Processive utilization of the human telomerase template: lack of a requirement for template switching.

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.

A universal telomerase RNA core structure includes structured motifs required for binding the telomerase reverse transcriptase protein.

Telomerase synthesizes telomeric DNA by copying a short template sequence within its telomerase RNA component. We delineated nucleotides and base-pairings within a previously mapped central domain of the Saccharomyces cerevisiae telomerase RNA (TLC1) that are important for telomerase function and for binding to the telomerase catalytic protein Est2p. Phylogenetic comparison of telomerase RNA sequences from several budding yeasts revealed a core structure common to Saccharomyces and Kluyveromyces yeast species.

Antitumor activity of systemically delivered ribozymes targeting murine telomerase RNA.

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.

Rapid inhibition of cancer cell growth induced by lentiviral delivery and expression of mutant-template telomerase RNA and anti-telomerase short-interfering RNA.

In human cancers, telomeres are commonly maintained by elevated levels of the ribonucleoprotein enzyme telomerase, which contains an intrinsic templating RNA moiety (human telomerase RNA; hTER) and the core protein (human telomerase reverse transcriptase). We developed a lentiviral system for efficient overexpression of mutant-template human telomerase RNA (MT-hTer) to add mutant DNA to telomeres in cancer cells. We show that such MT-hTer overexpression rapidly inhibits cell growth and induces apoptosis in telomerase-positive precancerous or cancer cells but not in telomerase-negative cells.

Co-expression of neuregulins 1, 2, 3 and 4 in human breast cancer.

We have produced antibodies to the NRG2-alpha, NRG2-beta, NRG3 and NRG4 proteins and used these, and previously described antibodies to NRG1-alpha and NRG1-beta, to detect expression of each ligand by immunocytochemical staining in a series of 45 breast cancers. Each protein was expressed in a proportion of cases. Statistical analysis suggested that expression of one factor was associated with a high probability that other members of the family were co-expressed.

Anatomy and dynamics of DNA replication fork movement in yeast telomeric regions.

Replication initiation and replication fork movement in the subtelomeric and telomeric DNA of native Y' telomeres of yeast were analyzed using two-dimensional gel electrophoresis techniques. Replication origins (ARSs) at internal Y' elements were found to fire in early-mid-S phase, while ARSs at the terminal Y' elements were confirmed to fire late. An unfired Y' ARS, an inserted foreign (bacterial) sequence, and, as previously reported, telomeric DNA each were shown to impose a replication fork pause, and pausing is relieved by the Rrm3p helicase.

Telomeres and telomerase.

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.

Reason as our guide.

Two scientist members of the President's Council on Bioethics express their concerns about two recently issued reports by the Council in which the science is presented incompletely and myths are perpetuated

Nucleolar protein PinX1p regulates telomerase by sequestering its protein catalytic subunit in an inactive complex lacking telomerase RNA.

Human TRF1-binding protein PinX1 inhibits telomerase activity. Here we report that overexpression of yeast PinX1p (yPinX1p) results in shortened telomeres and decreased in vitro telomerase activity. yPinX1p coimmunoprecipitated with yeast telomerase protein Est2p even in cells lacking the telomerase RNA TLC1, or the telomerase-associated proteins Est1p and Est3p.

Mutant telomere sequences lead to impaired chromosome separation and a unique checkpoint response.

Mutation of the template region in the RNA component of telomerase can cause incorporation of mutant DNA sequences at telomeres. We made all 63 mutant sequence combinations at template positions 474-476 of the yeast telomerase RNA, TLC1. Mutants contained faithfully incorporated template mutations, as well as misincorporated sequences in telomeres, a phenotype not previously reported for Saccharomyces cerevisiae telomerase template mutants.

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.

Comprehensive structure-function analysis of the core domain of human telomerase RNA.

Telomerase is a cellular reverse transcriptase that uses part of its integral RNA (called TER) as the template to synthesize telomeric DNA repeats. Vertebrate TERs are thought to share a conserved, highly structured core domain that includes the templating sequence and a pseudoknot, but not all features of the predicted core structure have been verified directly or shown to affect telomerase enzymatic activity. Here, we report a systematic mutational analysis of the core domain (residues 1 to 210) of human telomerase RNA (hTER).

Catalytically active human telomerase mutants with allele-specific biological properties.

Expression of the catalytic subunit of human telomerase, hTERT, extends human primary fibroblast life span. Such life span extension has generally been reported to be accompanied by net telomere lengthening, which led to the hypothesis that it is the telomere lengthening that causes the life span extension. Here we show that hTERT+C and hTERT-FlagC, mutant telomerase proteins with either 10 additional residues or a FLAG epitope added to the hTERT C-terminus, confer significant but limited life span extension to IMR90 human primary lung fibroblasts.

A novel pseudoknot element is essential for the action of a yeast telomerase.

Telomerase contains an essential RNA, which includes the template sequence copied by the reverse transcription action of telomerase into telomeric DNA. Using phylogenetic comparison, we identified seven conserved sequences in telomerase RNAs from Kluyveromyces budding yeasts. We show that two of these sequences, CS3 and CS4, are essential for normal telomerase function and can base-pair to form a putative long-range pseudoknot.

Telomerase and ATM/Tel1p protect telomeres from nonhomologous end joining.

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.

Molecular manifestations and molecular determinants of telomere capping.

Multiple interacting components of the telomere, together with telomerase (and sometimes recombination), determine whether a telomere will be functional, allowing cell proliferation. The various components reinforce each other, providing for a robust and resilient system of protection and replenishment of telomeres. A characteristic of a telomere is that its structural features elicit responses that allow it to be dynamically recapped.

A role for a novel 'trans-pseudoknot' RNA-RNA interaction in the functional dimerization of human telomerase.

The integral RNA (hTER) of the human telomerase ribonucleoprotein has a conserved secondary structure that contains a potential pseudoknot. Here we examine the role of an intermolecular hTER-hTER interaction in the previously reported functional dimerization of telomerase. We provide evidence that the two conserved, complementary sequences of one stem of the hTER pseudoknot domain can pair intermolecularly in vitro, and that formation of this stem as part of a novel "trans-pseudoknot" is required for telomerase to be active in its dimeric form.

Telomeric protein distributions and remodeling through the cell cycle in Saccharomyces cerevisiae.

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.

A molecular switch underlies a human telomerase disease.

Telomerase is a ribonucleoprotein (RNP) required for maintenance of telomeres. Although up-regulated telomerase activity is closely linked to the cellular immortality characteristic of late stage carcinogenesis, recently, mutations in the telomerase RNA gene in humans have been associated with dyskeratosis congenita and aplastic anemia, both typified by impaired haemopoietic function. These mutations include base changes in a highly conserved putative telomerase RNA pseudoknot.

A bulged stem tethers Est1p to telomerase RNA in budding yeast.

It is well established that the template for telomeric DNA synthesis is provided by the RNA subunit of telomerase; however, the additional functions provided by most of the rest of the RNA (>1000 nucleotides in budding yeast) are largely unknown. By alignment of telomerase RNAs of Saccharomyces cerevisiae and six Kluyveromyces species followed by mutagenesis of the S. cerevisiae RNA, we found a conserved region that is essential for telomere maintenance.

The genome-wide expression response to telomerase deletion in Saccharomyces cerevisiae.

Loss of the protective function of telomeres has previously been hypothesized to cause a DNA damage response. Here, we report a genome-wide expression response, the telomerase deletion response (TDR), that occurs when telomeres can no longer be maintained by telomerase. The TDR shares features with other DNA damage responses and the environmental stress response.