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

S. L. Weinrich, R. Pruzan, L. Ma, M. Ouellette, V. M. Tesmer, S. E. Holt, A. G. Bodnar, S. Lichtsteiner, N. W. Kim, J. B. Trager, R. D. Taylor, R. Carlos, W. H. Andrews, W. E. Wright, J. W. Shay, C. B. Harley, G. B. Morin

Research output: Contribution to journalArticle

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Abstract

The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA polymerases cannot fully replicate the ends of linear molecules. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tomours. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division. 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 proliferation. 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 enzyme, 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 (SpTrt1) telomerases, 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 homologues. 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.

Original languageEnglish (US)
Pages (from-to)498-502
Number of pages5
JournalNature Genetics
Volume17
Issue number4
StatePublished - 1997

Fingerprint

Telomerase
Protein Subunits
Catalytic Domain
Telomere
Cell Aging
RNA-Directed DNA Polymerase
telomerase RNA
Enzymes
Euplotes
Tetrahymena
Proteins
Schizosaccharomyces
DNA-Directed DNA Polymerase
Diploidy
Germ Cells
Cell Division
Saccharomyces cerevisiae
Carcinogenesis
Chromosomes
Maintenance

ASJC Scopus subject areas

  • Genetics(clinical)
  • Genetics

Cite this

Weinrich, S. L., Pruzan, R., Ma, L., Ouellette, M., Tesmer, V. M., Holt, S. E., ... Morin, G. B. (1997). Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nature Genetics, 17(4), 498-502.

Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. / Weinrich, S. L.; Pruzan, R.; Ma, L.; Ouellette, M.; Tesmer, V. M.; Holt, S. E.; Bodnar, A. G.; Lichtsteiner, S.; Kim, N. W.; Trager, J. B.; Taylor, R. D.; Carlos, R.; Andrews, W. H.; Wright, W. E.; Shay, J. W.; Harley, C. B.; Morin, G. B.

In: Nature Genetics, Vol. 17, No. 4, 1997, p. 498-502.

Research output: Contribution to journalArticle

Weinrich, SL, Pruzan, R, Ma, L, Ouellette, M, Tesmer, VM, Holt, SE, Bodnar, AG, Lichtsteiner, S, Kim, NW, Trager, JB, Taylor, RD, Carlos, R, Andrews, WH, Wright, WE, Shay, JW, Harley, CB & Morin, GB 1997, 'Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT', Nature Genetics, vol. 17, no. 4, pp. 498-502.
Weinrich SL, Pruzan R, Ma L, Ouellette M, Tesmer VM, Holt SE et al. Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nature Genetics. 1997;17(4):498-502.
Weinrich, S. L. ; Pruzan, R. ; Ma, L. ; Ouellette, M. ; Tesmer, V. M. ; Holt, S. E. ; Bodnar, A. G. ; Lichtsteiner, S. ; Kim, N. W. ; Trager, J. B. ; Taylor, R. D. ; Carlos, R. ; Andrews, W. H. ; Wright, W. E. ; Shay, J. W. ; Harley, C. B. ; Morin, G. B. / Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. In: Nature Genetics. 1997 ; Vol. 17, No. 4. pp. 498-502.
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