Telomerase Mechanism of Telomere Synthesis

Abstract

Telomerase is the essential reverse transcriptase required for linear chromosome maintenance in most eukaryotes. Telomerase supplements the tandem array of simple-sequence repeats at chromosome ends to compensate for the DNA erosion inherent in genome replication. The template for telomerase reverse transcriptase is within the RNA subunit of the ribonucleoprotein complex, which in cells contains additional telomerase holoenzyme proteins that assemble the active ribonucleoprotein and promote its function at telomeres. Telomerase is distinct among polymerases in its reiterative reuse of an internal template. The template is precisely defined, processively copied, and regenerated by release of single-stranded product DNA. New specificities of nucleic acid handling that underlie the catalytic cycle of repeat synthesis derive from both active site specialization and new motif elaborations in protein and RNA subunits. Studies of telomerase provide unique insights into cellular requirements for genome stability, tissue renewal, and tumorigenesis as well as new perspectives on dynamic ribonucleoprotein machines.

Keywords: DNA replication; reverse transcriptase; ribonucleoprotein biogenesis; telomere.

Figure 1

Telomere structures for end protection. The telomeric repeat DNA structure and telomere-associated proteins important for chromosome end protection are illustrated for (a,b) two ciliates, (c,d ) two yeasts, and (e) vertebrate cells. Typical telomeric repeat tract lengths in each organism are indicated as base pairs of duplex and nucleotides of 3′ overhang, highlighting the evolutionary divergence of overall telomere and overhang length. Telomere proteins in direct contact with double-stranded DNA are colored blue-green. Proteins other than in CST that are in direct contact with single-stranded DNA are colored brown. Proteins that are orthologous are similarly colored in both this figure and Figure 4 using a shared coloring scheme. Other subunits colored in gray are indirectly bound to DNA. Saccharomyces cerevisiae proteins with functions other than end protection are not labeled; these proteins contribute to telomere length regulation and heterochromatin assembly. Abbreviations: bp, base pair; Cdc, cell division cycle; Ccq, coiled-coil protein quantitatively enriched; CST, heterotrimer containing Stn1, Ten1, and variable third subunit; nt, nucleotide; Pat, Pot1-associated Tetrahymena; Pot, protection of telomeres; Poz, Pot1-associated protein; Rap1, repressor/activator protein 1; Stn, suppressor of Cdc13; Taz, telomere-associated in Schizosaccharomyces pombe; TEBP, telomere end–binding protein; Ten1, telomeric pathways in association with Stn1, number 1; TIN2, TRF1-interacting nuclear protein 2; TPP1, vertebrate shelterin protein designation for proteins, initially named TINT1/PTOP/PIP1; Tpt, TPP1/Tpz1 in Tetrahymena thermophila; Tpz, TPP1 homolog in S. pombe; TRF, telomeric repeat binding factor.

Figure 2

The telomerase catalytic core. (a) Schematic of human, Tetrahymena thermophila, and Saccharomyces cerevisiae TERTs. TERT is composed of the TEN domain, linker, and TERT ring containing TRBD, RT domain, and CTE/thumb domains. Domain boundaries are labeled according to the TERT amino acid sequence. The TERT T-motif and motifs 1, 2, 3, A, IFD, B, C, D, and E are conserved across evolution (38). (b) The 2.7 Å –resolution structure of Tribolium castaneum TERT ring with DNA and RNA base paired as a primer–template duplex in the active site (40). The illustration was rendered from Protein Data Bank 3KYL with RNA in green and DNA in dark blue. Each TERT domain was given a different color. Tribolium TERT lacks a TEN domain. (c) TERs share conserved functional motifs including the template, pseudoknot, TBE, and template-distal TERT-binding motif corresponding to CR 4/5 in hTR, stem IV loop in T. thermophila TER, and the TWJ in S. cerevisiae TLC1. Species-specific TER binding sites for proteins involved in RNP biogenesis are indicated in purple. Abbreviations: CR, conserved region; CTE, C-terminal extension; Est1, ever-shorter telomeres protein 1; H/ACA, hairpin-H box-hairpin-ACA motif; hTR, human TER; IFD, insertion in fingers domain; Ku, dimeric protein complex that binds to DNA double-stranded break ends; nt, nucleotide; Pop, processing of precursor RNAs; RNP, ribonucleoprotein; RT, reverse transcriptase; Sm, proteins identified by Sm serotype antibodies from patients with autoimmune disease; TBE, template boundary element; TEN, TERT N-terminal; TER, telomerase RNA; TERT, telomerase reverse transcriptase; TLC1, S. cerevisiae TER identified as telomerase component 1; TRBD, TERT-specific high-affinity RNA-binding domain; TRE, template recognition element; TWJ, three-way junction.

Figure 3

The telomerase catalytic cycle. The telomerase catalytic cycle begins with base pairing of DNA primer (blue) to the template 3′ end, while the template 5′ end loops out (top left). After binding the duplex and dNTP, the active site closes to form the elongation-competent conformation (step ❶). Elongation proceeds until the template 5′ boundary, which is typically defined by a steric barrier, is reached (steps ❷ and ❸). As the template 5′ region is reeled into the active site, the template 3′ region and flanking RNA are displaced. If only 5–7 base pairs are stabilized by the telomerase active site, the initial duplex displaced by new repeat synthesis may fray in a manner that affects the conformation and positioning of the template 3′ end and flanking RNA (step ❹). This fraying could occur during or after repeat synthesis; it is shown here as occurring after repeat synthesis for illustration simplicity. We suggest that the displaced template 3′ end and flanking RNA favor a substantial active site opening necessary for strand separation (step ❺). DNA previously base paired to the template could retain and/or form additional protein contacts with an SRS of TERT, which holds the newly synthesized repeat of DNA while the template translocates (step ❻). After template translocation, default placement of the template 3′ end near the DNA 3′ end would promote the formation of a short duplex (step ❼). If this short duplex is captured into the TERT ring central cavity by the conformational changes necessary to restore a functional active site, another round of repeat synthesis begins. Alternatively, product release could occur prior to reestablishment of the active site. Abbreviations: dNTP, deoxynucleotide triphosphate; SRS, single-stranded DNA retention surface; TER, telomerase RNA; TERT, telomerase reverse transcriptase.

Figure 4

Telomerase holoenzymes are illustrated for (a) Tetrahymena thermophila, (b) Saccharomyces cerevisiae, (c) Schizosaccharomyces pombe, and (d ) vertebrates. TERs are labeled TER, TLC1, or TER1; they are greatly simplified in secondary structure representation. TERT proteins are shaded blue and labeled TERT, Est2, or Trt1. Other colors group proteins with generally related biological functions. Green arrows indicate interactions important for G-strand synthesis, and black arrows indicate interactions thought to be important for C-strand synthesis. Abbreviations: Ccq1, coiled-coil protein quantitatively enriched 1; Cdc13, cell division cycle protein 13; CST, heterotrimer containing Stn1, Ten1, and variable third subunit; Est, ever-shorter telomeres; H/ACA, hairpin-H box-hairpin-ACA motif; Ku, dimeric protein complex that binds to DNA double-strand break ends; Lsm, like Sm; nt, nucleotide; PolαPrimase, polymerase α primase; p19-p45-p75, Tetrahymena telomerase CST with subunits of 19, 45, and 75 kDa; p65, Tetrahymena telomerase subunit of 65 kDa; Pop, processing of precursor RNAs; Sm proteins, proteins identified by Sm serotype antibodies from patients with autoimmune disease; Stn1, suppressor of cdc thirteen 1; TCAB1, telomerase and Cajal body protein 1; Teb1, telomeric repeat binding subunit 1; TEB, Teb1 heterotrimer complex; Ten1, telomeric pathways in association with Stn1, number 1; TER, telomerase RNA; TERT, telomerase reverse transcriptase; TLC1, S. cerevisiae TER identified as telomerase component 1; TPP1, vertebrate shelterin protein designation for proteins initially named TINT1/PTOP/PIP1; Tpz1, TPP1 homolog in S. pombe; Trt1, TERT of S. pombe.

Figure 5

Pathways of telomerase biogenesis. Cellular trafficking for biogenesis of (a) Saccharomyces cerevisiae and (b) human telomerase holoenzymes is illustrated using a light gray background for nuclear localization and a light blue background for cytoplasmic localization. TERT proteins are labeled TERT or Est2 and are shaded blue. Other colors group proteins with generally related biological functions. The 5′ TMG cap added to TLC1 and hTR is shown as a yellow hexagon. Individual steps of trafficking are described in the text. Biogenesis pathways are illustrated to finish with the nucleoplasmic localization of telomerase holoenzyme that precedes its binding to telomeres. Abbreviations: Est, ever-shorter telomeres; GAR1, glycine/arginine-rich domain protein 1; hTR, human telomerase RNA; Ku, dimeric protein complex that binds to DNA double-stranded break ends; NAF1, nuclear assembly factor 1; NHP2, nonhistone chromatin protein 2; Pop, processing of precursor RNAs; RNAP II, RNA polymerase II; Sm ring, heteroheptamer of Sm proteins; TCAB1, telomerase and Cajal body protein 1; TERT, telomerase reverse transcriptase; Tgs1, trimethylguanosine synthase 1; TLC1, S. cerevisiae TER identified as telomerase component 1; TMG, trimethylguanosine.