CONSERVATION OF TELOMERE FUNCTION AND THE
The concept of a healing factor for chromosome ends or telomeres was evoked80 years ago owing to the recognition by Barbara McClintock and Hermann Mullerthat the natural end of a linear intact chromosome differs from that of a brokenchromosome. Using fruit flies and corn as model organisms, they observed thatnatural chromosome ends, unlike broken ones, never fuse (McClintock, 1931;Muller, 1938). McClintock reported that during cell division in the embryo a brokenchromosome can permanently heal to acquire the functions of a natural chromosomeend (McClintock, 1939). One of the healing factors or mechanisms was identified50 years later, in 1985, by Carol Greider and Elizabeth Blackburn, in the ciliatedprotozoan, Tetrahymena thermophila, and named telomere terminal transferase ortelomerase (Greider and Blackburn, 1985).
While the function and essential nature of telomeres is conserved among eukar-
yotes, the DNA sequences, associated proteins and structures at telomeres, and modesof telomere maintenance vary. Recombination-based mechanisms of telomere main-
tenance have been reported in telomerase-negative immortalized alternative length-ening of telomere (ALT) human cancer cells and upon telomerase gene deletion inyeast, known as Type I, Type II, and heterochromatin amplification-mediated andtelomerase-independent (HAATI) (see Chapters 7, 10, 11, and subsequent sections ofthis chapter) (Cesare and Reddel, 2010; Jain et al., 2010). Recombination can occur
Telomerases: Chemistry, Biology, and Clinical Applications, First Edition.
Edited by Neal F. Lue and Chantal Autexier.
Ó 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.
between telomeric and telomeric, subtelomeric or heterochromatin sequences, andmay or may not lead to telomere elongation. In Drosophila melanogaster, one of thetwo organisms in which the special function of chromosome ends first becameevident, retrotransposons and specialized terminin proteins, which are structurallydistinct from the typical telomere nucleoprotein complex, are nevertheless capable ofsupplying the capping function at chromosome ends (see Chapters 7, 10, 11, andsubsequent sections of this chapter) (Mason et al., 2008; Raffa et al., 2009, 2010).
However, the most common mechanism for telomere maintenance is the enzyme
telomerase, which is almost universally conserved and active in eukaryotes includingciliated protozoa, yeasts, mammals, and plants (see Chapters 2 and 3) (Autexier andLue, 2006). Prior to the discovery of telomerase, the first telomere sequences had beenidentified in T. thermophila, by Elizabeth Blackburn and Joseph Gall, to consist ofrepeats of the hexanucleotide TTGGGG (Blackburn and Gall, 1978). Most eukaryoteswhich maintain telomeres by telomerase possess G-rich sequences at their chromo-some ends (see Chapter 7). The search for an enzyme that can maintain telomeres wasspurred by the recognition of the end replication problem by James Watson andAlexey Olovnikov in the 1970s (see Chapters 7 and 10) (Olovnikov, 1973; Wat-son, 1972). Based on the properties of the conventional DNA replication machinery,they postulated that DNA at chromosome ends could not be completely replicated andthat terminal sequences would be lost at each cell division. The identification of anenzymatic activity that adds G-rich DNA sequences to synthetic telomeric oligonu-cleotides in vitro led to the discovery of the first cellular reverse transcriptase, aribonucleoprotein (RNP) composed of both RNA and protein (Greider and Black-burn, 1985, 1987, 1989). Two factors were critical to the development of the activityassay: the use of synthetic oligonucleotides with G-rich telomere-like sequences assubstrates and the preparation of extracts from Tetrahymena as the source of enzyme.
The single-stranded G-rich oligonucleotides mimic the natural substrates for telo-merase and can be supplied at high concentrations to drive the reaction (Hendersonand Blackburn, 1989; McElligott and Wellinger, 1997). In addition, the enzyme isabundant in T. thermophila due to the large number of chromosome ends that aregenerated and which must be stabilized following the chromosome fragmentation andamplification that occurs during the development of the transcriptionally activesomatic macronucleus in this organism (Turkewitz et al., 2002).
The importance of telomere synthesis by telomerase is highlighted by the discovery
that this mode of replication at DNA ends is evolutionary conserved. Linear DNAexogenously introduced into yeast cells is typically degraded or rearranged. However,Elizabeth Blackburn and Jack Szostak performed what they later described as anoutlandish experiment. They attached T. thermophila telomeric sequences to the endsof a linear DNA prior to its introduction into yeast and discovered that the DNA wasmaintained in a stable linear form due to the addition of yeast telomeric sequences tothe T. thermophila sequences by a yeast cellular machinery (Blackburn et al., 2006;Szostak and Blackburn, 1982). Moreover, when telomerase activity was identified,Carol Greider and Elizabeth Blackburn also discovered that T. thermophila can add T.
thermophila telomeric sequences to a yeast telomeric substrate in vitro, emphasizingthe evolutionarily conserved nature of telomere synthesis by telomerase (Blackburn
THE DISCOVERY OF THE TWO MINIMAL TELOMERASE COMPONENTS
et al., 2006; Greider and Blackburn, 1985). For these pioneering and fundamentaldiscoveries, Blackburn, Greider, and Szostak were awarded the Nobel Prize inPhysiology and Medicine in 2009.
THE DISCOVERY OF THE TWO MINIMAL TELOMERASE
The RNA component of telomerase (referred to as TR or TER in general) contains ashort template region, which is repeatedly reverse transcribed into its complementarytelomeric DNA sequence (Table 1.1). Initial proof for this function was elucidatedusing in vitro experiments in which an oligonucleotide complementary to thetemplate region of the T. thermophila telomerase RNA was found to inhibittelomerase activity, as did the cleavage of the DNA–RNA hybrid at the RNA templateregion by RNase H (Greider and Blackburn, 1989). In T. thermophila cells,expression of mutant telomerase RNAs leads to the synthesis of the correspondinglymutated telomeric sequences at chromosome ends, confirming the function oftelomerase in telomere synthesis (Yu et al., 1990). Phenotypes elicited by thesynthesis of mutated telomere sequences include altered telomere length homeosta-sis, impaired cell division, severe delay or block in completing mitotic anaphase, andsenescence (Kirk et al., 1997; Yu et al., 1990). These phenotypes underscore thecritical nature of the sequence at the telomeres and the essential nature of telomeremaintenance for cell survival. Telomerase RNAs from other eukaryotes wereidentified using biochemical and genetic approaches, however, some RNAs, forexample, those from Schizosaccharomyces pombe and Arabidopsis thaliana, haveonly been recently discovered largely due to size divergence and weak primarysequence conservation (see Chapter 2) (Cifuentes-Rojas et al., 2011; Leonardiet al., 2008). Despite the large size variation of the telomerase RNAs (ranging from$150 nucleotides (nt) in ciliates to over 1300 nt in yeasts), the secondary structures oftelomerase RNAs are remarkably well conserved (see Chapter 2).
TABLE 1.1 Nomenclature for the Telomerase Catalytic and RNA Subunits in VariousOrganisms
The search for the protein component of telomerase (TERT) proved as daunting as
that of the RNA component. Eventually in 1997, sustained efforts by severallaboratories culminated in the identification of TERTs from multiple organisms,including Saccharomyces cerevisiae, Euplotes aediculatus, and human (originallynamed hTRT, hEST2, TP2, and hTCS1 in human) (Counter et al., 1997; Harringtonet al., 1997; Kilian et al., 1997; Lingner et al., 1997b; Meyerson et al., 1997;Nakamura et al., 1997) (Table 1.1). The S. cerevisiae TERT gene had, in fact, beenidentified in 1996 as EST2 (Ever Shorter Telomeres) in a genetic screen for mutantscausing senescence and shortening of telomere length (Lendvay et al., 1996). Geneticand biochemical analyses revealed that conserved amino acids within the reversetranscriptase motifs present in TERTare essential for telomerase activity and telomeresynthesis both in vitro and in vivo (Beattie et al., 1998; Counter et al., 1997;Harrington et al., 1997; Nakamura et al., 1997; Weinrich et al., 1997). More recently,several crystal structures of TERT or TERT domains from various organisms haveprovided a framework for interpreting existing biochemical and genetic data whileallowing further targeted experimentation on this protein (Gillis et al., 2008; Jacobset al., 2006; Mitchell et al., 2010; Rouda and Skordalakes, 2007) (see Chapter 2).
Expression of human TERT (hTERT) mRNA correlated with telomerase activity incell lines (the telomerase RNA component is constitutively expressed), and was foundto be upregulated in tumor cells and during immortalization. Hence, hTERT isbelieved to be the limiting factor for telomerase activity and to be regulated largelythrough transcription (see Chapter 5) (Feng et al., 1995; Meyerson et al., 1997). Theextent of regulation via posttranslational modification of telomerase by phosphor-ylation and ubiquitination is currently unclear (see Chapter 6). Nonetheless, inac-tivation of the c-Abl kinase leads to increased telomerase activity and telomerelengths, while overexpression or downregulation of the ubiquitin ligases Hdm2 andMKN1 alters telomerase activity, telomere lengths, and/or cellular resistance toapoptosis (Kharbanda et al., 2000; Kim et al., 2005; Oh et al., 2010).
Another relatively unexplored and poorly characterized aspect of telomerase
regulation is the potential contribution of alternatively spliced TERT variants (seeChapter 5). Analysis of the hTERT gene revealed the potential for complex splicingpatterns that may reflect a specific aspect of telomerase regulation in proliferation,differentiation, and apoptosis (Kilian et al., 1997; Sykorova and Fajkus, 2009). Anumber of alternatively-spliced TERT mRNAs have been identified in vertebrates andplants, yet their role in telomere maintenance and cell survival is poorly characterized.
In human development, the specific expression of hTERT splice variants that arepredicted to encode catalytically-defective telomerases correlates with telomereshortening, suggesting that these transcripts may have important physiological roles(Ulaner et al., 2001).
TELOMERASE BEYOND THE MINIMAL COMPONENTS:
TERT and TR are sufficient to form an active telomerase enzyme when expressedin a rabbit reticulocyte lysate-based transcription and translation system in vitro
TELOMERASE BEYOND THE MINIMAL COMPONENTS: ASSOCIATED PROTEINS
(Collins, 2006; Collins and Gandhi, 1998; Weinrich et al., 1997). However, a largenumber of telomerase-associated proteins have been identified in ciliates, yeast, andvertebrates (Autexier and Lue, 2006) (see Chapter 4). The proteins vary greatlybetween the species and very few are common to all telomerases. While many havebeen identified as components of a telomerase holoenzyme, some may be associatedonly transiently with the complex to regulate telomerase assembly and stability,trafficking, localization, posttranslational modification, and recruitment to andactivity at the telomere. Consequently, it is difficult to determine whether theholoenzyme has been described in its entirety. A molecular mass of $270 or
$500 kDa was determined by chromatography of endogenously assembled ciliatetelomerases using glycerol gradient sedimentation or gel filtration, respectively(Collins and Greider, 1993; Wang and Blackburn, 1997; Witkin and Collins, 2004).
Human and yeast telomerase complexes appear larger (0.6 MDa for yeast,0.65–2 MDa for human) possibly due to the larger size of RNAs in these organismsand their ability to act as scaffolds to build complex RNPs (Fu and Collins, 2007;Lingner et al., 1997a; Lustig, 2004; Venteicher et al., 2009).
Adding to the challenges of deciphering the components of the holoenzyme are the
difficulties encountered in the purification of telomerase protein complexes, typicallyin very low abundance in nonciliate organisms. Initial purification strategies based onthe use of template-complementary oligonucleotide hybridization in ciliates andhuman led to disruption of ribonucleoprotein assembly (Lingner and Cech, 1996;Schnapp et al., 1998). Recently, more gentle tandem affinity purification strategies, asfirst described by the group of Kathleen Collins, have yielded a more complete pictureof telomerase RNP organization (Fu and Collins, 2007; Venteicher et al., 2008, 2009;Witkin and Collins, 2004).
Telomerase-associated proteins have been best characterized in a single-celled
eukaryotes (Fu and Collins, 2007). The ciliate T. thermophila is a good model systemowing to its cellular structural and functional complexity, arguably comparable to thatof metazoans (Turkewitz et al., 2002). Although many of the fundamental discoveriesabout telomerase and telomere biology were made using T. thermophila, thisorganism’s telomerase appears to have a unique RNP biogenesis pathway thatinvolves the telomerase-specific proteins p65, p45, p75, and p20 (O’Connor andCollins, 2006; Witkin and Collins, 2004; Witkin et al., 2007). More recently, threeadditional holoenzyme proteins were identified, p19, p50, and p82 (Min andCollins, 2009). The p75, p45, and p19 form a telomere adaptor subcomplex, TASC,whose recruitment to the core enzyme (p65, TERT, and TER) is regulated by the p50subunit. The p82 subunit is a Replication Protein A (RPA)-related sequence-specificDNA-binding protein, which confers high repeat addition processivity to the telo-merase holoenzyme. The RNP biogenesis pathways of yeast and human telomeraseemploy a set of proteins shared with more abundant RNPs (Collins, 2006). Proteinsinvolved in yeast telomerase RNA processing, stability, trafficking, and biogenesisinclude importin B, which is involved in nuclear import of mRNA binding proteins, aswell as proteins involved in spliceosomal small nuclear (sn) RNP processing (Chaponet al., 1997; Ferrezuelo et al., 2002; Seto et al., 1999). Proteins involved in humantelomerase RNA processing and stability, and in RNP trafficking and biogenesisinclude proteins of H/ACA small nucleolar (sno) and small Cajal body (sca) CAB
box-containing RNPs, such as dyskerin, NHP2, NOP10, GAR1, and TCAB1(telomerase Cajal body protein 1), the chaperone proteins p23 and hsp90, theAAA þ ATPases pontin and reptin, the nucleolar acetyltransferase NAT10, and thenucleolar GTPase GNL3L (Cohen et al., 2007; Collins, 2008; Fu and Collins, 2007;Mitchell et al., 1999; Venteicher et al., 2008, 2009). Some of these proteins havebeen identified using tandem affinity purifications, and it has been proposed thattelomerase-associated proteins present at substoichiometric levels might be regu-latory as opposed to H/ACA proteins and hTERT, which are required for biologicalstability and catalytic activity, respectively (Fu and Collins, 2007).
In addition to telomeric proteins, which aid in the recruitment of telomerase to the
telomere (see below), a number of other proteins have been identified which have beenimplicated in the localization and recruitment of telomerase to the nucleus and to thetelomere. In yeast, these include Est1 and the Ku70/80 heterodimer, while in humanthe 14-3-3 regulator of intracellular protein localization, the telomerase inhibitorPinX1, and the heterogenous nuclear RNP family of proteins may regulate locali-zation of telomerase to the nucleus or recruitment to the telomere (Banik andCounter, 2004; Collins, 2006, 2008; Fisher et al., 2004; Ford et al., 2000; Fu andCollins, 2007; Hughes et al., 2000; LaBranche et al., 1998; Seimiya et al., 2000;Zappulla and Cech, 2004; Zhou and Lu, 2001).
REGULATION OF TELOMERASE BY TELOMERIC PROTEINS
Interestingly, the relationship between telomeres and telomerase extends beyond therole of telomeres as telomerase substrates (see Chapter 7). While disruption ofnumerous proteins leads to alterations of telomere homeostasis in mammalian cells,including many proteins involved in the maintenance of genomic integrity (e.g.,proteins affecting DNA replication, repair, recombination, and the DNA damageresponse), a six-protein complex known as the shelterin complex (TRF1, TRF2,hRAP1, TPP1, POT1, and TIN2), are directly responsible for the protection ofmammalian telomeres (d’Adda de Fagagna, 2008; Palm and de Lange, 2008;Slijepcevic, 2008). The shelterin proteins mediate the formation of a t-loop structureat telomeres, which prevents the recognition of the end of the chromosome as a DNAdouble-strand break and precludes engagement of a DNA damage response. Reg-ulation of telomerase by telomere binding proteins or proteins that associate withtelomeres can either be indirect or direct. Proteins that affect access of telomerase totelomeres, including proteins implicated in the generation of the single-stranded G-rich telomere overhang, can be viewed as indirect regulators, while those that recruittelomerase to the telomere and/or modulate telomerase activity are direct regulators.
A number of proteins, for example, budding yeast Rif1/2 and mammalian TRF1 andTRF2, regulate telomerase by altering telomere structure and/or length and byincreasing telomerase accessibility (see Chapter 7). TPP1 regulates telomeraserecruitment to the telomeres and, in concert with Pot1, also regulates activity oftelomerase at the telomere (Abreu et al., 2010; Latrick and Cech, 2010; Wanget al., 2007; Xin et al., 2007; Zaug et al., 2010). Similarly, Cdc13, one of the telomeric
TELOMERASE, TELOMERE MAINTENANCE, CANCER, AND AGING
proteins in budding yeast, participates in the recruitment of telomerase to telomeres,and evidently activates the enzyme as well (Pennock et al., 2001). In fission yeast,Tpz1 (orthologue of the mammalian TPP1) and the associated factors Poz1, Pot1, andCcq1, are also implicated in telomerase recruitment (Miyoshi et al., 2008; Tomita andCooper, 2008). Interestingly, TPP1 is a homologue of ciliate TEBP-b, one of the firsttelomere binding proteins to be identified (Price and Cech, 1989; Xin et al., 2007). Theinteraction between TPP1/TEBPb and telomerase appears to be one of the very fewconserved interactions between telomeric proteins and telomerase.
Another potentially significant regulator of telomerase at telomeres is the recently
discovered telomeric repeat containing RNA (TERRA). These noncoding RNAs aredetected at yeast, mammalian, and plant telomeres, and are transcribed from thesubtelomeric regions to the chromosome ends (Azzalin et al., 2007; Feuerhahnet al., 2010; Schoeftner and Blasco, 2008; Vrbsky et al., 2010) (see Chapter 6).
Interestingly in A. thaliana, antisense telomeric transcripts (ARRET) are alsoreported (Vrbsky et al., 2010). One of the postulated roles for TERRA for whichevidence is accumulating, is in the regulation of telomerase. TERRA can bind totelomerase and act as a potent competitive inhibitor for telomeric DNA (Redonet al., 2010; Schoeftner and Blasco, 2008). Increased levels of TERRA are alsocorrelated with shorter telomeres (Luke et al., 2008).
TELOMERASE, TELOMERE MAINTENANCE, CANCER, AND
In 1989, shortly following the identification of telomerase activity in the humancell line—HeLa, numerous studies were performed to assess the status of telo-merase activity and of telomere length in various types of human cells (Morin,1989). The telomere hypothesis of cellular aging and immortalization emerged as aconsequence of the correlation found in these studies between telomere lengthand telomerase activity in human cells (Harley, 1991) (see Chapter 10). Briefly,because telomerase was active in immortal, transformed human cells and in tumorcell lines, but not in normal somatic cells, and because telomere lengths weremaintained with increasing numbers of cell division in the former cells, but not inthe latter cells, it was postulated that telomere length serves as a mitotic clock innormal human somatic cells. Telomere shortening in normal human somatic cellsoccurs in a cell division-dependent fashion, eventually triggering replicativesenescence and exit from the cell cycle. The presence of telomerase and themaintenance of telomere length in immortal, transformed human cells and in tumorcell lines support the concept that telomere maintenance is a key requirement forunlimited replication of tumor cells (Hanahan and Weinberg, 2000; Harley, 1991).
In 1997, a survey of more than 3500 tumor and control samples showed thattelomerase is detected in approximately 85% of cancers, but is absent or weaklyexpressed in primary cells (Shay and Bacchetti, 1997). This and other studies, aswell as the telomere hypothesis for cellular aging and immortalization led totestable predictions, and to the identification of telomerase as an attractive targetfor anticancer therapy.
Addressing if telomere shortening is a cell division clock that limits cellular
lifespan became possible following the identification of hTERT. Elegant experimentsby the groups of Woodring Wright and Jerry Shay demonstrated that expression ofhTERT in normal human fibroblast cells with limited lifespan led to the induction oftelomerase activity, telomere maintenance, and extension of lifespan (Bodnaret al., 1998; Counter et al., 1998; Vaziri and Benchimol, 1998). Importantly, thecells did not adopt characteristics of cancer cells (Jiang et al., 1999; Moraleset al., 1999). It was noted however, that telomerase activation was not sufficient toimmortalize some normal human cell types, suggesting that other factors besidestelomere length, for example, the levels of the tumor suppressor p16, contributed toreplicative senescence in human cells (Kiyono et al., 1998). Several pioneering studiesaddressed the role of telomerase in tumorigenesis, and demonstrated that telomeraseactivation is essential but not sufficient for transformation of human cells (Hahnet al., 1999, 2002). In these experiments, normal human fibroblasts were converted totumorigenic cells capable of forming tumors in immunodeficient mice. This conver-sion required the expression of hTERT and alterations in key cellular genes includingthe tumor suppressors pRB, p53, the protooncogene Ras, and protein phosphatase 2A.
While the disruption of the telomerase RNA in ciliate and yeast model organisms
provided early evidence for an important role of telomerase in cell survival (Singerand Gottschling, 1994; Yu et al., 1990), the potential of telomerase inhibition as atherapeutic approach for treating human cancer was first demonstrated by theexpression of antisense hTR in immortal HeLa cells (Feng et al., 1995). Transfectionof HeLa cells with an antisense hTR led to loss of telomerase activity, telomereshortening, and cell death after 20–26 population doublings. Since then, severalapproaches for targeting telomerase and also telomeres have been developed andtested, with several ongoing clinical trials (see Chapter 10) (Harley, 2008).
The first evidence for a role of telomerase and telomere length in organismal aging
came from studies in telomerase knockout mouse models (see Chapter 9). Loss oftelomere function in aging late generation mTRÀ/À mice did not elicit a full spectrumof classical pathophysiological symptoms of aging. However, age-dependent telo-mere shortening and accompanying genetic instability were associated with short-ened life span, hair loss and graying, as well as a reduced capacity to respond tostresses such as wound healing and hematopoietic ablation (Rudolph et al., 1999).
Premature aging is also characteristic of patients with a rare multisystem disorder,dyskeratosis congenita (DC), who present with three distinctive clinical character-istics: abnormal skin pigmentation, nail dystrophy, and mucosal leukoplakia (Kirwanand Dokal, 2008, 2009). The underlying molecular defect in many DC patients turnsout to be abnormally short telomeres due to mutations in the telomerase holoenzymecomponents dyskerin, TERC, TERT, NOP10, and NHP2. Mutations in the shelterincomponent, TIN2, have also been identified. Three different subtypes have beendescribed: X-linked recessive, autosomal dominant, autosomal recessive, with themost common fatal complications related to bone marrow failure, pulmonary fibrosis,and cancer.
The link between telomerase and DC was first made in X-linked DC, which is
caused by mutations in the gene encoding dyskerin (Mitchell et al., 1999). Due to the
role of dyskerin in H/ACA snoRNP biogenesis, DC was initially believed to be due todefects in ribosomal RNA processing. However, dyskerin was found to bind to apreviously unidentified H/ACA RNA motif within hTR, and DC patients with mutantdyskerin have decreased hTR levels, decreased telomerase activity, and shortertelomeres.
Mutations in hTERT and hTERC have also been described in other diseases,
including other bone marrow failure syndromes such as aplastic anemia (AA),pancytopenia, and myelodysplastic syndrome (MDS), as well as in diseases nottypically associated with blood disorders, such as idiopathic pulmonary fibrosis (IPF)and liver disorders (Armanios, 2009; Armanios et al., 2007; Kirwan and Dokal, 2009;Savage and Alter, 2009).
The initially defined biological function of a protein may limit the identification orassessment of less well characterized roles for the protein (Blackburn, 2005). Firstidentified as having an essential role in the maintenance of telomere length andprotection of genetic information, it was not until the late 1990s that evidence ofadditional telomere synthesis-independent roles for telomerase began to emerge(Blackburn, 2000, 2005; Bollmann, 2008; Martinez and Blasco, 2011) (see Chapter8). TERT overexpression studies suggested a possible role for TERT in the promotionof tumorigenesis and tumor dissemination (Artandi et al., 2002; Canela et al., 2004;Gonzalez-Suarez et al., 2001, 2002), and in the resistance to cell inhibition and death,in certain instances, of postmitotic, nondividing cells (Lee et al., 2008; Rahmanet al., 2005). TERT overexpression leads to rapid induction of growth-promotinggenes (Smith et al., 2003), stimulation of hair follicle stem cell proliferation which insome studies was independent of the telomerase RNA component (Choi et al., 2008;Flores et al., 2005; Martinez and Blasco, 2011; Sarin et al., 2005), and activation of theMyc and Wnt pathways (Choi et al., 2008; Park et al., 2009). Park et al. showed thatTERT modulates Wnt/b-catenin signaling by serving as a cofactor in a b-catenintranscriptional complex, revealing yet another unanticipated role for the catalyticsubunit of telomerase. Alteration of histone modification and sensitization of humancells to DNA damage were observed in TERT small interfering (si) RNA knock-downstudies (Masutomi et al., 2005). Contrary to the evidence that TERT affects Wntsignaling, Vidal-Cardenas and Greider (2010) reported no change in gene expressionor DNA damage response in both mTRÀ/À G1 and mTERTÀ/À G1 mice with longtelomeres when compared to wild-type mice. More recently, Strong et al. (2011)failed to find evidence of altered Wnt signaling in various adult and embryonic tissuesof mTERT-deficient mice. Additional studies which aim to clarify the role of TERT inWnt signaling will be required. Other potential alternative roles of telomerase, forexample, in the mitochondria, continue to be investigated (see Chapter 8) (Martinezand Blasco, 2011).
Most recently, a novel RNA partner for hTERT was discovered, highlighting a new
role for telomerase. Maida et al. (2009) showed that hTERT interacts with the RNA
component of mitochondrial RNA processing endoribonuclease (RMRP). Together,they form a ribonucleoprotein complex that exhibits RNA-dependent RNA poly-merase (RdRP) activity, generating double-stranded RNAs that are processed in aDicer-dependent manner into siRNA. Mutations in RMRP are found in cartilage-hairhypoplasia (CHH) (Ridanpaa et al., 2001), suggesting a link between the integrity ofthe hTERT–RMRP complex and disease development and progression (Maidaet al., 2009).
Most cancers, which are characterized by high rates of proliferation and high rates ofgenomic instability, have adapted to the high rate of division by upregulatingtelomerase activity (Shay and Bacchetti, 1997). However, 10–15% of cancers areable to maintain their telomere lengths in the absence of telomerase, using one or morerecombination-based mechanisms referred to as ALT (Cesare and Reddel, 2010; Shayand Bacchetti, 1997). An additional alternate mode of telomerase-independenttelomere maintenance occurs in D. melanogaster via retrotransposon-type mechan-isms (Mason et al., 2008) (see Chapter 11).
While a recombination-mediated method to replicate telomeres was suggested by
Walmsley et al. (1984), the first evidence of a recombination-dependent telomerelength maintenance mechanism was described in survivors of an est1-null mutant ofS. cerevisiae (Bhattacharyya et al., 2010; Lundblad and Blackburn, 1993; Lundbladand Szostak, 1989). Yeasts that survive in the absence of telomerase holoenzymecomponents present different methods of survival (Lendvay et al., 1996; Lundbladand Blackburn, 1993; Singer and Gottschling, 1994; Teng and Zakian, 1999). Twoclasses of survivors were initially identified (Teng and Zakian, 1999). Those classifiedas Type I show drastically amplified Y’ DNA elements that are found in thesubtelomeric region of most chromosomes and retain very short terminal repeats,while Type II survivors have long heterogeneous telomere tracts, reminiscent of ALTin human cancer cells.
Fission yeast, on the other hand, survive in the absence of telomerase mainly via
circularization of their chromosomes. However, linear survivors, formed viarecombination between persisting telomere sequences, are also observed (Nakamuraet al., 1998). Most recently, an additional mode of telomerase-null linear survivorswas characterized in S. pombe (Jain et al., 2010). These cells survive the loss oftelomeric sequences by continually amplifying and rearranging heterochromaticsequences using the heterochromatin assembly machinery, and are thus referred to asHAATI. The linearity of HAATI chromosomes is preserved by Pot1 and itsinteracting partner Ccq1 (Jain et al., 2010; Miyoshi et al., 2008). Pot1 is able toconfer its essential end-protection function in the absence of its specific DNA bindingsequence, demonstrating that, as in D. melanogaster, telomere sequence is dispens-able for chromosome linearity in fission yeast (Jain et al., 2010).
Recombination at human telomeres was first proposed based on the observation of
rapid telomere lengthening and shortening in telomerase-negative cells (Cesare and
Reddel, 2010; Murnane et al., 1994). The telomeres of ALT cells retain featurescommon to those of telomerase-positive cells, including double- and single-strandedtelomeric repeats, the association of shelterin and other proteins, and the t-loopsstructures (Cesare and Reddel, 2010). However, ALT cells are characterized by theheterogeneous nature of their telomere lengths, ranging from <2 to > 50 kb (Bryanet al., 1995; Cesare and Reddel, 2008, 2010). Hallmarks of ALT include thegeneration of extrachromosomal telomeric DNA and ALT-associated promyelocyticleukemia bodies (APBs, sites of DNA synthesis and possibly recombination),although these features are also detectable in telomerase-positive cells that haveundergone trimming of over-lengthened telomeres (Cesare and Reddel, 2010; Dras-kovic et al., 2009; Nabetani et al., 2004; Yeager et al., 1999). There have also beenreports of telomerase-negative cancer cells that do not have all the characteristicstypically associated with ALT cells (Cerone et al., 2005; Fasching et al., 2005;Marciniak et al., 2005), highlighting the potential for complex and varied mechanismsof telomere maintenance. Recent studies by Henson et al. (2009) have shownextrachromosomal C-circles, consisting of a complete C-rich strand and an incom-plete G-rich strand, to be the best indicator of whether ALT activity is present. Threesuggested mechanisms of telomere elongation in ALT cells, which are not mutuallyexclusive, include telomere sister chromatid exchanges (T-SCEs), homologousrecombination-dependent telomere copying, and t-loop junction resolution (Cesareand Reddel, 2008, 2010).
Unlike most organisms, the telomere elongation and capping functions are naturally
uncoupled in D. melanogaster (Rong, 2008). A distinctive feature of the fruit fly is thatit has no telomerase. Instead, its telomere structure is comprised of head-to-tail arraysof three different telomere-specific non-long-terminal-repeat (non-LTR) retrotrans-posons, HeT-A, TART, and TAHRE found only at the chromosome ends (Masonet al., 2008; Rong, 2008) (see Chapter 11). All organisms possess an end-cappingcomplex to protect the chromosome end from being recognized as a double-strandedbreak by the DNA repair machinery. D. melanogaster uses a sequence-independentmechanism, contrary to the short repeats employed by most organisms. While anumber of telomere-capping proteins prevent chromosome end-to-end fusions inD. melanogaster, only three proteins have been found to localize exclusively attelomeres and function solely in telomere maintenance. These are the HP1/ORC2-associated protein (HOAP), modigliani (moi), and Verrocchio (Ver) (Cenci et al., 2003;Perrini et al., 2004; Raffa et al., 2009, 2010). These proteins are functional equivalentsof the shelterin complex and have been collectively given the name terminin(Raffa et al., 2009, 2010). Modigliani encodes a novel protein that binds both HOAPand the heterochromatin protein HP1, which efficiently binds and stabilizes ssDNAmuch like POT1.
Although the telomerase-based telomere elongation system enhances telomere
stability and length control efficacy, the survival of organisms utilizing various formsof ALT and recombination mechanisms suggests that adaptation is possible. Alter-native telomere maintenance mechanisms have been observed after telomeraseinhibition (Bechter et al., 2004) or genetic deletion of telomerase (Changet al., 2003; Hande et al., 1999; Morrish and Greider, 2009; Niida et al., 2000).
These observations potentially complicate the development of treatments that targettelomerase or telomere function. Studies in model organisms, including yeast andmice reveal increased telomeric recombination after induction of telomere dysfunc-tion through mutation or deletion of telomere-capping proteins (Bechard et al., 2009;Celli et al., 2006; Grandin et al., 2001; He et al., 2006; Iyer et al., 2005; Tenget al., 2000; Underwood et al., 2004; Wu et al., 2006). Recently telomeric recom-bination was also observed following the induction of telomere dysfunction intelomerase-positive cells, suggesting that telomeric recombination may be a potentialadaptation mechanism in response to telomere dysfunction in mammalian cells(Brault and Autexier, 2010).
The discovery of telomerase was the result of a quest to understand a basic biologicalquestion: how are the ends of a linear chromosome replicated? The success of thisquest led to a range of experimental questions touching on fundamental aspects of cellfunction and regulation. Even though quite unanticipated at the outset, the study oftelomerase also provided critical insights on aging and cancer. The full significanceand implication of the discovery of telomerase are only now becoming clear, as thecontributions of Elizabeth Blackburn, Carol Greider, and Jack Szostak to theadvancement of our knowledge in this field were recognized by the Nobel Foundationin 2009. Our understanding of telomerase regulation and function remains far fromcomplete. The next few years will surely witness new and exciting developments inthe field with regard to fundamental mechanisms of telomerase regulation andfunction. These developments should in turn provide the foundation for designingspecific and effective therapeutic strategies to modulate telomerase in disease.
Chantal Autexier acknowledges support from the Canadian Institutes of HealthResearch, the Canadian Cancer Society and Le Fonds en Recherche en Sante´ duQue´bec.
Abreu E, Aritonovska E, Reichenbach P, Cristofari G, Culp B, Terns RM, Lingner J, Terns MP.
(2010) TIN2-tethered TPP1 recruits human telomerase to telomeres in vivo. Mol. Cell Biol.
Armanios M. (2009) Syndromes of telomere shortening. Annu. Rev. Genomics Hum. Genet. 10:
Armanios MY, Chen JJ, Cogan JD, Alder JK, Ingersoll RG, Markin C, Lawson WE, Xie M,
Vulto I, Phillips JA, 3rd, Lansdorp PM, Greider CW, Loyd JE. (2007) Telomerase mutationsin families with idiopathic pulmonary fibrosis. N. Engl. J. Med. 356(13): 1317–1326.
Artandi SE, Alson S, Tietze MK, Sharpless NE, Ye S, Greenberg RA, Castrillon DH, Horner
JW, Weiler SR, Carrasco RD, DePinho RA. (2002) Constitutive telomerase expressionpromotes mammary carcinogenesis in aging mice. Proc. Natl. Acad. Sci. USA 99:8191–8196.
Autexier C, Lue N. (2006) The structure and function of telomerase reverse transcriptase. Ann.
Azzalin CM, Reichenbach P, Khoriauli L, Giulotto E, Lingner J. (2007) Telomeric repeat
containing RNA and RNA surveillance factors at mammalian chromosome ends. Science318(5851): 798–801.
Banik SSR, Counter CM. (2004) Characterization of interactions between PinX1 and human
telomerase subunits hTERT and hTR. J. Biol. Chem. 279: 51745–51748.
Beattie TL, Zhou W, Robinson MO, Harrington L. (1998) Reconstitution of human telomerase
activity in vitro. Curr. Biol. 8: 177–180.
Bechard LH, Butuner BD, Peterson GJ, McRae W, Topcu Z, McEachern MJ. (2009) Mutant
telomeric repeats in yeast can disrupt the negative regulation of recombination-mediatedtelomere maintenance and create an alternative lengthening of telomeres-like phenotype.
Mol. Cell Biol. 29(3): 626–639.
Bechter OE, Zou Y, Walker W, Wright WE, Shay JW. (2004) Telomeric recombination in
mismatch repair deficient human colon cancer cells after telomerase inhibition. Cancer Res.
Bhattacharyya S, Sandy A, Groden J. (2010) Unwinding protein complexes in ALTernative
telomere maintenance. J. Cell Biochem. 109(1): 7–15.
Blackburn EH. (2000) Telomere states and cell fates. Nature 408(6808): 53–56.
Blackburn EH. (2005) Cell biology: Shaggy mouse tales. Nature 436(7053): 922–923.
Blackburn EH, Gall JG. (1978) A tandemly repeated sequence at the termini of the extra-
chromosomal ribosomal RNA genes in Tetrahymena. J. Mol. Biol. 120: 33–53.
Blackburn EH, Greider CW, Szostak JW. (2006) Telomeres and telomerase: the path from
maize, Tetrahymena and yeast to human cancer and aging. Nat. Med. 12(10): 1133–1138.
Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu C-P, Morin GB, Harley CB, Shay JW,
Lichtsteiner S, Wright WE. (1998) Extension of life-span by introduction of telomerase intonormal human cells. Science 279: 349–352.
Bollmann FM. (2008) The many faces of telomerase: emerging extratelomeric effects.
Brault ME, Autexier C. (2010) Telomeric recombination induced by dysfunctional telomeres.
Bryan TM, Englezou A, Gupta J, Bacchetti S, Reddel RR. (1995) Telomere elongation in
immortal human cells without detectable telomerase activity. EMBO J. 14: 4240–4248.
Canela A, Martin-Caballero J, Flores JM, Blasco MA. (2004) Constitutive expression of tert in
thymocytes leads to increased incidence and dissemination of T-cell lymphoma in Lck-Tertmice. Mol. Cell Biol. 24: 4275–4293.
Celli GB, Denchi EL, de Lange T. (2006) Ku70 stimulates fusion of dysfunctional telomeres
yet protects chromosome ends from homologous recombination. Nat. Cell Biol. 8(8):885–890.
Cenci G, Siriaco G, Raffa GD, Kellum R, Gatti M. (2003) The Drosophila HOAP protein is
required for telomere capping. Nat. Cell Biol. 5(1): 82–84.
Cerone MA, Autexier C, Londono-Vallejo J-A, Bacchetti S. (2005) A human cell line that
maintains telomeres in the absence of telomerase and of key markers of ALT. Oncogene 24:7893–7901.
Cesare AJ, Reddel RR (2008) Telomere uncapping and alternative lengthening of telomeres.
Mech. Ageing Dev. 129(1, 2): 99–108.
Cesare AJ, Reddel RR. (2010) Alternative lengthening of telomeres: models, mechanisms and
implications. Nat. Rev. Genet. 11(5): 319–330.
Chang S, Khoo CM, Naylor ML, Maser RS, DePinho RA (2003) Telomere-based crisis:
functional differences between telomerase activation and ALT in tumor progression. GenesDev. 17: 88–100.
Chapon C, Cech TR, Zaug AJ (1997) Polyadenylation of telomerase RNA in budding yeast.
Choi J, Southworth LK, Sarin KY, Venteicher AS, Ma W, Chang W, Cheung P, Jun S, Artandi
MK, Shah N, Kim SK, Artandi SE. (2008) TERT promotes epithelial proliferation throughtranscriptional control of a Myc- and Wnt-related developmental program. PLoS Genet.
Cifuentes-Rojas C, Kannan K, Tseng L, Shippen DE (2011) Two TER subunits for
Arabidopsis telomerase and the TER binding activity of POT1a. Proc. Natl. Acad. Sci.
U.S.A. 108: 73–78.
Cohen SB, Graham ME, Lovrecz GO, Bache N, Robinson PJ, Reddel RR. (2007) Protein
composition of catalytically active human telomerase from immortal cells. Science 315:1850–1853.
Collins K. (2006) The biogenesis and regulation of telomerase holoenzymes. Nat. Rev. Mol.
Collins K. (2008) Physiological assembly and activity of human telomerase complexes. Mech.
Collins K, Gandhi L. (1998) The reverse transcriptase component of the Tetrahymena
telomerase ribonucleoprotein complex. Proc. Natl. Acad. Sci. U.S.A. 95: 8485–8490.
Collins K, Greider CW. (1993) Tetrahymena telomerase catalyzes nucleolytic cleavage and
non-processive elongation. Genes Dev. 7: 1364–1376.
Counter CM, Meyerson M, Eaton EN, Ellisen LW, Dickinson Caddle S, Haber DA, Weinberg
RA. (1998) Telomerase activity is restored in human cells by ectopic expression of hTERT(hEST2), the catalytic subunit of telomerase. Oncogene 16: 1217–1222.
Counter CM, Meyerson M, Eaton EN, Weinberg RA. (1997) The catalytic subunit of yeast
telomerase. Proc. Natl. Acad. Sci. U.S.A. 94: 9202–9207.
d’Adda de Fagagna F. (2008) Living on a break: cellular senescence as a DNA-damage
response. Nat. Rev. Cancer 8: 512–522.
Draskovic I, Arnoult N, Steiner V, Bacchetti S, Lomonte P, Londono-Vallejo A. (2009) Probing
PML body function in ALT cells reveals spatiotemporal requirements for telomere recom-bination. Proc. Natl. Acad. Sci. U.S.A. 106(37): 15726–15731.
Fasching CL, Bower K, Reddel RR. (2005) Telomerase-independent telomere length main-
tenance in the absence of alternative lengthening of telomeres-associated promyelocyticleukemia bodies. Cancer Res. 65: 2722–2729.
Feng J, Funk WD, Wang S-S, Weinrich SL, Avilion AA, Chiu C-P, Adams RR, Chang E,
Allsopp RC, Yu J, Le S, West MD, Harley CB, Andrews WH, Greider CW, Villeponteau B.
(1995) The RNA component of human telomerase. Science 269: 1236–1241.
Ferrezuelo F, Steiner B, Aldea M, Futcher B. (2002) Biogenesis of yeast telomerase depends on
the importin mtr10. Mol. Cell Biol. 22(17): 6046–6055.
Feuerhahn S, Iglesias N, Panza A, Porro A, Lingner J. (2010) TERRA biogenesis, turnover and
implications for function. FEBS Lett. 584(17): 3812–3818.
Fisher TS, Taggart AKP, Zakian V. (2004) Cell cycle-dependent regulation of yeast telomerase
by Ku. Nat. Struct. Mol. Biol. 11: 1198–1205.
Flores I, Cayuela ML, Blasco MA. (2005) Effects of telomerase and telomere length on
epidermal stem cell behavior. Science 309(5738): 1253–1256.
Ford LP, Suh JM, Wright WE, Shay JW. (2000) Heterogeneous nuclear ribonucleoproteins C1
and C2 associate with the RNA component of human telomerase. Mol. Cell Biol. 20:9084–9091.
Fu D, Collins K. (2007) Purification of human telomerase complexes identifies factors involved
in telomerase biogenesis and telomere length regulation. Mol. Cell 28: 773–785.
Gillis AJ, Schuller AP, Skordalakes E. (2008) Structure of the Tribolium castaneum telomerase
catalytic subunit TERT. Nature 455(7213): 633–637.
Gonzalez-Suarez E, Flores JM, Blasco MA. (2002) Cooperation between p53 mutation and
high telomerase transgenic expression in spontaneous cancer development. Mol. Cell Biol.
Gonzalez-Suarez E, Samper E, Ramirez A, Flores JM, Martin-Caballero J, Jorcano JL, Blasco
MA. (2001) Increased epidermal tumors and increased skin wound healing in transgenicmice overexpressing the catalytic subunit of telomerase, mTERT, in basal keratinocytes.
EMBO J. 20: 2619–2630.
Grandin N, Damon C, Charbonneau M. (2001) Cdc13 prevents telomere uncapping and Rad50-
dependent homologous recombination. EMBO J. 20(21): 6127–6139.
Greider CW, Blackburn EH. (1985) Identification of a specific telomere terminal transferase
activity in Tetrahymena extracts. Cell 43: 405–413.
Greider CW, Blackburn EH. (1987) The telomere terminal transferase of Tetrahymena is a
ribonucleoprotein enzyme with two kinds of primer specificity. Cell 51: 887–898.
Greider CW, Blackburn EH. (1989) A telomeric sequence in the RNA of Tetrahymena
telomerase required for telomere repeat synthesis. Nature 337: 331–337.
Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg RA.
(1999) Creation of human tumour cells with defined genetics elements. Nature 400: 464–468.
Hahn WC, Dessain SK, Brooks MW, King JE, Elenbaas B, Sabatini DM, DeCaprio JA,
Weinberg RA. (2002) Enumeration of the simian virus early region elements necessary forhuman cell transformation. Mol. Cell Biol. 22: 2111–2123.
Hanahan D, Weinberg RA. (2000) The hallmarks of cancer. Cell 100(1): 57–70.
Hande MP, Samper E, Lansdorp P, Blasco MA. (1999) Telomere length dynamics and
chromosomal instability in cells derived from telomerase null mice. J. Cell Biol. 144:589–601.
Harley CB. (1991) Telomere loss: mitotic clock or genetic time bomb? Mutat. Res. 256:
Harley CB. (2008) Telomerase and cancer therapeutics. Nat. Rev. Cancer 8(3): 167–179.
Harrington L, Zhou W, McPhail T, Oulton R, Yeung DSK, Mar V, Bass MB, Robinson MO.
(1997) Human telomerase contains evolutionarily conserved catalytic and structural sub-units. Genes Dev. 11: 3109–3115.
He H, Multani AS, Cosme-Blanco W, Tahara H, Ma J, Pathak S, Deng Y, Chang S. (2006)
POT1b protects telomeres from end-to-end chromosomal fusions and aberrant homologousrecombination. EMBO J. 25(21): 5180–5190.
Henderson E, Blackburn EH. (1989) An overhang 30 terminus is a conserved feature of
telomeres. Mol. Cell Biol. 9: 345–348.
Henson JD, Cao Y, Huschtscha LI, Chang AC, Au AY, Pickett HA, Reddel RR. (2009) DNA C-
circles are specific and quantifiable markers of alternative-lengthening-of-telomeres activity.
Nat. Biotechnol. 27(12): 1181–1185.
Hughes TR, Evans SK, Weilbaecher RG, Lundblad V. (2000) The Est3 protein is a subunit of
yeast telomerase. Curr. Biol. 10: 809–812.
Iyer S, Chadha AD, McEachern MJ. (2005) A mutation in the STN1 gene triggers an alternative
lengthening of telomere-like runaway recombinational telomere elongation and rapiddeletion in yeast. Mol. Cell Biol. 25(18): 8064–8073.
Jacobs SA, Podell ER, Cech TR. (2006) Crystal structure of the essential N-terminal domain of
telomerase reverse transcriptase. Nat. Struct. Mol. Biol. 13: 218–225.
Jain D, Hebden AK, Nakamura TM, Miller KM, Cooper JP. (2010) HAATI survivors replace
canonical telomeres with blocks of generic heterochromatin. Nature 467: 223–227.
Jiang X-R, Jimenez G, Chang E, Frolkis M, Kusler B, Sage M, Beeche M, Bodnar AG, Wahl
GM, Tisty TD, Chiu C-P. (1999) Telomerase expression in human somatic cells does notinduce changes associated with a transformed phenotype. Nat. Genet. 21: 111–114.
Kharbanda S, Kumar V, Dhar S, Pandey P, Chen C, Majumder P, Yuan Z-M, Whang Y, Strauss
W, Pandita TK, Weaver D, Kufe D. (2000) Regulation of the hTERT telomerase catalyticsubunit by the c-Abl tyrosine kinase. Curr. Biol. 10: 568–575.
Kilian A, Bowtell DDL, Abud HE, Hime GR, Venter DJ, Keese PK, Duncan EL, Reddel RR,
Jefferson RA. (1997) Isolation of a candidate human telomerase catalytic subunit gene, whichreveals complex splicing patterns in different cell types. Hum. Mol. Genet. 6: 2011–2019.
Kim JH, Park SM, Kang MR, Oh SY, Lee TH, Muller MT, Chung IK. (2005) Ubiquitin ligase
MKRN1 modulates telomere length homeostasis through a proteolysis of hTERT. GenesDev. 19(7): 776–781.
Kirk KE, Harmon BP, Reichardt IK, Sedat JW, Blackburn EH. (1997) Block in anaphase
chromosome separation caused by a telomerase template mutation. Science 275: 1478–1481.
Kirwan M, Dokal I. (2008) Dyskeratosis congenita: a genetic disorder of many faces. Clin.
Kirwan M, Dokal I. (2009) Dyskeratosis congenita, stem cells and telomeres. Biochim.
Kiyono T, Foster SA, Koop JI, McDougall JK, Galloway DA, Klingelhutz AJ. (1998) Both Rb/
p16INK4a inactivation and telomerase activity are required to immortalize human epithelialcells. Nature 396: 84–88.
LaBranche H, Dupuis S, Ben-David Y, Bani M-R, Wellinger RJ, Chabot B. (1998) Telomere
elongation by hnRNPA1 and a derivative that interacts with telomeric repeats and telome-rase. Nat. Genet. 19: 199–202.
Latrick CM, Cech TR. (2010) POT1-TPP1 enhances telomerase processivity by slowing primer
dissociation and aiding translocation. EMBO J. 29(5): 924–933.
Lee J, Sung YH, Cheong C, Choi YS, Jeon HK, Sun W, Hahn WC, Ishikawa F, Lee H-W. (2008)
TERT promotes cellular and organismal survival independently of telomerase activity.
Oncogene 27: 3754–3760.
Lendvay TS, Morris DK, Sah J, Balasubramanian B, Lundblad V. (1996) Senescence mutants of
Saccharomyces cerevisiae with a defect in telomere replication identify three additional ESTgenes. Genetics 144: 1399–1412.
Leonardi J, Box JA, Bunch JT, Baumann P. (2008) TER1, the RNA subunit of fission yeast
telomerase. Nat. Struct. Mol. Biol. 15(1): 26–33.
Lingner J, Cech TR. (1996) Purification of telomerase from Euplotes aediculatus: requirement
of a 30 overhang. Proc. Natl. Acad. Sci. U.S.A. 93: 10712–10717.
Lingner J, Cech TR, Hughes TR, Lundblad V. (1997a) Three ever shorter telomere (EST) genes
are dispensable for in vitro yeast telomerase activity. Proc. Natl. Acad. Sci. U.S.A. 94:11190–11195.
Lingner J, Hughes TR, Shevchenko A, Mann M, Lundblad V, Cech TR. (1997b) Reverse
transcriptase motifs in the catalytic subunit of telomerase. Science 276: 561–567.
Luke B, Panza A, Redon S, Iglesias N, Li Z, Lingner J. (2008) The Rat1p 50 to 30 exonuclease
degrades telomeric repeat-containing RNA and promotes telomere elongation in Saccha-romyces cerevisiae. Mol. Cell 32(4): 465–477.
Lundblad V, Blackburn EH. (1993) An alternative pathway for yeast telomere maintenance
rescues est1-senescence. Cell 73: 347–360.
Lundblad V, Szostak JW. (1989) A mutant with a defect in telomere elongation leads to
senescence in yeast. Cell 57: 633–643.
Lustig AJ. (2004) Telomerase RNA: a flexible RNA scaffold for telomerase biosynthesis. Curr.
Maida Y, Yasukawa M, Furuuchi M, Lassmann T, Possemato R, Okamoto N, Kasim V,
Hayashizaki Y, Hahn WC, Masutomi K. (2009) An RNA-dependent RNA polymeraseformed by TERT and the RMRP RNA. Nature 461(7261): 230–235.
Marciniak RA, Cavazos D, Montellano R, Chen Q, Guarente L, Johnson FB. (2005) A novel
telomere structure in a human alternative lengthening of telomeres cell line. Cancer Res. 65:2730–2737.
Martinez P, Blasco MA. (2011) Telomeric and extra-telomeric roles for telomerase and the
telomere-binding proteins. Nat. Rev. Cancer 11(3): 161–176.
Mason JM, Frydrychova RC, Biessmann H. (2008) Drosophila telomeres: an exception
providing new insights. Bioessays 30(1): 25–37.
Masutomi K, Possemato R, Wong JM, Currier JL, Tothova Z, Manola JB, Ganesan S,
Lansdorp PM, Collins K, Hahn WC. (2005) The telomerase reverse transcriptase regulateschromatin state and DNA damage responses. Proc. Natl. Acad. Sci. U.S.A. 102(23):8222–8227.
McClintock B. (1931) Cytological observations of deficiencies involving known
genes, translocations and an inversion in Zea mays. Mo. Agric. Exp. Res. Stn. Res. Bull.
McClintock B. (1939) The behavior in successive nuclear divisions of a chromosome broken at
meiosis. Proc. Natl. Acad. Sci. U.S.A. 25: 405–416.
McElligott R, Wellinger RJ. (1997) The terminal DNA structure of mammalian chromosomes.
Meyerson M, Counter CM, Eaton EN, Ellisen LW, Steiner P, Dickinson SC, Ziaugra L,
Beijersbergen RL, Davidoff MJ, Liu Q, Bacchetti S, Haber DA, Weinberg RA. (1997)hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cellsand during immortalization. Cell 90: 785–795.
Min B, Collins K. (2009) An RPA-related sequence-specific DNA-binding subunit of telo-
merase holoenzyme is required for elongation processivity and telomere maintenance. Mol.
Cell 36(4): 609–619.
Mitchell JR, Wood E, Collins K. (1999) A telomerase component is defective in the human
disease dyskeratosis congenita. Nature 402: 551–555.
Mitchell M, Gillis A, Futahashi M, Fujiwara H, Skordalakes E. (2010) Structural basis for
telomerase catalytic subunit TERT binding to RNA template and telomeric DNA. Nat.
Struct. Mol. Biol. 17(4): 513–518.
Miyoshi T, Kanoh J, Saito M, Ishikawa F. (2008) Fission yeast Pot1-Tpp1 protects telomeres
and regulates telomere length. Science 320(5881): 1341–1344.
Morales CP, Holt SE, Ouellette M, Kaur KJ, Yan Y, Wilson KS, White MA, Wright WE, Shay
JW. (1999) Absence of cancer-associated changes in human fibroblasts immortalized withtelomerase. Nat. Genet. 21: 115–118.
Morin GB. (1989) The human telomere terminal transferase enzyme is a ribonucleoprotein that
synthesizes TTAGGG repeats. Cell 59: 521–529.
Morrish TA, Greider CW. (2009) Short telomeres initiate telomere recombination in primary
and tumor cells. PLoS Genet. 5(1): e1000357.
Muller HJ. (1938) The remaking of chromosomes. Collecting Net 13: 181–198.
Murnane JP, Sabatier L, Marder BA, Morgan WF. (1994) Telomere dynamics in an immortal
human cell line. EMBO J. 13: 4953–4962.
Nabetani A, Yokoyama O, Ishikawa F. (2004) Localization of hRad9, hHus1, hRad1, and
hRad17 and caffeine-sensitive DNA replication at the alternative lengthening of telomeres-associated promyelocytic leukemia body. J. Biol. Chem. 279(24): 25849–25857.
Nakamura TM, Cooper JP, Cech TR. (1998) Two modes of survival of fission yeast without
Nakamura TM, Morin GB, Chapman KB, Weinrich SL, Andrews WH, Lingner J, Harley CB,
Cech TR. (1997) Telomerase catalytic subunit homologs from fission yeast and human.
Science 277: 955–959.
Niida H, Shinkai Y, Hande MP, Matsumoto T, Takehara S, Tachibana M, Oshimura M, Lansdorp
PM, Furuichi Y. (2000) Telomere maintenance in telomerase-deficient mouse embryonicstem cells: characterization of an amplified telomeric DNA. Mol. Cell Biol. 20(11):4115–4127.
O’Connor CM, Collins K. (2006) A novel RNA binding domain in tetrahymena telomerase
p65 initiates hierarchical assembly of telomerase holoenzyme. Mol. Cell Biol. 26(6):2029–2036.
Oh W, Lee EW, Lee D, Yang MR, Ko A, Yoon CH, Lee HW, Bae YS, Choi CY, Song J. (2010)
Hdm2 negatively regulates telomerase activity by functioning as an E3 ligase of hTERT.
Oncogene 29(28): 4101–4112.
Olovnikov AM. (1973) A theory of marginotomy. J. Theor. Biol. 41: 181–190.
Palm W, de Lange T. (2008) How shelterin protects mammalian telomeres. Annu. Rev. Genet.
Park JI, Venteicher AS, Hong JY, Choi J, Jun S, Shkreli M, Chang W, Meng Z, Cheung P, Ji H,
McLaughlin M, Veenstra TD, Nusse R, McCrea PD, Artandi SE. (2009) Telomerase modulatesWnt signalling by association with target gene chromatin. Nature 460(7251): 66–72.
Pennock E, Buckley K, Lundblad V. (2001) Cdc13 delivers separate complexes to the telomere
for end protection and replication. Cell 104: 387–396.
Perrini B, Piacentini L, Fanti L, Altieri F, Chichiarelli S, Berloco M, Turano C, Ferraro A,
Pimpinelli S. (2004) HP1 controls telomere capping, telomere elongation, and telomeresilencing by two different mechanisms in Drosophila. Mol. Cell 15(3): 467–476.
Price CM, Cech TR. (1989) Properties of the telomeric DNA binding protein from Oxytricha
Raffa GD, Raimondo D, Sorino C, Cugusi S, Cenci G, Cacchione S, Gatti M, Ciapponi L.
(2010) Verrocchio, a Drosophila OB fold-containing protein, is a component of the terminintelomere-capping complex. Genes Dev. 24(15): 1596–1601.
Raffa GD, Siriaco G, Cugusi S, Ciapponi L, Cenci G, Wojcik E, Gatti M. (2009) The Drosophila
modigliani (moi) gene encodes a HOAP-interacting protein required for telomere protection.
Proc. Natl. Acad. Sci. U. S. A. 106(7): 2271–2276.
Rahman R, Latonen L, Wiman KG. (2005) hTERT antagonizes p53-induced apoptosis
independently of telomerase activity. Oncogene 24: 1320–1327.
Redon S, Reichenbach P, Lingner J. (2010) The non-coding RNATERRA is a natural ligand and
direct inhibitor of human telomerase. Nucleic Acids Res. 38(17): 5797–5806.
Ridanpaa M, van Eenennaam H, Pelin K, Chadwick R, Johnson C, Yuan B, vanVenrooij W,
Pruijn G, Salmela R, Rockas S, Makitie O, Kaitila I, de la Chapelle A. (2001) Mutations inthe RNA component of RNase MRP cause a pleiotropic human disease, cartilage-hairhypoplasia. Cell 104(2): 195–203.
Rong YS. (2008) Telomere capping in Drosophila: dealing with chromosome ends that most
resemble DNA breaks. Chromosoma 117(3): 235–242.
Rouda S, Skordalakes E. (2007) Structure of the RNA-binding domain of telomerase:
implications for RNA recognition and binding. Structure 15(11): 1403–1412.
Rudolph KL, Chang S, Lee H-W, Blasco M, Gottlieb GJ, Greider C, DePinho RA.
(1999) Longevity, stress response and cancer in aging telomerase-deficient mice. Cell96: 701–712.
Sarin KY, Cheung P, Gilison D, Lee E, Tennen RI, Wang E, Artandi MK, Oro AE, Artandi SE.
(2005) Conditional telomerase induction causes proliferation of hair follicle stem cells.
Nature 436: 1048–1052.
Savage SA, Alter BP. (2009) Dyskeratosis congenita. Hematol. Oncol. Clin. North Am. 23(2):
Schnapp G, Rodi H-P, Rettig WJ, Schnapp A, Damm K. (1998) One-step affinity purification
protocol for human telomerase. Nucl. Acids Res. 26: 3311–3313.
Schoeftner S, Blasco MA. (2008) Developmentally regulated transcription of mammalian
telomeres by DNA-dependent RNA polymerase II. Nat. Cell Biol. 10(2): 228–236.
Seimiya H, Sawada H, Muramatsu Y, Shimizu M, Ohko K, Yamane K, Tsuruo T. (2000)
Involvement of 14-3-3 proteins in nuclear localization of telomerase. EMBO J. 19:2652–2661.
Seto AG, Zaug AJ, Sobel SG, Wolin SL, Cech TR. (1999) Saccharomyces cerevisiae telomerase
is an Sm small nuclear ribonucleoprotein particle. Nature 401: 177–180.
Shay JW, Bacchetti S. (1997) A survey of telomerase activity in human cancer. Eur. J. Cancer
Singer MS, Gottschling DE. (1994) TLC1: template RNA component of Saccharomyces
cerevisiae telomerase. Science 266: 404–409.
Slijepcevic P. (2008) DNA damage response, telomere maintenance and ageing in light of the
integrative model. Mech. Ageing Dev. 129(1, 2): 11–16.
Smith LL, Coller HA, Roberts JM. (2003) Telomerase modulates expression of growth-
controlling genes and enhances cell proliferation. Nat. Cell Biol. 5: 474–479.
Strong MA, Vidal-Cardenas SL, Karim B, Yu H, Guo N, Greider CW. (2011) Phenotypes in
mTERTþ/À and mTERTÀ/À mice are due to short telomeres, not telomere-independentfunctions of TERT. Mol. Cell Biol. 31(12): 2369–2379.
Sykorova E, Fajkus J. (2009) Structure–function relationships in telomerase genes. Biol. Cell
Szostak JW, Blackburn EH. (1982) Cloning yeast telomeres on linear plasmid vectors. Cell 29:
Teng S, Zakian V. (1999) Telomere-telomere recombination is an efficient bypass pathway for
telomere maintenance in Saccharomyces cerevisiae. Mol. Cell Biol. 19: 8083–8093.
Teng SC, Chang J, McCowan B, Zakian VA. (2000) Telomerase-independent lengthening of
yeast telomeres occurs by an abrupt Rad50p-dependent, Rif-inhibited recombinationalprocess. Mol. Cell 6(4): 947–952.
Tomita K, Cooper JP. (2008) Fission yeast Ccq1 is telomerase recruiter and local checkpoint
controller. Genes Dev. 22(24): 3461–3474.
Turkewitz AP, Orias E, Kapler G. (2002) Functional genomics: the coming of age for
Tetrahymena thermophila. Trends Genet. 18(1): 35–40.
Ulaner GA, Hu J-F, Vu TH, Ciudice LC, Hoffman AR. (2001) Tissue-specific alternate splicing
of human telomerase reverse transcriptase (hTERT) influences telomere lengths duringhuman development. Int. J. Cancer 91: 644–649.
Underwood DH, Carroll C, McEachern MJ. (2004) Genetic dissection of the Kluyveromyces
lactis telomere and evidence for telomere capping defects in TER1 mutants with longtelomeres. Eukaryot. Cell 3(2): 369–384.
Vaziri H, Benchimol S. (1998) Reconstitution of telomerase activity in normal human cells
leads to elongation of telomeres and extended replicative life span. Curr. Biol. 8: 279–282.
Venteicher AS, Abreu EB, Meng Z, McCann KE, Terns RM, Veenstra TD, Terns MP, Artandi
SE. (2009) A human telomerase holoenzyme protein required for Cajal body localization andtelomere synthesis. Science 323(5914): 644–648.
Venteicher AS, Meng Z, Mason PJ, Veenstra TD, Artandi SE. (2008) Identification of ATPases
pontin and reptin as telomerase components essential for holoenzyme assembly. Cell 132:945–957.
Vidal-Cardenas SL, Greider CW. (2010) Comparing effects of mTR and mTERT deletion on
gene expression and DNA damage response: a critical examination of telomere lengthmaintenance-independent roles of telomerase. Nucleic Acids Res. 38(1): 60–71.
Vrbsky J, Akimcheva S, Watson JM, Turner TL, Daxinger L, Vyskot B, Aufsatz W, Riha K.
(2010) siRNA-mediated methylation of Arabidopsis telomeres. PLoS Genet. 6(6):e1000986.
Walmsley RW, Chan CSM, Tye B-K, Petes TD. (1984) Unusual DNA sequences associated
with the ends of yeast chromosomes. Nature 310: 157–160.
Wang F, Podell ER, Zaug AJ, Yang Y, Baciu P, Cech TR, Lei M. (2007) The Pot1-TPP1 telomere
complex is a telomerase processivity factor. Nature 445: 506–510.
Wang H, Blackburn EH. (1997) De novo telomere addition by Tetrahymena telomerase in vitro.
Watson JD. (1972) Origin of concatameric T4 DNA. Nat. New Biol. 239: 197–201.
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 componenthTR and the catalytic protein subunit hTRT. Nat. Genet. 17: 498–502.
Witkin KL, Collins K. (2004) Holoenzyme proteins required for physiological assembly and
activity of telomerase. Genes Dev. 18: 1107–1118.
Witkin KL, Prathapam R, Collins K. (2007) Positive and negative regulation of Tetrahymena
telomerase holoenzyme. Mol. Cell Biol. 27(6): 2074–2083.
Wu L, Multani AS, He H, Cosme-Blanco W, Deng Y, Deng JM, Bachilo O, Pathak S, Tahara H,
Bailey SM, Behringer RR, Chang S. (2006) Pot1 deficiency initiates DNA damagecheckpoint activation and aberrant homologous recombination at telomeres. Cell 126(1):49–62.
Xin H, Liu D, Wan M, Safari A, Kim H, Sun W, O’Connor MS, Songyang Z. (2007) TPP1 is a
homologue of ciliate TEBP-b and interacts with Pot1 to recruit telomerase. 445: 559–562.
Yeager TR, Neumann AA, Englezou A, Huschtscha LI, Noble JR, Reddel RR. (1999)
Telomerase-negative immortalized human cells contain a novel type of promyelocyticleukemia (PML) body. Cancer Res. 59(17): 4175–4179.
Yu G-L, Bradley JD, Attardi LD, Blackburn EH. (1990) In vivo alteration of telomere sequences
and senescence caused by mutated Tetrahymena telomerase RNAs. Nature 344: 126–132.
Zappulla DC, Cech TR. (2004) Yeast telomerase RNA: a flexible scaffold for protein subunits.
Proc. Natl. Acad. Sci. U. S. A. 101(27): 10024–10029.
Zaug AJ, Podell ER, Nandakumar J, Cech TR. (2010) Functional interaction between telomere
protein TPP1 and telomerase. Genes Dev. 24(6): 613–622.
Zhou XZ, Lu KP. (2001) The Pin2/TRF1-interacting protein PinX1 is a potent telomerase
Studie über Herpesbläschen-Patches Wirksam und gut verträglich bei Herpes- Infektionen Herpes simplex labialis – kurz Herpes genannt - ist weit verbreitet und tritt bei fast 40% der Erwachsenen auf. Bisher wurde die Krankheit, die sich durch brennende und juckende Fieberbläschen bemerkbar macht, mit antiviralen Cremes oder Salben therapiert. Seit einiger Zeit sind spezielle He
Which of the following divisions of the trigeminal nerve passes through the Which of the following extraocular muscles has a primary function of intorsion Secondary deviation is always greater than primary deviation in Noncomitant strabismus (paralytic strabismus)Concomitant strabismus (non-paralytic strabismus)Hypertropia (vertical misalignment of eyes)