Catalytic protein subunits of telomerase from the ciliate Euplotes aediculatus and the yeast Saccharomyces cerevisiae contain reverse transcriptase motifs. Here the homologous genes from the fission yeast Schizosaccharomyces pombe and human are identified. Disruption of the S. pombe gene resulted in telomere shortening and senescence, and expression of mRNA from the human gene correlated with telomerase activity in cell lines. Sequence comparisons placed the telomerase proteins in the reverse transcriptase family but revealed hallmarks that distinguish them from retroviral and retrotransposon relatives. Thus, the proposed telomerase catalytic subunits are phylogenetically conserved and represent a deep branch in the evolution of reverse transcriptases.

https://science.sciencemag.org/content/sci/277/5328/955.full.pdf

Telomerase catalytic subunit homologs from fission yeast and human

Telomere Shortening and Tumor Formation by Mouse Cells Lacking Telomerase RNA

https://courses.washington.edu/gs466/readings/meyerson.pdf

hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization

Telomerase is a ribonucleoprotein enzyme essential for the replication of chromosome termini in most eukaryotes. Telomerase RNA components have been identified from many organisms, but no protein component has been demonstrated to catalyze telomeric DNA extension. Telomerase was purified from Euplotes aediculatus, a ciliated protozoan, and one of its proteins was partially sequenced by nanoelectrospray tandem mass spectrometry. Cloning and sequence analysis of the corresponding gene revealed that this 123-kilodalton protein (p123) contains reverse transcriptase motifs. A yeast (Saccharomyces cerevisiae) homolog was found and subsequently identified as EST2 (ever shorter telomeres), deletion of which had independently been shown to produce telomere defects. Introduction of single amino acid substitutions within the reverse transcriptase motifs of Est2 protein led to telomere shortening and senescence in yeast, indicating that these motifs are important for catalysis of telomere elongation in vivo. In vitro telomeric DNA extension occurred with extracts from wild-type yeast but not from est2 mutants or mutants deficient in telomerase RNA. Thus, the reverse transcriptase protein fold, previously known to be involved in retroviral replication and retrotransposition, is essential for normal chromosome telomere replication in diverse eukaryotes.

https://science.sciencemag.org/content/sci/276/5312/561.full.pdf

Reverse Transcriptase Motifs in the Catalytic Subunit of Telomerase

The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA poly-merases cannot fully replicate the ends of linear molecules1–4. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumours5,6. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division7–9. It has been proposed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation10. In addition to the human telomerase RNA component (hTR; ref. 11), TP1/TLP1 (refs 12,13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme14, has been identified in humans. More recently, the human telomerase reverse transcriptase (hTRT; refs 15, 16), which is homologous to the reverse transcriptase (RT)-like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosaccharomyces pombe (SpTrt!) telomerases15,17, has been reported to be a telomerase protein subunit. A catalytic function has been demonstrated for Est2p in the RT-like class but not for p80 or its homologues17,18. We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activity that exhibits enzymatic properties like those of the native enzyme. Single amino-acid changes in conserved telomerase-specific and RT motifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase. Normal human diploid cells transiently expressing hTRT possessed telomerase activity, demonstrating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells. The ability to reconstitute telomerase permits further analysis of its biochemical and biological roles in cell aging and carcinogenesis.

Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT

The telomerase ribonucleoprotein catalyzes the addition of new telomeres onto chromosome ends A gene encoding a mammalian telomerase homolog called TP1 (telomerase-associated protein 1) was identified and cloned TP1 exhibited extensive amino acid similarity to the Tetrahymena telomerase protein p80 and was shown to interact specifically with mammalian telomerase RNA Antiserum to TP1 immunoprecipitated telomerase activity from cell extracts, suggesting that TP1 is associated with telomerase in vivo The identification of TP1 suggests that telomerase-associated proteins are conserved from ciliates to humans

http://science.sciencemag.org/content/sci/275/5302/973.full.pdf

Amgen Est Program

Papers

A mammalian telomerase-associated protein.