Tetrahymena telomerase catalyzes nucleolytic cleavage and nonprocessive elongation

Telomerase is a ribonucleoprotein enzyme that adds telomeric repeats to chromosomes, maintaining telomere length and stabilizing chromosome ends. In vitro, telomerase from the ciliate Tetrahymena elongates single-stranded, guanosine-rich DNA primers by adding repeats of the Tetrahymena telomeric sequence, dT2G4. We have identified two activities of Tetrahymena telomerase in addition to the previously described processive elongation reaction: a 3'-5' nucleolytic cleavage of primer or product DNA and a nonprocessive mode of elongation. The nucleolytic cleavage activity removed residues not conforming to the telomeric repeat sequence from a primer 3' end, eliminating mismatch between DNA primer and RNA template sequences. Template-matched residues were also cleaved from primer or product DNA. Specific primer lengths, sequences, and concentrations stimulated cleavage and processive or nonprocessive elongation differentially. These newly identified activities suggest that telomerase may catalyze a range of telomere synthesis and repair functions and suggest mechanistic similarities between telomerase and RNA polymerase enzymes. On the basis of our results, we propose a model for telomerase primer binding, cleavage, and elongation

Boundary elements of the Tetrahymena telomerase RNA template and alignment domains

Telomerase is a DNA polymerase fundamental to the replication and maintenance of telomere sequences at chromosome ends. The RNA component of telomerase is essential for the synthesis of telomere repeats. In vitro, the template domain (5'-CAACCCCAA-3') of the Tetrahymena telomerase RNA dictates the addition of Tetrahymena-specific telomere repeats d(TTGGGG)n, onto the 3' end of G-rich or telomeric substrates that are base-paired with the template and alignment regions of the RNA. Using a reconstituted in vitro system, we determined that altering the sequence of the alignment and template domains affects processivity of telomerase without abolishing telomerase activity. These results suggest that alternative template/alignment regions may be functional. In the ciliate telomerase RNAs, there is a conserved sequence 5'-(CU)GUCA-3', located two residues upstream of the template domain. The location and sequence of this conserved domain defined the 5' boundary of the template region. These data provide insights into the regulation of telomere synthesis by telomerase

A sequence-dependent exonuclease activity from Tetrahymena thermophila

<p>Abstract</p> <p>Background</p> <p>Telomere function requires a highly conserved G rich 3'- overhang. This structure is formed by 5'-resection of the C-rich telomere strand. However, while many nucleases have been suggested to play a role in processing, it is not yet clear which nucleases carry out this 5'-resection.</p> <p>Results</p> <p>We used biochemical purification to identify a sequence-dependent exonuclease activity in <it>Tetrahymena thermophila </it>cell extracts. The nuclease activity showed specificity for 5'-ends containing AA or AC sequences, unlike Exo1, which showed sequence-independent cleavage. The <it>Tetrahymena </it>nuclease was active on both phosphorylated and unphosphorylated substrates whereas Exo1 requires a 5'-phosphate for cleavage.</p> <p>Conclusions</p> <p>The specificities of the enzyme indicate that this novel <it>Tetrahymena </it>exonuclease is distinct from Exo1 and has properties required for 3'-overhang formations at telomeres.</p

Leadership, the logic of sufficiency and the sustainability of education

The notion of sufficiency has not yet entered mainstream educational thinking, and it still has to make its mark upon educational leadership. However, a number of related concepts – particularly those of sustainability and complexity theory – are beginning to be noticed. This article examines these two concepts and uses them to critique the quasi-economic notion of efficiency, before arguing that the concept of sufficiency arises naturally from this discussion. This concept, originally derived from environmental thinking, has both metaphorical and practical impact for educational organizations and their leadership. An examination of three possible meanings suggests that while an embrace of an imperative concept of sufficiency seems increasingly necessary, its adoption would probably lead to a number of other problems, as it challenges some fundamental societal values and assumptions. Nevertheless, the article argues that these need to be addressed for the sake of both sustainable leadership and a sustainable planet

Extreme Telomere Length Dimorphism in the Tasmanian Devil and Related Marsupials Suggests Parental Control of Telomere Length

Telomeres, specialised structures that protect chromosome ends, play a critical role in preserving chromosome integrity. Telomere dynamics in the Tasmanian devil (Sarcophilus harrisii) are of particular interest in light of the emergence of devil facial tumour disease (DFTD), a transmissible malignancy that causes rapid mortality and threatens the species with extinction. We used fluorescent in situ hybridisation to investigate telomere length in DFTD cells, in healthy Tasmanian devils and in four closely related marsupial species. Here we report that animals in the Order Dasyuromorphia have chromosomes characterised by striking telomere length dimorphism between homologues. Findings in sex chromosomes suggest that telomere length dimorphism may be regulated by events in the parental germlines. Long telomeres on the Y chromosome imply that telomere lengthening occurs during spermatogenesis, whereas telomere diminution occurs during oogenesis. Although found in several somatic cell tissue types, telomere length dimorphism was not found in DFTD cancer cells, which are characterised by uniformly short telomeres. This is, to our knowledge, the first report of naturally occurring telomere length dimorphism in any species and suggests a novel strategy of telomere length control. Comparative studies in five distantly related marsupials and a monotreme indicate that telomere dimorphism evolved at least 50 million years ago. © 2012 Bender et al

Angular Dependence of Neutrino Flux in KM3 Detectors in Low Scale Gravity Models

Cubic kilometer neutrino telescopes are capable of probing fundamental questions of ultra-high energy neutrino interactions. There is currently great interest in neutrino interactions caused by low-scale, extra dimension models. Above 1 PeV the cross section in low scale gravity models rises well above the total Standard Model cross section. We assess the observability of this effect in the 1 PeV - 100 PeV energy range of kilometer-scale detectors with several new points of emphasis that hinge on enhanced neutral current cross sections. A major point is the importance of ``feed-down'' regeneration of upward neutrino flux, driven by new-physics neutral current interactions in the flux evolution equations. Feed-down is far from negligible, and it is essential to include its effect. We then find that the angular distribution of events has high discriminating value in separating models. In particular the ``up-to-down'' ratio between upward and downward-moving neutrino fluxes is a practical diagnostic tool which can discriminate between models in the near future. The slope of the angular distribution, in the region of maximum detected flux, is also substantially different in low-scale gravity and the Standard Model. These observables are only weakly dependent on astrophysical flux uncertainties. We conclude that angular distributions can reveal a breakdown of the Standard Model and probe the new physics beyond, as soon as data become available.Comment: 25 pages, 6 figures, discussion of calculations expanded, references adde

Functional reconstitution of wild-type and mutant Tetrahymena telomerase

Telomerase is a ribonucleoprotein that catalyzes telomere elongation in vitro and in vivo. The 159-nucleotide RNA component of Tetrahymena telomerase contains the sequence 5'-CAACCCCAA-3' ("template region"), which serves as a template for the addition of the sequence d(TTGGGG)n to Tetrahymena telomeres. To dissect the Tetrahymena telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential telomerase RNA by micrococcal nuclease digestion of partially purified telomerase, the addition of in vitro-transcribed telomerase RNA reconstituted telomerase activity. The reconstituted activity was processive and showed the same primer specificities as native telomerase. Mutants in the RNA template region were tested in reconstitution assays to determine the role of the residues in this region in primer recognition and elongation. Two template mutants, encoding the sequences 5'-UAACCCCAA-3' and 5'-UAACCCUAA-3', specified the incorporation of dATP into the sequence d(TTAGGG). Telomerase reconstituted with a template mutant encoding the sequence 5'-CAACCCUAA-3' did not specify dATP incorporation and elongation by this mutant was not terminated by the addition of ddATP. In addition, a template mutant encoding the sequence 5'-CGGCCCCAA-3' specified the incorporation of ddCTP but not ddTTP while a mutant encoding the sequence 5'-CAACCCCGG-3' specified the incorporation of ddTTP but not ddCTP. These data suggest that only the most 5' six residues of the template region dictate the addition of telomeric repeats

The RNA component of human telomerase

Eukaryotic chromosomes are capped with repetitive telomere sequences that protect the ends from damage and rearrangements. Telomere repeats are synthesized by telomerase, a ribonucleic acid (RNA)-protein complex. Here, the cloning of the RNA component of human telomerase, termed hTR, is described. The template region of hTR encompasses 11 nucleotides (5'-CUAACCCUAAC) complementary to the human telomere sequence (TTAGGG)n. Germline tissues and tumor cell lines expressed more hTR than normal somatic cells and tissues, which have no detectable telomerase activity. Human cell lines that expressed hTR mutated in the template region generated the predicted mutant telomerase activity. HeLa cells transfected with an antisense hTR lost telomeric DNA and began to die after 23 to 26 doublings. Thus, human telomerase is a critical enzyme for the long-term proliferation of immortal tumor cells

The Telomere Terminal Transferase of Tetrahymena Is a Ribonucleoprotein Enzyme with Two Kinds of Primer Specificity

We have analyzed the de novo telomere synthesis catalyzed by the enzyme telomere terminal transferase (telomerase) from Tetrahymena. Oligonucleotides representing the G-rich strand of telomeric sequences from five different organisms specifically primed the addition of TTGGGG repeats in vitro, suggesting that primer recognition may involve a DNA structure unique to these oligonucleotides. The sequence at the 3' end of the oligonucleotide primer specified the first nucleotide added in the reaction. Furthermore, the telomerase was shown to be a ribonucleoprotein complex whose RNA and protein components were both essential for activity. After extensive purification of the enzyme by a series of five different chromatographic steps, a few small low abundance RNAs copurified with the activity