POT1 recruits and regulates CST-Polα/primase at human telomeres.

Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP).

Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq.

Telomerase is a specialized reverse transcriptase that uses an intrinsic RNA subunit as the template for telomeric DNA synthesis. Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-containing pseudoknot (t/PK) and the three-way junction (CR4/5). These two hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are thus essential for telomerase catalytic activity.

Reconstitution of a telomeric replicon organized by CST.

Telomeres, the natural ends of linear chromosomes, comprise repeat-sequence DNA and associated proteins. Replication of telomeres allows continued proliferation of human stem cells and immortality of cancer cells. This replication requires telomerase extension of the single-stranded DNA (ssDNA) of the telomeric G-strand ((TTAGGG)); the synthesis of the complementary C-strand ((CCCTAA)) is much less well characterized.

CST does not evict elongating telomerase but prevents initiation by ssDNA binding.

The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol α-primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation.

The structure of human CST reveals a decameric assembly bound to telomeric DNA.

The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo-electron microscopy structure of human CST bound to telomeric single-stranded DNA (ssDNA), which assembles as a decameric supercomplex.

Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres.

Telomerase maintains genome integrity by adding repetitive DNA sequences to the chromosome ends in actively dividing cells, including 90% of all cancer cells. Recruitment of human telomerase to telomeres occurs during S-phase of the cell cycle, but the molecular mechanism of the process is only partially understood. Here, we use CRISPR genome editing and single-molecule imaging to track telomerase trafficking in nuclei of living human cells.

A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression.

Background: To facilitate indefinite proliferation, stem cells and most cancer cells require the activity of telomerase, which counteracts the successive shortening of telomeres caused by incomplete DNA replication at the very end of each chromosome. Human telomerase activity is often determined by the expression level of telomerase reverse transcriptase (TERT), the catalytic subunit of the ribonucleoprotein complex. The low expression level of TERT and the lack of adequate antibodies have made it difficult to study telomerase-related processes in human cells.

Cancer. TERT promoter mutations and telomerase reactivation in urothelial cancer.

Reactivation of telomerase, the chromosome end-replicating enzyme, drives human cell immortality and cancer. Point mutations in the telomerase reverse transcriptase (TERT) gene promoter occur at high frequency in multiple cancers, including urothelial cancer (UC), but their effect on telomerase function has been unclear. In a study of 23 human UC cell lines, we show that these promoter mutations correlate with higher levels of TERT messenger RNA (mRNA), TERT protein, telomerase enzymatic activity, and telomere length.

Disease mutant analysis identifies a new function of DAXX in telomerase regulation and telomere maintenance.

Most human cancers depend on the telomerase to maintain telomeres; however, about 10% of cancers are telomerase negative and utilize the alternative lengthening of telomeres (ALT) mechanism. Mutations in the DAXX gene have been found frequently in both telomerase-positive and ALT cells, and how DAXX mutations contribute to cancers remains unclear. We report here that endogenous DAXX can localize to Cajal bodies, associate with the telomerase and regulate telomerase targeting to telomeres.

Many disease-associated variants of hTERT retain high telomerase enzymatic activity.

Mutations in the gene for telomerase reverse transcriptase (hTERT) are associated with diseases including dyskeratosis congenita, aplastic anemia, pulmonary fibrosis and cancer. Understanding the molecular basis of these telomerase-associated diseases requires dependable quantitative measurements of telomerase enzyme activity. Furthermore, recent findings that the human POT1-TPP1 chromosome end-binding protein complex stimulates telomerase activity and processivity provide incentive for testing variant telomerases in the presence of these factors.

The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity.

Human chromosome ends are capped by shelterin, a protein complex that protects the natural ends from being recognized as sites of DNA damage and also regulates the telomere-replicating enzyme, telomerase. Shelterin includes the heterodimeric POT1-TPP1 protein, which binds the telomeric single-stranded DNA tail. TPP1 has been implicated both in recruiting telomerase to telomeres and in stimulating telomerase processivity (the addition of multiple DNA repeats after a single primer-binding event).

Telomere shortening and loss of self-renewal in dyskeratosis congenita induced pluripotent stem cells.

The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons, muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood, pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis.

Functional interaction between telomere protein TPP1 and telomerase.

Human chromosome end-capping and telomerase regulation require POT1 (Protection of Telomeres 1) and TPP1 proteins, which bind to the 3' ssDNA extension of human telomeres. POT1-TPP1 binding to telomeric DNA activates telomerase repeat addition processivity. We now provide evidence that this POT1-TPP1 activation requires specific interactions with telomerase, rather than it being a DNA substrate-specific effect.

Mutation in TERT separates processivity from anchor-site function.

Telomerase shows repeat-addition processivity (RAP): synthesis of multiple telomeric DNA repeats without primer dissociation. Leu14 mutants in the telomerase essential N-terminal domain of Tetrahymena thermophila telomerase reverse transcriptase retain full activity and anchor-site function but lose RAP, suggesting models for how this domain facilitates DNA translocation.

The POT1-TPP1 telomere complex is a telomerase processivity factor.

Telomeres were originally defined as chromosome caps that prevent the natural ends of linear chromosomes from undergoing deleterious degradation and fusion events. POT1 (protection of telomeres) protein binds the single-stranded G-rich DNA overhangs at human chromosome ends and suppresses unwanted DNA repair activities. TPP1 is a previously identified binding partner of POT1 that has been proposed to form part of a six-protein shelterin complex at telomeres.

Human POT1 disrupts telomeric G-quadruplexes allowing telomerase extension in vitro.

The POT1 (protection of telomeres 1) protein binds the ssDNA overhangs at the ends of chromosomes in diverse eukaryotes. POT1 is essential for chromosome end-protection, as best demonstrated in fission yeast. In human cells, hPOT1 is also involved in telomere-length regulation.

Switching human telomerase on and off with hPOT1 protein in vitro.

POT1 (protection of telomeres 1) protein binds the G-rich single-stranded telomeric DNA at the ends of chromosomes. In human cells hPOT1 is involved in telomere length regulation, but the mechanism of this regulation remains unknown. Examination of the high-resolution crystal structure of the hPOT1-TTAGGGTTAG complex suggested that it would not be extended by telomerase, a hypothesis that we confirm by in vitro assays with recombinant telomerase.

A template-proximal RNA paired element contributes to Saccharomyces cerevisiae telomerase activity.

The ribonucleoprotein complex telomerase is critical for replenishing chromosome-end sequence during eukaryotic DNA replication. The template for the addition of telomeric repeats is provided by the RNA component of telomerase. However, in budding yeast, little is known about the structure and function of most of the remainder of the telomerase RNA.

Yeast telomerase is specialized for C/A-rich RNA templates.

Telomeres, the protective caps of eukaryotic chromosomes, are maintained by the enzyme telomerase. This telomere-specific reverse transcriptase (RT) uses a small region of its RNA subunit as template to synthesize telomeric DNA, which is generally G/T rich in the strand that contains the 3' end. To further our understanding of why telomeres are usually G/T rich, we screened Saccharomyces cerevisiae telomerase RNA (TLC1) libraries with randomized template sequences for complementation of a tlc1 deletion and decapping of existing telomeres.

Essential regions of Saccharomyces cerevisiae telomerase RNA: separate elements for Est1p and Est2p interaction.

The Saccharomyces cerevisiae telomerase RNA subunit is encoded by the TLC1 gene. A selection for viable alleles of TLC1 RNA from a large library of random deletion alleles revealed that less than half (approximately 0.5 kb of the approximately 1.