of stem cells is directly induced by primary derangement of telomere maintenance mechanisms. The newly modified telomere complex may promote survival of cancer stem cells, independently of telomere maintenance. An inherent resistance of stem cells to transformation may be linked to specific, robust mechanisms that help maintain telomere integrity.

POP1 promotes the progression of breast cancer through maintaining telomere integrity.

Breast cancer is one of the most common and disastrous neoplasm for women worldwide, especially triple negative breast cancer (TNBC). Emerging evidences have demonstrated that RNase subunits are closely related to the occurrence and development of malignant tumors. However, the functions and underlying molecular mechanisms of Processing of Precursor 1 (POP1), a core component of RNase subunits, in breast cancer development have not been fully defined. Our study identified the POP1was upregulated in breast cancer cell lines and tissues and patients with higher POP1 expression were associated with poor outcomes. Overexpression of POP1 promoted cell progression in breast cancer cells, whereas silencing of POP1 induced cell cycle arrest. Moreover, Xenograft model reproduced its growth regulatory roles in breast cancer in vivo. Mechanistically, POP1 interacted and activated the telomerase complex by stabilizing the telomerase RNA component (TERC), thus protecting telomeres from shortening during division. Collectively, our findings demonstrate POP1 may as a novel prognostic marker and a therapeutic target for the management of breast cancer.

Altered Nucleotide Insertion Mechanisms of Disease-Associated TERT Variants.

Telomere biology disorders (TBDs) are a spectrum of diseases that arise from mutations in genes responsible for maintaining telomere integrity. Human telomerase reverse transcriptase (hTERT) adds nucleotides to chromosome ends and is frequently mutated in individuals with TBDs. Previous studies have provided insight into how relative changes in hTERT activity can lead to pathological outcomes. However, the underlying mechanisms describing how disease-associated variants alter the physicochemical steps of nucleotide insertion remain poorly understood. To address this, we applied single-turnover kinetics and computer simulations to the Tribolium castaneum TERT (tcTERT) model system and characterized the nucleotide insertion mechanisms of six disease-associated variants. Each variant had distinct consequences on tcTERT's nucleotide insertion mechanism, including changes in nucleotide binding affinity, rates of catalysis, or ribonucleotide selectivity. Our computer simulations provide insight into how each variant disrupts active site organization, such as suboptimal positioning of active site residues, destabilization of the DNA 3' terminus, or changes in nucleotide sugar pucker. Collectively, this work provides a holistic characterization of the nucleotide insertion mechanisms for multiple disease-associated TERT variants and identifies additional functions of key active site residues during nucleotide insertion.

Comparative Cytogenetics of Lace Bugs (Tingidae, Heteroptera): New Data and a Brief Overview.

Simple SummaryTingidae, or lace bugs, is a family of herbivorous true bugs, with approximately 2600 identified species in 318 genera classified in two or sometimes three subfamilies, among which the largest subfamily Tinginae comprises about 2500 species. Here, an account is given of the karyotypes of 16 lace bug species studied using conventional chromosomal techniques and FISH with two repetitive DNA probes, 18S rDNA and (TTAGG)n. We also summarize and analyze all information accumulated to date on karyotypes of lace bugs. In general, such information is available for 60 species and 22 genera of Tinginae. We show that lace bugs are characterized by: (1) very conservative karyotypes, with six pairs of autosomes in a haploid set; (2) either an XY or X(0) sex chromosome system; (3) a conventional sequence of sex chromosome divisions in male meiosis; (4) absence of the (TTAGG)n mechanism for maintaining telomere integrity; (5) four discrete patterns of 18S rDNA chromosomal localization. We conclude that the search for chromosomal landmarks is of paramount importance for the characterization of the lace bug cytogenetics in more detail.The lace bug family Tingidae comprises more than 2600 described species in 318 genera that are classified into the subfamilies Tinginae (about 2500 species and 300 genera), Cantacaderinae, and Vianadinae. We provide data on karyotypes of 16 species belonging to 10 genera of the tribes Tingini and Acalyptaini (Tinginae) studied using conventional chromosome staining and FISH. The species of Tingini possess 2n = 12A + XY, whereas those of Acalyptaini have 2n = 12A + X(0). FISH for 18S rDNA revealed hybridization signals on one of the medium-sized bivalents in species of both tribes. FISH with a telomeric probe TTAGG produced no signals in any species. In addition, we provide a list of all data obtained to date on Tingidae karyotypes, which includes 60 species from 22 genera of Tinginae. The subfamily is highly conservative in relation to the number and size of autosomes, whereas it shows diversity in the number and chromosomal distribution of the rDNA arrays, which may be located either on a pair of autosomes (the predominant and supposedly ancestral pattern), on one or both sex chromosomes, or on an autosome pair and the X. The absence of the “insect” telomeric sequence TTAGG in all species implies that Tinginae have some other, yet unknown, telomere organization.

MutSβ regulates G4-associated telomeric R-loops to maintain telomere integrity in ALT cancer cells.

Up to 15% of human cancers maintain their telomeres through a telomerase-independent mechanism, termed "alternative lengthening of telomeres" (ALT) that relies on homologous recombination between telomeric sequences. Emerging evidence suggests that the recombinogenic nature of ALT telomeres results from the formation of RNA:DNA hybrids (R-loops) between telomeric DNA and the long-noncoding telomeric repeat-containing RNA (TERRA). Here, we show that the mismatch repair protein MutSβ, a heterodimer of MSH2 and MSH3 subunits, is enriched at telomeres in ALT cancer cells, where it prevents the accumulation of telomeric G-quadruplex (G4) structures and R-loops. Cells depleted of MSH3 display increased incidence of R-loop-dependent telomere fragility and accumulation of telomeric C-circles. We also demonstrate that purified MutSβ recognizes and destabilizes G4 structures invitro. These data suggest that MutSβ destabilizes G4 structures in ALT telomeres to regulate TERRA R-loops, which is a prerequisite for maintenance of telomere integrity during ALT.

TbTRF suppresses the TERRA level and regulates the cell cycle-dependent TERRA foci number with a TERRA binding activity in its C-terminal Myb domain.

Telomere repeat-containing RNA (TERRA) has been identified in multiple organisms including Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. T. brucei regularly switches its major surface antigen, VSG, to evade the host immune response. VSG is expressed exclusively from subtelomeric expression sites, and we have shown that telomere proteins play important roles in the regulation of VSG silencing and switching. In this study, we identify several unique features of TERRA and telomere biology in T. brucei. First, the number of TERRA foci is cell cycle-regulated and influenced by TbTRF, the duplex telomere DNA binding factor in T. brucei. Second, TERRA is transcribed by RNA polymerase I mainly from a single telomere downstream of the active VSG. Third, TbTRF binds TERRA through its C-terminal Myb domain, which also has the duplex DNA binding activity, in a sequence-specific manner and suppresses the TERRA level without affecting its half-life. Finally, levels of the telomeric R-loop and telomere DNA damage were increased upon TbTRF depletion. Overexpression of an ectopic allele of RNase H1 that resolves the R-loop structure in TbTRF RNAi cells can partially suppress these phenotypes, revealing an underlying mechanism of how TbTRF helps maintain telomere integrity.

Abstract PO-020: Telomerase reverse transcriptase inhibitor NU-1 enhances radiation and chemotherapy sensitivity by disrupting DNA repair and accelerating cellular senescence

Abstract Upon reactivation in cancer, telomerase adds TTAGGG DNA repeats to the ends of chromosomes to prevent telomere erosion and senescence despite deregulated cell proliferation. The telomerase catalytic core consists of telomerase reverse transcriptase (TERT) and the template RNA, telomerase RNA (TR). While TR is broadly expressed, TERT is silenced in somatic cells, leading to progressive telomere shortening with each cell division leading to replicative senescence or apoptosis. That most cancers can maintain telomere integrity despite continued cell division has made TERT an attractive target for cancer therapy. We recently reported NU-1, a novel covalent telomerase inhibitor derived from the natural product chrolactomycin, that irreversibly blocks the telomerase activity of TERT. In examining mechanisms of action, our RNA-seq analysis found that NU-1 affects not only telomere maintenance but also DNA damage response (DDR) pathways. TERT has previously been implicated in multiple cancer-related pathways, including cell survival and repair of DNA double strand breaks (DSBs). To further explore the effects of TERT inhibition on the DDR, we used the human ER+ breast cancer cell line MCF7 to examine radiation response in the presence or absence of TERT inhibitors, including NU-1, chrolactomycin, BIBR1532 and MST-312. TERT inhibition was sufficient to delay both the resolution of γH2AX foci, a marker for DDR signaling, and DSB repair based on neutral comet assay. Surviving cells displayed increased cellular senescence after TERT inhibition. Importantly, only a minor fraction of the persistent γH2AX foci induced by TERT inhibitors colocalized with telomeres, based on binding of PNA probes. Examining mechanisms of DSB repair, NU-1 or BIBR1532 appeared to primarily inhibit the non-homologous end joining (NHEJ) DSB repair pathway versus less effect on homologous recombination (HR) repair, using Traffic Light Reporter assays. Similarly, NU-1 enhanced loss of cell proliferation after treatment with irinotecan, doxorubicin, etoposide, or paclitaxel in multiple TERT-expressing cancer cell lines. This effect was absent in the TERT negative Saos2 cell line. These results establish NU-1 as a chemical probe to explore TERT catalytic activity as a determinant of DNA damage tolerance and suggest telomerase inhibitors as tumor-specific radio/chemo-sensitizers. Citation Format: Yue Liu, Stephen J. Kron, Rick C. Betori, Karl A. Scheidt, Grant Frost, Ding Wu, Don J. Wolfgeher, Elena V. Efimova, Scott B. Cohen. Telomerase reverse transcriptase inhibitor NU-1 enhances radiation and chemotherapy sensitivity by disrupting DNA repair and accelerating cellular senescence [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-020.

Telomere Replication: Solving Multiple End Replication Problems.

Eukaryotic genomes are highly complex and divided into linear chromosomes that require end protection from unwarranted fusions, recombination, and degradation in order to maintain genomic stability. This is accomplished through the conserved specialized nucleoprotein structure of telomeres. Due to the repetitive nature of telomeric DNA, and the unusual terminal structure, namely a protruding single stranded 3′ DNA end, completing telomeric DNA replication in a timely and efficient manner is a challenge. For example, the end replication problem causes a progressive shortening of telomeric DNA at each round of DNA replication, thus telomeres eventually lose their protective capacity. This phenomenon is counteracted by the recruitment and the activation at telomeres of the specialized reverse transcriptase telomerase. Despite the importance of telomerase in providing a mechanism for complete replication of telomeric ends, the majority of telomere replication is in fact carried out by the conventional DNA replication machinery. There is significant evidence demonstrating that progression of replication forks is hampered at chromosomal ends due to telomeric sequences prone to form secondary structures, tightly DNA-bound proteins, and the heterochromatic nature of telomeres. The telomeric loop (t-loop) formed by invasion of the 3′-end into telomeric duplex sequences may also impede the passage of replication fork. Replication fork stalling can lead to fork collapse and DNA breaks, a major cause of genomic instability triggered notably by unwanted repair events. Moreover, at chromosomal ends, unreplicated DNA distal to a stalled fork cannot be rescued by a fork coming from the opposite direction. This highlights the importance of the multiple mechanisms involved in overcoming fork progression obstacles at telomeres. Consequently, numerous factors participate in efficient telomeric DNA duplication by preventing replication fork stalling or promoting the restart of a stalled replication fork at telomeres. In this review, we will discuss difficulties associated with the passage of the replication fork through telomeres in both fission and budding yeasts as well as mammals, highlighting conserved mechanisms implicated in maintaining telomere integrity during replication, thus preserving a stable genome.

Pan-cancer analyses reveal regulation and clinical outcome association of the shelterin complex in cancer.

Shelterin, a protective complex at telomeres, plays essential roles in cancer. In addition to maintain telomere integrity, shelterin functions in various survival pathways. However, the detailed mechanisms of shelterin regulation in cancer remain elusive. Here, we perform a comprehensive analysis of shelterin in 9125 tumor samples across 33 cancer types using multi-omic data from The Cancer Genome Atlas, and validate some findings in Chinese Glioma Genome Atlas and cancer cell lines from Cancer Cell Line Encyclopedia. In the genomic landscape, we identify the amplification of TRF1 and POT1, co-amplification/deletion of TRF2-RAP1-TPP1 as the dominant alteration events. Clustering analysis based on shelterin expression reveals three cancer clusters with different degree of genome instability. To measure overall shelterin activity in cancer, we derive a shelterin score based on shelterin expression. Pathway analysis shows shelterin is positively correlated with E2F targets, while is negatively correlated with p53 pathway. Importantly, shelterin links to tumor immunity and predicts response to PD-1 blockade immune therapy. In-depth miRNA analysis reveals a miRNA-shelterin interaction network, with p53 regulated miRNAs targeting multiple shelterin components. We also identify a significant amount of lncRNAs regulating shelterin expression. In addition, we find shelterin expression could be used to predict patient survival in 24 cancer types. Finally, by mining the connective map database, we discover a number of potential drugs that might target shelterin. In summary, this study provides broad molecular signatures for further functional and therapeutic studies of shelterin, and also represents a systemic approach to characterize key protein complex in cancer.

RTEL1 Regulates G4/R-Loops to Avert Replication-Transcription Collisions

SummaryRegulator of telomere length 1 (RTEL1) is an essential helicase that maintains telomere integrity and facilitates DNA replication. The source of replication stress in Rtel1-deficient cells remains unclear. Here, we report that loss of RTEL1 confers extensive transcriptional changes independent of its roles at telomeres. The majority of affected genes in Rtel1−/− cells possess G-quadruplex (G4)-DNA-forming sequences in their promoters and are similarly altered at a transcriptional level in wild-type cells treated with the G4-DNA stabilizer TMPyP4 (5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine). Failure to resolve G4-DNAs formed in the displaced strand of RNA-DNA hybrids in Rtel1−/− cells is suggested by increased R-loops and elevated transcription-replication collisions (TRCs). Moreover, removal of R-loops by RNaseH1 overexpression suppresses TRCs and alleviates the global replication defects observed in Rtel1−/− and Rtel1PIP_box knockin cells and in wild-type cells treated with TMPyP4. We propose that RTEL1 unwinds G4-DNA/R-loops to avert TRCs, which is important to prevent global deregulation in both transcription and DNA replication.

Telomerase, the recombination machinery and Rap1 play redundant roles in yeast telomere protection.

Telomeres are specialized nucleoprotein complexes that protect the ends of eukaryotic chromosomes and distinguish them from broken DNA ends. Disruption of telomere protection may cause aging-associated pathologies and cancer. Here, we examined what makes telomere protection durable and resistant to perturbations using a budding yeast model organism. The protein Rap1 binds the telomeric repeats, negatively regulates telomere length, and protects telomeres by repressing homologous recombination and non-homologous end joining (NHEJ). A single-nucleotide mutation in the Kluyveromyces lactis telomerase RNA (TER1) template, ter1-16T, is incorporated into the telomeric repeats, disrupting the binding of Rap1 and causing dramatic telomere elongation. However, cell viability is not significantly affected, suggesting the existence of additional mechanism(s) for telomere protection. To examine this hypothesis, we explored the contribution of the recombination factor Rad52 and telomerase to telomere protection in the background of ter1-16T. To disrupt the function of telomerase, we exploited small mutations in a stem-loop domain of TER1 (Reg2), which result in short but stable telomeres. We generated K. lactis strains with combinations of three different mutations: ter1-16T, RAD52 deletion, and a two-nucleotide substitution in Reg2. Our results show that upon Rap1 depletion from telomeres, telomerase and the recombination machinery compensate for the loss of Rap1 protection and play redundant but critical roles in preventing NHEJ and maintaining telomere integrity and cell viability. These results demonstrate how redundant pathways make the essential role of telomeres-protecting our genome integrity and preventing cancer-more robust and resistant to assaults and perturbations.

P.026 Role of orexin-1 receptor within medial prefrontal cortex in acquisition and expression of morphine- and natural induced conditioned place preference in rats

The ends of eukaryotic chromosomes typically contain a 3′ ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing–based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.

An ordered assembly of MYH glycosylase, SIRT6 protein deacetylase, and Rad9-Rad1-Hus1 checkpoint clamp at oxidatively damaged telomeres.

In the base excision repair pathway, MYH/MUTYH DNA glycosylase prevents mutations by removing adenine mispaired with 8-oxoG, a frequent oxidative lesion. MYH glycosylase activity is enhanced by Rad9-Rad1-Hus1 (9-1-1) checkpoint clamp and SIRT6 histone/protein deacetylase. Here, we show that MYH, SIRT6, and 9-1-1 are recruited to confined oxidatively damaged regions on telomeres in mammalian cells. Using different knockout cells, we show that SIRT6 responds to damaged telomeres very early, and then recruits MYH and Hus1 following oxidative stress. However, the recruitment of Hus1 to damaged telomeres is partially dependent on SIRT6. The catalytic activities of SIRT6 are not important for SIRT6 response but are essential for MYH recruitment to damaged telomeres. Compared to wild-type MYH, the recruitment of hMYHV315A mutant (defective in both SIRT6 and Hus1 interactions), but not hMYHQ324H mutant (defective in Hus1 interaction only), to damaged telomeres is severely reduced. The formation of MYH/SIRT6/9-1-1 complex is of biological significance as interrupting their interactions can increase cell’s sensitivity to H2O2 and/or elevate cellular 8-oxoG levels after H2O2 treatment. Our results establish that SIRT6 acts as an early sensor of BER enzymes and both SIRT6 and 9-1-1 serve critical roles in DNA repair to maintain telomere integrity.

RTEL Resolves G4/R-Loops to Avert Replication-Transcription Collisions

RTEL1 is an essential helicase that maintains telomere integrity and facilitates DNA replication. The source of replication stress in Rtel1 deficient cells remains unclear. Here, we report that loss of RTEL1 confers extensive transcriptional changes independent of its roles at telomeres. The majority of affected genes in Rtel1-/- cells possess G4-DNA forming sequences in their promoters and are similarly altered at a transcriptional level in wild-type cells treated with the G4-DNA stabilizer TMPyP4. Failure to resolve G4-DNAs formed in the displaced strand of RNA-DNA hybrids in Rtel1-/- cells is suggested by increased R-loops and elevated transcription-replication collisions (TRCs). Moreover, removal of R-loops by RNaseH1 overexpression suppresses TRCs and alleviates the global replication defects observed in Rtel1-/-, Rtel1PIP_box knock-in cells and in wild type cells treated with TMPyP4. We propose that RTEL1 unwinds G4-DNA/R-loops to avert TRCs, which is important to prevent global deregulation in both transcription and DNA replication.

Role of folding kinetics of secondary structures in telomeric G-overhangs in the regulation of telomere maintenance in Saccharomyces cerevisiae

The ends of eukaryotic chromosomes typically contain a 3′ ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing–based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.

Mechanisms of nucleotide selection by telomerase.

Telomerase extends telomere sequences at chromosomal ends to protect genomic DNA. During this process it must select the correct nucleotide from a pool of nucleotides with various sugars and base pairing properties, which is critically important for the proper capping of telomeric sequences by shelterin. Unfortunately, how telomerase selects correct nucleotides is unknown. Here, we determined structures of Tribolium castaneum telomerase reverse transcriptase (TERT) throughout its catalytic cycle and mapped the active site residues responsible for nucleoside selection, metal coordination, triphosphate binding, and RNA template stabilization. We found that TERT inserts a mismatch or ribonucleotide ~1 in 10,000 and ~1 in 14,000 insertion events, respectively. At biological ribonucleotide concentrations, these rates translate to ~40 ribonucleotides inserted per 10 kilobases. Human telomerase assays determined a conserved tyrosine steric gate regulates ribonucleotide insertion into telomeres. Cumulatively, our work provides insight into how telomerase selects the proper nucleotide to maintain telomere integrity.

The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans

Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3' long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.

Pwp1 regulates telomere length by stabilizing shelterin complex and maintaining histone H4K20 trimethylation

Telomere maintenance is critical for chromosome stability. Here we report that periodic tryptophan protein 1 (PWP1) is involved in regulating telomere length homeostasis. Pwp1 appears to be essential for mouse development and embryonic stem cell (ESC) survival, as homozygous Pwp1-knockout mice and ESCs have never been obtained. Heterozygous Pwp1-knockout mice had shorter telomeres and decreased reproductive capacity. Pwp1 depletion induced rapid telomere shortening accompanied by reduced shelterin complex and increased DNA damage in telomeric regions. Mechanistically, PWP1 bound and stabilized the shelterin complex via its WD40 domains and regulated the overall level of H4K20me3. The rescue of telomere length in Pwp1-deficient cells by PWP1 overexpression depended on SUV4-20H2 co-expression and increased H4K20me3. Therefore, our study revealed a novel protein involved in telomere homeostasis in both mouse and human cells. This knowledge will improve our understanding of how chromatin structure and histone modifications are involved in maintaining telomere integrity.