Chromosome End Maintenance by Telomerase*

Telomeres, protein-DNA complexes at the ends of eukaryotic linear chromosomes, are essential for genome stability. The accumulation of chromosomal abnormalities in the absence of proper telomere function is implicated in human aging and cancer. Repetitive telomeric sequences are maintained by telomerase, a ribonucleoprotein complex containing a reverse transcriptase subunit, a template RNA, and accessory components. Telomere elongation is regulated at multiple levels, including assembly of the telomerase holoenzyme, recruitment of telomerase to the chromosome terminus, and telomere accessibility. This minireview provides an overview of telomerase structure, function, and regulation and the role of telomerase in human disease.

cialized higher order complex (reviewed in Ref. 4). In mammalian cells, the G-tails invade the duplex portion of the telomere to form a t-loop structure (5). A six-subunit complex consisting of TRF1, TRF2, TIN2, Rap1, TPP1, and POT1, termed shelterin, is responsible for formation of the t-loop and protection of telomeres (4,6).
The complete replication of linear DNA molecules is problematic for the eukaryotic DNA replication machinery. This "end replication problem," originally proposed by James Watson (7) and Alexey Olovnikov (8) in the early 1970s, poses that removal of the terminal RNA primer on the lagging strand leaves an unreplicated gap, resulting in loss of terminal sequences. The discovery that chromosome termini have a 3Ј-overhang requires re-examination of this problem. Because the longer, G-rich strand is the template for discontinuous replication, removal of the RNA primer should recreate a G-tail, although the observation that most C-rich strands end in the same terminal nucleotide suggests that some additional processing does occur (9). On the shorter leading strand, replication to the extreme terminus creates a blunt-ended molecule that must be processed to create a single-stranded overhang structure, resulting in net sequence loss (Fig. 1A) (see Ref. 10). Processing of both strands is tightly regulated, but the final overhang length and sequence vary among yeast, ciliates, and humans (9), suggesting mechanistic differences.

Telomerase: A Reverse Transcriptase with an Intrinsic RNA Template
Given the loss of essential genetic information predicted by the end replication problem, eukaryotic cells require a mechanism to maintain telomeres. A series of biochemical and genetic experiments performed in the 1980s identified and characterized this evolutionarily conserved mechanism: the enzyme telomerase.
Biochemical and Genetic Approaches to Identify Telomerase Components-Pioneering studies demonstrating that linear DNA molecules capped with Tetrahymena telomeres were stable in yeast and acquired the characteristic yeast heterogeneous telomeric repeat first suggested the existence of a telomere maintenance enzyme (1). In 1985, Carol Greider and Elizabeth Blackburn reported the discovery of an activity in Tetrahymena nuclear extracts capable of elongating a synthetic telomeric (TTGGGG) 4 oligonucleotide (11). They named this enzyme "telomere terminal transferase," later shortened to "telomerase." Treatment with RNase inactivated telomerase, suggesting that an RNA molecule provided the template for nucleotide addition (12). When the gene encoding the RNA was cloned in 1989, the presence of sequence CAACCCCAA (complementary to the Tetrahymena telomeric repeat) provided evidence of this mechanism (13). The catalytic protein component was identified through a convergence of biochemistry and genetics. Joachim Lingner and Thomas Cech identified two proteins (p123 and p43) that copurified with the ciliate telomerase RNA (14). At nearly the same time, a yeast genetic screen performed by Victoria Lundblad's group yielded several genes that caused an EST (ever-shorter telomere) phenotype when mutated (15). Cloning of EST2 and p123 revealed homologous proteins with motifs similar to known RTs 2 (16). One year later, the catalytic subunit was identified in humans (hTERT) by multiple groups (see Ref. 17).
In the wake of these groundbreaking experiments, the telomerase field has expanded rapidly, with the identification of telomerase in many organisms, including plants (18). Here, we draw from examples in mammals, ciliates, and yeast, highlighting those organisms in which telomerase function and regulation are best understood.
Mechanism of Telomerase Action-Even though the catalytic protein had not yet been identified, the general model of telomerase action originally proposed by Greider and Blackburn in 1989 (13) was remarkably accurate. Telomerase utilizes an intrinsic RNA molecule (TER) as the template for nucleotide addition to the chromosome terminus by a catalytic RT (TERT) (Fig. 1B). Complementarity between the chromosome terminus and the telomerase RNA facilitates alignment of the DNA terminus adjacent to a short RNA template sequence. After reverse transcription of the template, repeated cycles of alignment and extension endow telomeres with their characteristic G-rich repeat arrays. The ability of telomerase to mediate a single round of synthesis is described as nucleotide addition processivity, whereas realignment of the same enzyme for a second round of addition is described as repeat addition processivity ( Fig. 1B) (19). After telomerase-mediated extension of the 3Ј-end, the lagging-strand synthesis machinery fills in the opposite strand.
Catalytic Protein Subunit of Telomerase-Significant effort has been invested to understand the structure and function of TERT, homologs of which have been identified from a large number of organisms. With few exceptions, these homologs share a common domain structure (Fig. 1C) (see Ref. 19). The central region of TERT contains homology to other RTs, including seven canonical motifs that contribute to catalytic activity (17). Like other polymerases, TERT contains a triad of absolutely conserved aspartic acid residues thought to be essential for metal binding and enzyme chemistry, mutation of which destroys TERT catalytic activity (16). A recently published crystal structure of TERT from the beetle Tribolium castaneum reveals the right-handed "fingers, palm, and thumb" domain structure characteristic of all nucleic acid polymerases (20). The fingers and palm are contributed by the highly conserved RT motifs, whereas the less conserved region C-terminal to the RT domain (the CTE) forms the thumb. Yeast TERT from which the CTE has been deleted maintains short but stable telomeres in vivo (although enzyme processivity is reduced in vitro), and Caenorhabditis elegans TERT lacks this domain entirely (19). In contrast, some mutations in the hTERT CTE impair telomere maintenance while retaining catalytic activity, suggesting that this domain may have a telomere maintenance role distinct from enzymatic function (19).
The region of TERT located N-terminal to the RT domain contributes to properties unique to telomerase, including association with the intrinsic RNA template, binding of additional protein components, and modulation of processivity. Sequence alignments and mutagenesis have identified several functionally important N-terminal regions termed GQ, CP, QFP, and T ( Fig. 1C) (see Ref. 19 for alternative nomenclature). The CP, QFP, and T regions contribute to high affinity interactions with the telomerase RNA. The most N-terminal region (GQ; RID1 in hTERT) displays low affinity RNA interactions but may also mediate association with other telomerase components (e.g. Est3 in S. cerevisiae) and provide an "anchor site" at which A.

B.
C. FIGURE 1. The end replication problem and telomerase. A, end replication problem. Replication of a telomere containing a 3Ј-overhang on the laggingstrand template (dark blue) and a recessed 5Ј terminus on the leading-strand template (red) is diagrammed. The RNA primer is depicted as a dotted line. B, general model of telomerase function. The template of the telomerase RNA (CA-rich sequence; shown from Tetrahymena) is complementary to the 3Ј-terminal overhang. TERT protein (blue oval) adds nucleotides (red) until the template boundary is reached. Translocation and repositioning of the 3Ј terminus allow addition of a second repeat (green). C, generalized domain structure of TERT. Conserved aspartic acid residues (D) necessary for catalysis are indicated. The N terminus contains regions that confer high affinity (green) and low affinity (blue) RNA binding. For details, see Ref. 19. telomerase associates with the DNA primer (19). Such protein/ DNA contact may facilitate repeat addition processivity by allowing telomerase to remain associated with the telomere while repositioning the 3Ј-DNA terminus within the active site. A crystal structure of the most N-terminal structural domain (TEN domain) containing the GQ motif of Tetrahymena thermophila revealed a putative primer-binding surface and identified residues contributing to DNA interaction (21). This domain is lacking in T. castaneum TERT (20), so the structural relationship of the TEN domain to the catalytic domain is unknown.
Telomerase RNA Component-The RNA component of telomerase (TER) has been cloned from many different organisms and shows great variability in length, sequence, and structure (22). In common between all TERs is a short template sequence located on an unpaired region of the RNA and complementary to the telomeric repeat. In contrast to other RTs, reverse transcription is constrained to this short template. In both human and yeast TERs, a stem-loop structure prevents telomerase extension past the end of the template (23,24), whereas 5Ј-boundary definition in Tetrahymena may require interaction between TERT and an unpaired sequence located immediately 5Ј of the template (25).
Those TERs that have been extensively studied bind proteins involved in RNP biogenesis. hTR is transcribed by RNA polymerase II and stabilized by association with a group of proteins (dyskerin, GAR1, NOP10, and NHP2) involved in the accumulation of small nucleolar and small Cajal body RNAs (26). Two ATPases, pontin and reptin, contribute to RNA stability and telomerase assembly (27). The S. cerevisiae telomerase RNA (TLC1 (telomerase component 1)) is stabilized by association with the Sm proteins, a complex of seven polypeptides that contribute to the assembly of RNPs necessary for pre-mRNA splicing (28). The detailed mechanism of telomerase assembly and intracellular trafficking has been reviewed recently (26) and remains an area of active research.
Additional Protein Components Contribute to the Activity of Telomerase-Telomerase activity from several different organisms can be reconstituted in rabbit reticulocyte lysate by expression of TERT and TER, indicating that these subunits comprise the catalytic core of the enzyme (19). Numerous proteins associate with telomerase, many of which, as discussed above, contribute to RNP biogenesis. However, some factors contribute directly to telomere replication in vivo.
The role of such proteins has been most extensively studied in S. cerevisiae. The Est1 and Est3 proteins are essential for telomere elongation in vivo (15,29), but extract prepared from cells lacking either protein retains telomerase activity, suggesting a regulatory role (30). Est1 binds the telomerase RNA via a bulged stem structure (31) and associates directly with Cdc13 (32), a protein that binds the single-stranded telomeric overhang. Direct fusion of Est2 to Cdc13 bypasses the requirement for Est1 in telomere maintenance (3), suggesting that the Est1/Cdc13 interaction recruits telomerase to telomeres (see below). Two human homologs, EST1A and EST1B, also associate with telomerase (33,34). Although EST1A uncaps chromosomes when overexpressed (34), a role in telomerase recruitment has not been established.
EST1A may promote telomere integrity by modulating the accumulation of RNA transcripts at telomeres (35).
Like Est1, the Est3 protein co-immunoprecipitates with the catalytic core of telomerase (36), but its function is poorly understood. Structural modeling suggests that Est3 is homologous to mammalian TPP1 (37,38), a component of the shelterin complex. This evolutionary relationship raises questions about the function of this accessory protein and its differential association with the telomerase complex (in yeast) or the telomerebinding complex (in higher organisms).

Regulation of Telomerase Activity
The extent and timing of telomere replication are regulated by several mechanisms, including transcriptional and posttranscriptional regulation of TERT, recruitment of telomerase to the chromosome terminus, and control of telomerase accessibility by telomeric binding factors.
Transcriptional and Post-transcriptional Regulation of TERT-Transcriptional regulation of hTERT is a major factor limiting telomerase activity in human cells. hTERT expression is low or absent in most somatic cells but is activated during embryonic development, in some stem cells, and in most tumors ( Fig. 2) (39). Expression of hTR also limits telomerase function in some cell types (40). Post-transcriptional events, including alternative splicing of TERT, phosphorylation and ubiquitination of the enzyme, and intracellular trafficking of telomerase components, add additional layers of complexity to the regulation of telomerase (40,41). The relative contributions of these activities to telomerase function remain incompletely understood.
Recruitment of Telomerase to Telomeres-The activity of telomerase at the telomere is modulated, at least in part, by regulating its recruitment to the extreme chromosome terminus. This event is separable from telomerase association with telomeric chromatin. These mechanisms are best characterized in S. cerevisiae. Est2 (yeast TERT) is constitutively associated with telomeric DNA, although telomeres are extended only during S phase. Telomeric enrichment is high in G 1 phase, decreases in early S phase, and rises again in late S phase (42). G 1 association depends upon interaction of a 48-nucleotide stemloop of the TLC1 RNA with Ku, a heterodimeric complex that binds at the junction of single-and double-stranded telomeric DNAs, whereas the peak of Est2 enrichment in late S phase requires Cdc13 (3).
In contrast to Est2, the telomerase accessory factor Est1 is only telomere-associated in late S through G 2 phases (42). During G 1 phase, Est2 may be sequestered from its substrate by the interaction of TLC1 RNA with the Ku complex. As the cell cycle progresses, several events facilitate the productive recruitment of telomerase to its site of action. First, Est1 expression increases as cells enter S phase. Second, progression to late S phase results in telomerase-independent lengthening of the single-stranded telomeric overhang (43), allowing increased association by Cdc13 (42). Third, phosphorylation of Cdc13 by the cyclin-dependent kinase CDK1 facilitates interaction with Est1 (44). Mutations that prevent phosphorylation of Cdc13 by CDK1 reduce but do not abolish telomerase recruitment and activity (44), suggesting that additional mechanisms contribute to this regulation (see below).
The association of mammalian TERT with the chromosome terminus is also cell cycle-regulated (detected only in S phase) (45), and some mutations in TERT can be rescued by fusion to telomere-binding proteins (46). Although the mechanism of telomerase recruitment to human telomeres is an area of further research, this evidence suggests that recruitment of the enzyme is an important regulatory step.
Control of Telomerase Accessibility to the Chromosome Terminus-The extent of telomerase activity in cells is significantly regulated through the modulation of telomerase access to telomeres by telomere-binding proteins. In S. cerevisiae, sequence-specific binding of Rap1 protein to the doublestranded telomeric repeats "measures" telomere length through a mechanism in which the number of Rap1 molecules bound to a telomere inversely correlates with the action of telomerase at that telomere (4,47). In Schizosaccharomyces pombe and human cells, Rap1 homologs contribute to negative telomere length regulation, although in both cases, Rap1 associates indirectly with telomeric DNA (through Taz1 or TRF2, respectively). Like TRF2 and Rap1, other components of the telomere-binding shelterin complex act as negative regulators of telomerase in mammals (4).
In S. cerevisiae, the pattern of telomere addition has been analyzed during a single S phase (48). Intriguingly, telomerase does not act on every telomere in each cell cycle but preferentially extends the shortest telomeres. At wild-type length, only 6 -8% of telomeres are extended, whereas the frequency increases to 50% upon shortening of telomeres to 100 nucleotides (48). These data suggest that the telomere switches between telomerase-extendible and telomerase-nonextendible states in a manner influenced by telomere length. The frequency of telomere elongation increases when Rap1 function is compromised, implying that Rap1 facilitates formation of a state that discourages telomerase action (48).
A series of recent publications point to a central role of the yeast homolog of the ATM (ataxia telangiectasia mutated) kinase, Tel1, in promoting telomerase activity at short telomeres (reviewed in Ref. 49). Tel1 preferentially associates with short telomeres in vivo and is required for telomerase association with, and activity on, these short telomeres. Cdc13 is a substrate of Tel1 (50), raising the possibility that Tel1-dependent phosphorylation of Cdc13 contributes to the regulation of telomerase recruitment. Telomerase also preferentially elongates critically short telomeres in mouse cells (51), suggesting that similar regulatory mechanisms may protect mammalian genome stability in the face of net telomere shortening.

Role of Telomerase in Disease
Because telomerase activity is low or absent in most human cells, telomeres shorten with each division. When telomeres reach a threshold length, checkpointmediated cellular senescence is initiated, halting cellular division ( Fig. 2A). As a result, telomere length serves as a cellular "clock" to limit proliferation. This limitation may contribute to the aging process in both normal and disease states, whereas inappropriate activation of telomerase allows unregulated cell growth (Fig. 2) (52).
Role of Telomerase in Degenerative Diseases-Much of our understanding of the role of telomerase in human health comes from studies of diseases of telomerase deficiency. Dyskeratosis congenita (a disease associated with progressive bone marrow failure and defects in other highly proliferative tissues) results from mutations affecting the stability or activity of telomerase ( Fig. 2A) (53). Autosomal dominant dyskeratosis congenita is associated with haploinsufficiency of hTR or hTERT, whereas some recessive forms are linked to genes with roles in hTR maturation and stability (dyskerin, NHP2, and NOP10) (53)(54)(55). The recent demonstration that aplastic anemia and idiopathic pulmonary fibrosis can be caused by telomerase dysfunction provides further evidence of the critical role of telomerase in cell proliferation (53). Many of these diseases involve phenotypes typically associated with aging, suggesting that limitations on tissue renewal imposed by telomere shortening may contribute to the normal aging process (53).
Telomerase and Cancer-In humans, the endogenous limit imposed on proliferation may serve an important tumor-suppressive role (Fig. 2B). Rarely, a checkpoint-deficient cell continues dividing, causing telomeres to uncap. This phase (crisis) is characterized by frequent chromosomal fusions and rearrangements. The massive genome instability that results from these processes is usually lethal. Occasionally, telomerase can be reactivated to restore telomeres, thereby stabilizing the genome and allowing cancer cells to escape normal limits on cell proliferation (Fig. 2B) (39). Telomerase reactivation occurs in at least 85% of human tumors. Telomeres in the remaining tumors are maintained by an alternative, recombination-based mechanism termed ALT (56).

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B.

FIGURE 2.
Telomere dynamics during normal and abnormal cell growth. A, in general, germ cells express telomerase and maintain long telomeres (light gray), whereas absent or low expression of telomerase in somatic cells and adult stem cells is insufficient to prevent telomere shortening (medium gray). Cell proliferation slows or ceases when cell cycle checkpoints are activated by short telomeres (dashed line), perhaps contributing to normal aging. Individuals with reduced telomerase activity exhaust telomere reserves more rapidly (dark gray), leading to premature aging, particularly in highly proliferative tissues. B, shown is a model of telomerase activation during tumorigenesis. When cell cycle checkpoints fail, cells divide in the face of severe telomere loss. Telomere uncapping causes genome instability and lethality (crisis). Reactivation of telomerase (or initiation of ALT) allows cells to exit crisis and proliferate with short but stable telomeres.
Transcriptional regulation of hTERT is a major determinant of telomerase activity in cancer cells, but multiple other mechanisms for the tumor-specific up-regulation of telomerase have been proposed, such as gene amplification, alternative splicing, and changes in subcellular localization and phosphorylation of the enzyme (41). The relative importance of each of these mechanisms is incompletely understood.
Because of the near-ubiquitous activation of telomerase during oncogenesis, telomerase is an attractive target for anticancer drugs and gene therapy. Telomerase inhibitors can trigger apoptotic cell death when telomeres reach a critical length. In addition, hTR and hTERT regulatory sequences can be used to drive selective expression of cytotoxic agents in tumor cells. Telomerase is a candidate antigen for cancer vaccines aimed at harnessing the immune system to selectively target TERT-positive cancer cells. Clinical trials testing telomerase inhibitors and vaccine-type approaches are currently under way (57).

Summary
By distinguishing normal and broken chromosome ends and providing a mechanism for complete replication, telomeres play an essential role in safeguarding genomic integrity. The enzyme telomerase, a unique RT with an intrinsic RNA template, is responsible for synthesizing telomeric DNA. A myriad of regulatory events ensure that the shortest telomeres are specifically targeted for replication by telomerase and that telomere length is appropriately modulated. Future work will continue to address the full complement of proteins that comprise the telomerase complex and the mechanisms that regulate telomerase access to telomeres. Such understanding is critical to our ability to manipulate telomerase function in disease where both inappropriate up-and down-regulation of telomerase have detrimental consequences.