A Personal Account of the Discovery of Telomerase · known biochemical needs of bacterial or bacteriophage DNA replication A Personal Account of the Discovery of Telomerase 553 Telomeres,

A Personal Account of the Discovery of Telomerase

Elizabeth H. BlackburnDepartment of Biochemistry and BiophysicsUniversity of California, San FranciscoSan Francisco, California 94143–2200

DID A NEW ENZYME EXIST IN CELLS THAT COULD SYNTHESIZE TELOMERIC DNA?

The molecular features of telomeric DNA were not consistent with anyof the properties expected from the models for telomere replication thathad become current in the 1970s. In the early 1980s, four lines of evidence led me to think that a new enzyme activity might work ontelomeres to elongate them. Each consisted of molecular observations ontelomeric DNA that could not be readily explained otherwise.First, telomeric GGGGTT repeat tracts on minichromosomes in the

ciliates Tetrahymena thermophila and Glaucoma chattoni were heteroge-neous in length (Blackburn and Gall 1978; Yao et al. 1979; Katzen et al.1981). Second, telomeric GGGGTT repeat tract DNA was found addedto various sequences in ciliate minichromosomes as a result of the processby which new telomeres formed on chromosomes during developmentof the somatic nucleus. This stage in the ciliate life cycle had an intrigu-ing parallel in the postfertilization phase when maize chromosomes couldheal and when Ascarid chromosomes fragmented to form new chromo-somes with stable ends. Meng-Chao Yao, working in Martin Gorovsky’slab and then Joe Gall’s lab, had found such telomeric DNA acquisition for Tetrahymena rDNA minichromosomes, and in my lab at the Universityof California at Berkeley, we had made similar observations for other,non-rDNA telomeres of the somatic nucleus (Yao et al. 1979; King andYao 1982; Blackburn et al. 1983).

Telomeres, 2nd Ed. ©2006 Cold Spring Harbor Laboratory Press 0-87969-734-2 551

Simultaneously, David Prescott’s group in Colorado had made thecorresponding observation for the telomeres of somatic minichromosomesin a ciliate of a different class, the hypotrichous ciliate Oxytricha (Boswellet al. 1982). Third, Piet Borst and his collaborators in Holland had foundthat a gene of trypanosomes encoding one of its variant surface antigens—a group of antigens that has important roles in the parasite’s ability toevade the host’s immune response—was located on a telomeric restrictionfragment. Following this gene’s restriction fragment over time, they observed that the telomeric DNA apparently gradually grew longer as trypanosome cells multiplied (Bernards et al. 1983). Fourth, sequencingby graduate student Janis Shampay in my lab (Fig. 1) had demonstratedthat yeast telomeric TG1-3 repeat DNA was added directly to the ends ofTetrahymena T2G2 repeat telomeres maintained in yeast (Szostak andBlackburn 1982; Shampay et al. 1984), as described in Chapter 1.Finally, a letter to me from Barbara McClintock in 1983 spurred

my thinking that a new activity might exist and act on telomeres. Init, she told that long ago she had identified a maize mutant that, by cytogenetic criteria, had lost the normal capacity to heal broken chromosome ends early in plant development (B. McClintock, pers.comm.; McClintock’s carbon copy of this letter is reproduced inhttp://profiles.nlm.nih.gov/LL/B/B/D/W/). I was struck by the implicationthat a fully functional telomere (“healed end”) was generated from a brokenchromosome end not just by chance, but by an active, developmentallycontrolled process. This suggested that there is a gene (which by definition

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Figure 1. (Left to right) Peter Challoner, Janis Shampay, and Carol Greider, at thetime of the research described in the text.

Telomeres, 2nd Ed. ©2006 Cold Spring Harbor Laboratory Press 0-87969-734-2

could be mutated) associated with the ability to heal. Together, I believedthat these lines of evidence were pointing to the possibility that a new enzyme activity might exist that could add telomeric DNA to DNA ends.My conviction—that an activity that could make telomeric DNA

should be sought—was further galvanized by a conversation on this topicI had with Barbara McClintock on a visit to Cold Spring Harbor Laboratory, at around the time when the other lines of evidence were accumulating. Thus, I became determined to look for such an enzyme.I was also encouraged by a more biochemically concrete suggestion of anenzyme activity in Tetrahymena that could make telomeric DNA longer.This had come from a serendipitous result obtained in 1982 by one ofmy first graduate students, Peter Challoner (Fig. 1). Peter was doing experiments in which he incubated DNA molecules in extracts of matingTetrahymena cells, prepared at the stage when telomeres might be addedto newly forming somatic chromosomal ends.The most obvious time for telomere addition, I reasoned, would be

at a particular stage after mating, when Tetrahymena was in the processof generating its hundreds of new telomeres in the newly forming somatic nucleus, and also replicating its rDNA minichromosomes intothousands of copies. If there were an enzyme that synthesized telomericDNA, this would be a time when great demands would be placed on it,and it presumably would be ramped up to high levels. In addition,through the work of David Nanney, Peter Bruns, Ed Orias, Sally Allen,and their colleagues in the ciliate genetics field, Tetrahymena had beenmade into a powerful experimental system in which the whole cell population could be synchronized for studying developmental processesfollowing mating. We already knew when the rDNA was first amplifiedand when the DNA fragmentation that leads to new telomere synthesiswas occurring. In contrast to the need to wait for many generations before one could observe yeast transformants, these aspects of ciliate cellreproduction took place over approximately 1 hour. That was the time tocatch Tetrahymena cells and make extracts to look for any of this hypothetical activity.In such extracts, Peter Challoner had incubated 1.6-kb “mini-rDNA”

molecules he had constructed in vitro, which had tracts of CCCCAA/GGGGTT repeats at both ends. He adapted a reaction mix that Tom Cechand colleagues (Zaug and Cech 1980) had used to study rRNA tran-scription and processing in vitro (and with which they discoveredself-splicing RNA). Peter had wanted to see if he could persuade thesemini-rDNAs to turn into palindromic molecules. Drawing from theknown biochemical needs of bacterial or bacteriophage DNA replication

A Personal Account of the Discovery of Telomerase 553


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Figure 2. Figure and text reprinted, with permission, from Peter B. Challoner’sthesis (Challoner 1984).


in vitro and the knowledge that DNA replication involves discontinuoussynthesis by a primase-initiated process, in which short RNA primers areused to start each growing DNA polynucleotide chain, he included allfour dNTPs as well as rNTPs. Although he did not find palindromic molecules, intriguingly he did note that after 180 minutes of incubation,the terminal restriction fragment bands (which ended in a tract of CCCCAA/GGGGTT repeats) got fuzzier-looking when fractionated bygel electrophoresis; thus, they had become heterogeneous during thecourse of the incubation with mated cell extract (Fig. 2). They also migrated slower, as though they were an average of about 100 bp longerthan the original input telomeric fragments (Challoner 1984). Althoughnot unambiguous evidence of addition of telomeric DNA, a lead hadnonetheless been inadvertently provided by these seemingly unrelated experiments.I decided first to look for more direct evidence that telomeric repeats

were added to DNA fragments in such extracts. It was logical to presumethat a still-hypothetical telomere-addition activity was acting in a settingin which DNA synthesis was occurring, because both DNA strands of the


Figure 2. (Continued)


telomeres would ultimately have to be made. Also, DNA replication needsenergy, so I included an ATP-regenerating enzyme system as well as allfour dNTPs and rNTPs. Finally, my idea was that telomeric DNA wouldbe best added to preexisting telomeres, but perhaps also to the nontelomericends, because they are the substrates for telomere addition during somatic nuclear development. I decided to cover all bases by starting witha mixture of DNA restriction fragments that had both a telomeric endand various nontelomeric ends as substrates.I incubated extracts made from freshly mated Tetrahymena cells

with the mixture of DNA restriction fragment substrates, and otherdNTPs, rNTPs, Mg�� ion and other salts, buffers, and the ATP-regenerating enzyme and its substrate. Then, after different times of incubation, the DNA products were purified, fractionated by gel elec-trophoresis, and analyzed by Southern blotting with probes for theinput restriction fragments and, most critically, with a probe for telomericCCCCAA repeats. Two things emerged. Several of the fragments acquired CCCCAA hybridization. Even better, at least one new band thathybridized with the CCCCAA probe appeared. It was fuzzy, with an appearance reminiscent of telomeric DNA fragments, just as one wouldexpect if newly added telomeric DNA were being added to a cleavage orrearrangement product of a restriction fragment that I had put into themix. Its intensity increased with time of incubation with the extract, con-sistent with enzyme action in a biochemical experiment. The next monthI showed the resulting autoradiograms in a slide at the April 1984 UCLAKeystone Symposium on Genome Rearrangements in Steamboat Springs,Colorado (Fig. 3).In April 1984, Carol Greider joined the lab as a graduate student, and

to my delight, she was willing and eager to start work on this project(see Fig. 1). As recounted more fully elsewhere (Greider and Blackburn2004), the assay for a telomere synthesis enzyme had to be greatly stream-lined by laborious trial and error. It was necessary to identify the optimalDNA substrate for a potential telomere synthesis enzyme and to test otherTetrahymena extract and reaction conditions. The innovation of usingDNA sequencing-type gels to resolve the reaction products greatlyhelped, as well as including a radiolabeled dNTP, ultimately [32P]dGTP,as the labeled nucleoside triphosphate substrate.December of 1984 brought the clearest sign to date of a potential

telomerase activity (although it was not yet named telomerase, a namecoined by Claire Wyman, who was another graduate student in the lab).A synthetic DNA oligonucleotide of the telomere G-strand TTGGGG wastested. Such a DNA primer substrate could now be added at micromolar

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concentrations, rather than the nanomolar concentrations which had beenthe highest concentrations that could be achieved with restriction frag-ments. This (TTGGGG)4 oligomer primed synthesis of DNA products thatformed a repeating pattern, with an apparent six-base periodicity visible,extending up to the top of the gel—precisely what was expected if atelomere synthesis enzyme had been acting in the assay (shown schemat-ically in Fig. 4A). Furthermore, the activity was stronger when the extractswere made from mated cells during the period when telomeric repeats wereadded to the newly fragmenting chromosomes, although repeat synthesiswas also clearly evident in vegetatively growing cells.Several months of work ruled out all the potential artifacts we could

think of that might have been potential confounding explanations forthese novel findings and established that the added repeats were indeedof the sequence (GGGGTT)n. In addition, a yeast telomeric sequenceoligonucleotide worked well as a substrate for a Tetrahymena extract activity in vitro (Fig. 4B). As described in Chapter 1, we knew thatin vivo, yeast added yeast repeats to linear minichromosomes carrying heterologous (ciliate) telomeres. We did not have yeast telomerase activity


Figure 3. Early experiments by E.H.B. incubating restriction fragments withTetrahymena extracts, showing acquisition of CCCCAA repeat hybridization. FragmentA was a DNA fragment from a bacterial plasmid.


in vitro then (this had to await my postdoctoral fellow Marita Cohn’ssubsequent development of an activity assay for this species’ telomerase[Cohn and Blackburn 1995]). Therefore, we devised the converse in vitroreaction: A yeast sequence DNA oligomer primer was added to theTetrahymena extract. Strikingly, this TG-rich yeast sequence oligonu-cleotide was elongated, and tandem TTGGGG hexanucleotide repeatswere added, just as they were to the TTGGGG repeat primers. We likedthe symmetry of this test of the idea that the basis for the functional conservation of telomeric DNA in vivo was that yeast telomerase hadelongated the foreign telomere, extending it with yeast repeats.The yeast primer experiment had another bonus. Whereas a

Tetrahymena oligonucleotide primer ending in four Gs at its 3� end was

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Figure 4. The Tetrahymena telomerase reaction, shown schematically. (A) Assayusing a Tetrahymena telomeric sequence primer. (B) Assay using a yeast telomericsequence primer (Greider and Blackburn 1985, 1987).


elongated starting with the apparent addition of two Ts, the yeast primer,which happened to end in three Gs, but had the same length as the all-Tetrahymena sequence primer, primed a product that started with anadded G, then two Ts, making a product pattern that was offset by onebase (Fig. 4). This observation was critical in the recognition that the sequence at the 3� end of the oligonucleotide determined which bases inthe TTGGGG repeat would be added and pointed to what we eventuallyfound was the RNA template in the RNA moiety of telomerase.In the meantime, the discovery of this new telomeric DNA-

synthesizing activity was published in December 1985 (Greider andBlackburn 1985). Excitingly, RNase inactivated the activity. During thenext 2 years, the activity was partially purified and its reaction propertieswere extensively characterized (Greider and Blackburn 1987). However,the RNA was a low-abundance species in the cell and difficult to obtain,and although partial sequence information was obtained by direct RNAsequencing, cloning the gene that encoded the RNA was not accom-plished until Carol had left Berkeley and become a Cold Spring HarborLaboratory Fellow. By 1989, the presence of a 9-nucleotide sequence,5�-CAACCCCAA-3�, within the RNA had suggested a template mechanism,which was confirmed by biochemical experiments (Fig. 5) (Greider andBlackburn 1989).

Tetrahymena telomerase, we found, added tandem repeats of theTetrahymena telomeric DNA sequence, TTGGGG, onto the 3� end of anyG-rich strand telomeric oligonucleotide primer, independently of an exogenously added nucleic acid template, from nearly all eukaryotestested (Greider and Blackburn 1985, 1987; Blackburn et al. 1989). Astelomerase synthesized only the G-rich strand of telomeres, we proposedthat synthesis of the complementary C-rich strand is carried out by primase-polymerase-mediated discontinuous synthesis, typical of semiconservativeDNA replication mechanisms, using the extended G-rich strand as a template (Shampay et al. 1984; Greider and Blackburn 1985).Similar findings were subsequently made for telomerase activities

from the ciliates Oxytricha and Euplotes by Dorothy Shippen-Lentz in mylab and by Alan Zahler in David Prescott’s lab, respectively (Zahler andPrescott 1988; Shippen-Lentz and Blackburn 1989), and from humancells by Gregg Morin in Joan Steitz’s lab (Morin 1989). Each telomerase synthesized its own species-specific sequence—GGGGTTTT repeats forthe hypotrichous ciliates and AGGGTT for human—and had primer-recognition properties similar to those we had found for the Tetrahymenatelomerase. Like the ciliate telomerase activities, the human cell activitywas also ribonuclease-sensitive (Morin 1989), suggesting that it too was



a ribonucleoprotein. Identification of telomerase activity in human cells,in particular, highlighted the generality of this enzyme in eukaryotes.The RNA moiety of the telomerase of the ciliate Euplotes was quickly

identified (Shippen-Lentz and Blackburn 1990). It too had a counterpartof the CAACCCCAA sequence of Tetrahymena telomerase RNA (Greiderand Blackburn 1989), 5�-CAAAACCCCAAAA-3�, which Dorothy Shippenshowed by functional analyses in vitro was the templating domain forsynthesis of GGGGTTTT repeats (Shippen-Lentz and Blackburn 1990),the telomeric sequence of Euplotes.These findings established telomerase as a widespread cellular reverse

transcriptase, unusual in synthesizing short repeats and in carrying itsown internal RNA template for DNA synthesis. The presence of such anRNA component prompted the speculation that telomerase may be a relic

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Figure 5. A model for the action of the Tetrahymena telomerase. (Reprinted, withpermission, from Greider and Blackburn 1989 [©Nature Publishing Group].)


from the time of the evolutionary transition from RNA to DNA genomes(Blackburn 1990). The analogy was drawn to the ability of the self-splicingribozyme of Tetrahymena to polymerize RNA, using an internal RNAtemplate (Zaug and Cech 1986). This suggested the possibility that telomerase is a relic of an RNA replicase ribozyme that became able tosynthesize DNA. In this model, as protein components took over this catalytic role, they gave rise to the purely protein reverse transcriptasesand DNA-dependent DNA polymerases found in modern eukaryotes andprokaryotes, but in the case of telomerase, the template RNA was retainedand is still used to maintain the ends of eukaryotic chromosomes.

DEMONSTRATION OF THE ROLE OF TELOMERASE IN VIVO

We now had good evidence that the enzyme telomerase could synthesizethe G-rich strand of telomeric DNA. Proof that the 5�-CAACCCCAA-3�sequence in the Tetrahymena telomerase RNA gene is the template fortelomere synthesis came from site-directed mutagenesis of this sequenceand overexpression of the mutated gene in Tetrahymena cells. These exper -iments, which established the in vivo role of telomerase, were done byGuo-Liang Yu in my lab, with help from his fellow graduate studentsLaura Attardi and John Bradley. Guo-Liang injected gene constructs containing template mutant versions of the telomerase RNA gene (TER),borne on a special vector system that he had devised for Tetrahymenatransformation (Yu et al. 1988). Altered telomere repeats specified by themutant gene appeared in the telomeres that he cloned out of the trans-formant cells. This proved that telomerase is indeed the cellular reversetranscriptase enzyme that synthesizes telomeres in cells by copying itsown internal RNA template within the TER moiety of the enzyme complex. Importantly, one template mutation caused failure to add anyof the predicted sequence DNA onto telomeric ends. Instead, the telomeresprogressively shortened as the cells grew for about 20 to 25 more cell divisions, and then the cells senesced. This showed that interference withthe telomerase function in the otherwise effectively immortal Tetrahymenacells could limit their life span. It established the first role for telomerasein cellular life span and the first indication that its presence was necessaryfor the replicative immortality of cells (Yu et al. 1990).Telomerase action, then, could explain how replication of the chro-

mosomal DNA could continue without the progressive loss of terminalsequences predicted to result from normal semiconservative DNA replication; addition of telomeric DNA to the chromosomal ends bytelomerase could counterbalance this terminal DNA attrition. Evidence



for such attrition had been previously seen for broken Drosophila chromosomes that lost terminal DNA sequences progressively (Biessmannand Mason 1988; Levis 1989), and in the dynamic behavior of individualSaccharomyces cerevisiae telomeres in vivo (Shampay and Blackburn 1988).The senescence phenotypes of the Tetrahymena telomerase RNA mutants(Yu et al. 1990) provided an experimental basis for the suggestion thatcell senescence in other organisms could be related to similar attrition oftelomeres.

ACKNOWLEDGMENTS

I am grateful for support over the years from many members of my laboratory who have contributed to the work cited here. I appreciativelyacknowledge funding support from the National Institutes of Healthsince 1978 and more recently from the Steven and Michele Kirsch Foundation and the Osher Foundation.

REFERENCES

Bernards A., Michels P.A., Lincke C.R., and Borst P. 1983. Growth of chromosome endsin multiplying trypanosomes. Nature 303: 592–597.

Biessmann H. and Mason J.M. 1988. Progressive loss of DNA sequences from terminalchromosome deficiencies in Drosophila melanogaster. EMBO J. 7: 1081–1086.

Blackburn E.H. 1990. Telomeres and their synthesis. Science 249: 489–490.Blackburn E.H. and Gall J.G. 1978. A tandemly repeated sequence at the termini of theextrachromosomal ribosomal RNA genes in Tetrahymena. J. Mol. Biol. 120: 33–53.

Blackburn E.H., Greider C.W., Henderson E., Lee M.S., Shampay J., and Shippen-Lentz D.1989. Recognition and elongation of telomeres by telomerase. Genome 31: 553–560.

Blackburn E.H., Budarf M.L., Challoner P.B., Cherry J.M., Howard E.A., Katzen A.L., PanW.C., and Ryan T. 1983. DNA termini in ciliate macronuclei. Cold Spring Harbor Symp.Quant. Biol. 47: 1195–1207.

Boswell R.E., Klobutcher L.A., and Prescott D.M. 1982. Inverted terminal repeats areadded to genes during macronuclear development in Oxytricha nova. Proc. Natl. Acad.Sci. 79: 3255–3259.

Challoner P.B. 1984. “The organization of the ribosomal RNA genes of Tetrahymenids.”Ph. D. thesis, University of California, Berkeley.

Cohn M. and Blackburn E.H. 1995. Telomerase in yeast. Science 269: 396–400.Greider C.W. and Blackburn E.H. 1985. Identification of a specific telomere terminaltransferase activity in Tetrahymena extracts. Cell 43: 405–413.

———. 1987. The telomere terminal transferase of Tetrahymena is a ribonucleoproteinenzyme with two kinds of primer specificity. Cell 51: 887–898.

———. 1989. A telomeric sequence in the RNA of Tetrahymena telomerase required fortelomere repeat synthesis. Nature 337: 331–337.

———. 2004. Tracking telomerase. Cell 116: S83–S86, S87.

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Katzen A.L., Cann G.M., and Blackburn E.H. 1981. Sequence-specific fragmentation ofmacronuclear DNA in a holotrichous ciliate. Cell 24: 313–320.

King B.O. and Yao M.C. 1982. Tandemly repeated hexanucleotide at Tetrahymena rDNAfree end is generated from a single copy during development. Cell 31: 177–182.

Levis R.W. 1989. Viable deletions of a telomere from a Drosophila chromosome. Cell 58:791–801.

Morin G.B. 1989. The human telomere terminal transferase enzyme is a ribonucleoproteinthat synthesizes TTAGGG repeats. Cell 59: 521–529.

Shampay J. and Blackburn E.H. 1988. Generation of telomere-length heterogeneity in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. 85: 534–538.

Shampay J., Szostak J.W., and Blackburn E.H. 1984. DNA sequences of telomeres maintainedin yeast. Nature 310: 154–157.

Shippen-Lentz D. and Blackburn E.H. 1989. Telomere terminal transferase activity fromEuplotes crassus adds large numbers of TTTTGGGG repeats onto telomeric primers.Mol. Cell. Biol. 9: 2761–2764.

———. 1990. Functional evidence for an RNA template in telomerase. Science 247:546–552.

Szostak J.W. and Blackburn E.H. 1982. Cloning yeast telomeres on linear plasmid vectors.Cell 29: 245–255.

Yao M.C., Blackburn E., and Gall J.G. 1979. Amplification of the rRNA genes in Tetrahymena. Cold Spring Harbor Symp. Quant. Biol. 43: 1293–1296.

Yu G.L., Hasson M., and Blackburn E.H. 1988. Circular ribosomal DNA plasmids transform Tetrahymena thermophila by homologous recombination with endogenousmacronuclear ribosomal DNA. Proc. Natl. Acad. Sci. 85: 5151–5155.

Yu G.L., Bradley J.D., Attardi L.D., and Blackburn E.H. 1990. In vivo alteration of telomeresequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature344: 126–132.

Zahler A.M. and Prescott D.M. 1988. Telomere terminal transferase activity in the hypotrichous ciliate Oxytricha nova and a model for replication of the ends of linearDNA molecules. Nucleic Acids Res. 16: 6953–6972.

Zaug A.J. and Cech T.R. 1980. In vitro splicing of the ribosomal RNA precursor in nucleiof Tetrahymena. Cell 19: 331–338.

———. 1986. The intervening sequence RNA of Tetrahymena is an enzyme. Science 231:470–475.



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A Personal Account of the Discovery of Telomerase · known biochemical needs of bacterial or bacteriophage DNA replication A Personal Account of the Discovery of Telomerase 553 Telomeres,