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. 1998 Feb 16;17(4):1152–1160. doi: 10.1093/emboj/17.4.1152

A novel specificity for the primer-template pairing requirement in Tetrahymena telomerase.

H Wang 1, D Gilley 1, E H Blackburn 1
PMCID: PMC1170463  PMID: 9463392

Abstract

Telomerase is a specialized reverse transcriptase with a built-in RNA template. Base pairing between the templating domain of telomerase RNA and a telomeric DNA primer is normally a characteristic of elongation of telomeric DNA. Here we demonstrate the mechanism by which Tetrahymena telomerase bypasses a requirement for template-primer pairing in order to add telomeric DNA de novo to completely non-telomeric DNA primers. We show that this reaction initiates by copying the template residue at the 3' boundary of the telomerase RNA template sequence. Unexpectedly, as the RNA template moves through the telomerase catalytic center, the number of required potential Watson-Crick base pairs between RNA template and DNA primer increases from zero to five. We propose that this unprecedented position specificity of a base pairing potential requirement in a polymerase underlies the chromosome healing mechanism of telomerase, and reflects constraints inherent in an internal template.

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Selected References

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  1. Bednenko J., Melek M., Greene E. C., Shippen D. E. Developmentally regulated initiation of DNA synthesis by telomerase: evidence for factor-assisted de novo telomere formation. EMBO J. 1997 May 1;16(9):2507–2518. doi: 10.1093/emboj/16.9.2507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blackburn E. H. Telomerases. Annu Rev Biochem. 1992;61:113–129. doi: 10.1146/annurev.bi.61.070192.000553. [DOI] [PubMed] [Google Scholar]
  3. Cohn M., Blackburn E. H. Telomerase in yeast. Science. 1995 Jul 21;269(5222):396–400. doi: 10.1126/science.7618104. [DOI] [PubMed] [Google Scholar]
  4. Collins K., Greider C. W. Tetrahymena telomerase catalyzes nucleolytic cleavage and nonprocessive elongation. Genes Dev. 1993 Jul;7(7B):1364–1376. doi: 10.1101/gad.7.7b.1364. [DOI] [PubMed] [Google Scholar]
  5. Fan Q., Yao M. New telomere formation coupled with site-specific chromosome breakage in Tetrahymena thermophila. Mol Cell Biol. 1996 Mar;16(3):1267–1274. doi: 10.1128/mcb.16.3.1267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Flint J., Craddock C. F., Villegas A., Bentley D. P., Williams H. J., Galanello R., Cao A., Wood W. G., Ayyub H., Higgs D. R. Healing of broken human chromosomes by the addition of telomeric repeats. Am J Hum Genet. 1994 Sep;55(3):505–512. [PMC free article] [PubMed] [Google Scholar]
  7. Forney J. D., Blackburn E. H. Developmentally controlled telomere addition in wild-type and mutant paramecia. Mol Cell Biol. 1988 Jan;8(1):251–258. doi: 10.1128/mcb.8.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gilley D., Blackburn E. H. Specific RNA residue interactions required for enzymatic functions of Tetrahymena telomerase. Mol Cell Biol. 1996 Jan;16(1):66–75. doi: 10.1128/mcb.16.1.66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gilley D., Lee M. S., Blackburn E. H. Altering specific telomerase RNA template residues affects active site function. Genes Dev. 1995 Sep 15;9(18):2214–2226. doi: 10.1101/gad.9.18.2214. [DOI] [PubMed] [Google Scholar]
  10. Greider C. W., Blackburn E. H. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature. 1989 Jan 26;337(6205):331–337. doi: 10.1038/337331a0. [DOI] [PubMed] [Google Scholar]
  11. Greider C. W., Blackburn E. H. The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell. 1987 Dec 24;51(6):887–898. doi: 10.1016/0092-8674(87)90576-9. [DOI] [PubMed] [Google Scholar]
  12. Hammond P. W., Lively T. N., Cech T. R. The anchor site of telomerase from Euplotes aediculatus revealed by photo-cross-linking to single- and double-stranded DNA primers. Mol Cell Biol. 1997 Jan;17(1):296–308. doi: 10.1128/mcb.17.1.296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Johnson K. A. Conformational coupling in DNA polymerase fidelity. Annu Rev Biochem. 1993;62:685–713. doi: 10.1146/annurev.bi.62.070193.003345. [DOI] [PubMed] [Google Scholar]
  14. Kramer K. M., Haber J. E. New telomeres in yeast are initiated with a highly selected subset of TG1-3 repeats. Genes Dev. 1993 Dec;7(12A):2345–2356. doi: 10.1101/gad.7.12a.2345. [DOI] [PubMed] [Google Scholar]
  15. Lamb J., Harris P. C., Wilkie A. O., Wood W. G., Dauwerse J. G., Higgs D. R. De novo truncation of chromosome 16p and healing with (TTAGGG)n in the alpha-thalassemia/mental retardation syndrome (ATR-16). Am J Hum Genet. 1993 Apr;52(4):668–676. [PMC free article] [PubMed] [Google Scholar]
  16. Lee M. S., Blackburn E. H. Sequence-specific DNA primer effects on telomerase polymerization activity. Mol Cell Biol. 1993 Oct;13(10):6586–6599. doi: 10.1128/mcb.13.10.6586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lee M. S., Gallagher R. C., Bradley J., Blackburn E. H. In vivo and in vitro studies of telomeres and telomerase. Cold Spring Harb Symp Quant Biol. 1993;58:707–718. doi: 10.1101/sqb.1993.058.01.078. [DOI] [PubMed] [Google Scholar]
  18. Lingner J., Hughes T. R., Shevchenko A., Mann M., Lundblad V., Cech T. R. Reverse transcriptase motifs in the catalytic subunit of telomerase. Science. 1997 Apr 25;276(5312):561–567. doi: 10.1126/science.276.5312.561. [DOI] [PubMed] [Google Scholar]
  19. Melek M., Greene E. C., Shippen D. E. Processing of nontelomeric 3' ends by telomerase: default template alignment and endonucleolytic cleavage. Mol Cell Biol. 1996 Jul;16(7):3437–3445. doi: 10.1128/mcb.16.7.3437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Melek M., Shippen D. E. Chromosome healing: spontaneous and programmed de novo telomere formation by telomerase. Bioessays. 1996 Apr;18(4):301–308. doi: 10.1002/bies.950180408. [DOI] [PubMed] [Google Scholar]
  21. Meyerson M., Counter C. M., Eaton E. N., Ellisen L. W., Steiner P., Caddle S. D., Ziaugra L., Beijersbergen R. L., Davidoff M. J., Liu Q. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell. 1997 Aug 22;90(4):785–795. doi: 10.1016/s0092-8674(00)80538-3. [DOI] [PubMed] [Google Scholar]
  22. Nakamura T. M., Morin G. B., Chapman K. B., Weinrich S. L., Andrews W. H., Lingner J., Harley C. B., Cech T. R. Telomerase catalytic subunit homologs from fission yeast and human. Science. 1997 Aug 15;277(5328):955–959. doi: 10.1126/science.277.5328.955. [DOI] [PubMed] [Google Scholar]
  23. Nevinsky G. A., Veniaminova A. G., Levina A. S., Podust V. N., Lavrik O. I., Holler E. Structure-function analysis of mononucleotides and short oligonucleotides in the priming of enzymatic DNA synthesis. Biochemistry. 1990 Feb 6;29(5):1200–1207. doi: 10.1021/bi00457a016. [DOI] [PubMed] [Google Scholar]
  24. Petruska J., Goodman M. F., Boosalis M. S., Sowers L. C., Cheong C., Tinoco I., Jr Comparison between DNA melting thermodynamics and DNA polymerase fidelity. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6252–6256. doi: 10.1073/pnas.85.17.6252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pologe L. G., Ravetch J. V. Large deletions result from breakage and healing of P. falciparum chromosomes. Cell. 1988 Dec 2;55(5):869–874. doi: 10.1016/0092-8674(88)90142-0. [DOI] [PubMed] [Google Scholar]
  26. Scherf A., Carter R., Petersen C., Alano P., Nelson R., Aikawa M., Mattei D., Pereira da Silva L., Leech J. Gene inactivation of Pf11-1 of Plasmodium falciparum by chromosome breakage and healing: identification of a gametocyte-specific protein with a potential role in gametogenesis. EMBO J. 1992 Jun;11(6):2293–2301. doi: 10.1002/j.1460-2075.1992.tb05288.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Scherf A., Mattei D. Cloning and characterization of chromosome breakpoints of Plasmodium falciparum: breakage and new telomere formation occurs frequently and randomly in subtelomeric genes. Nucleic Acids Res. 1992 Apr 11;20(7):1491–1496. doi: 10.1093/nar/20.7.1491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Strahl C., Blackburn E. H. The effects of nucleoside analogs on telomerase and telomeres in Tetrahymena. Nucleic Acids Res. 1994 Mar 25;22(6):893–900. doi: 10.1093/nar/22.6.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wang H., Blackburn E. H. De novo telomere addition by Tetrahymena telomerase in vitro. EMBO J. 1997 Feb 17;16(4):866–879. doi: 10.1093/emboj/16.4.866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wicky C., Villeneuve A. M., Lauper N., Codourey L., Tobler H., Müller F. Telomeric repeats (TTAGGC)n are sufficient for chromosome capping function in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8983–8988. doi: 10.1073/pnas.93.17.8983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wilkie A. O., Lamb J., Harris P. C., Finney R. D., Higgs D. R. A truncated human chromosome 16 associated with alpha thalassaemia is stabilized by addition of telomeric repeat (TTAGGG)n. Nature. 1990 Aug 30;346(6287):868–871. doi: 10.1038/346868a0. [DOI] [PubMed] [Google Scholar]
  32. Yao M. C., Zheng K., Yao C. H. A conserved nucleotide sequence at the sites of developmentally regulated chromosomal breakage in Tetrahymena. Cell. 1987 Mar 13;48(5):779–788. doi: 10.1016/0092-8674(87)90075-4. [DOI] [PubMed] [Google Scholar]
  33. Yu G. L., Blackburn E. H. Developmentally programmed healing of chromosomes by telomerase in Tetrahymena. Cell. 1991 Nov 15;67(4):823–832. doi: 10.1016/0092-8674(91)90077-c. [DOI] [PubMed] [Google Scholar]
  34. Zakian V. A. Telomeres: beginning to understand the end. Science. 1995 Dec 8;270(5242):1601–1607. doi: 10.1126/science.270.5242.1601. [DOI] [PubMed] [Google Scholar]
  35. Zaug A. J., Cech T. R. Analysis of the structure of Tetrahymena nuclear RNAs in vivo: telomerase RNA, the self-splicing rRNA intron, and U2 snRNA. RNA. 1995 Jun;1(4):363–374. [PMC free article] [PubMed] [Google Scholar]

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