Downloaded from http://cshperspectives.cshlp.org/ on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Replication of Telomeres and the Regulation of Telomerase Verena Pfeiffer and Joachim Lingner Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Frontiers in Genetics National Center of Competence in Research, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), 1015 Lausanne, Switzerland Correspondence: joachim.lingner@epfl.ch Telomeres are the physical ends of eukaryotic chromosomes. They protect chromosome ends from DNA degradation, recombination, and DNA end fusions, and they are important for nuclear architecture. Telomeres provide a mechanism for their replication by semiconserva- tive DNA replication and length maintenance by telomerase. Through telomerase repression and induced telomere shortening, telomeres provide the means to regulate cellular life span. In this review, we introduce the current knowledge on telomere composition and structure. We then discuss in depth the current understanding of how telomere components mediate their function during semiconservative DNA replication and how telomerase is regulated at the end of the chromosome. We focus our discussion on the telomeres from mammals and the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. erman Muller and Barbara McClintock Barbara McClintock analyzed maize strains in Hwere probably the first scientists to recog- which dicentric chromosomes were produced nize that the ends of eukaryotic chromosomes with high frequency. Dicentric chromosomes have special properties and crucial functions break when the two centromeres are pulled (reviewed by Blackburn 2006). Herman Muller toward opposite poles of the mitotic spindle studied Drosophila chromosomes and found during anaphase of the cell cycle. The broken that X rays could induce chromosome rear- chromosomal ends were unstable and fused rangements. However, he never observed chro- with other broken ends with which they came mosomes carrying terminal deletions (Muller in contact. However, when dicentric chromo- 1938). These results led him to conclude that somes were present in embryonic cells, the bro- “the terminal gene must have a special function, ken ends were somehow “healed” (McClintock that of sealing the end of the chromosome, so to 1941). speak,” and that “for some reason, a chromo- The molecular sequence of telomeric DNA some cannot persist indefinitely without having was first determined by Elizabeth Blackburn and its ends thus sealed.” Muller coined the term Joseph Gall in the ciliated protozoan Tetrahy- “telomere” for this terminal gene (from the mena thermophila (Blackburn and Gall 1978). Greek words telos ¼ end and meros ¼ part). These experiments revealed that the DNA ends Editors: Stephen D. Bell, Marcel Me´chali, and Melvin L. DePamphilis Additional Perspectives on DNA Replication available at www.cshperspectives.org Copyright # 2013 Cold Spring Harbor Laboratory Press; all rights reserved. Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a010405 1 Downloaded from http://cshperspectives.cshlp.org/ on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press V. Pfeiffer and J. Lingner 0 0 0 in this organism consist of 5 -T2G4-3 /5 -C4A2- identification of the first Tert subunits opened 30 telomeric repeats with the G-rich strand run- the door to identify the corresponding genes ning toward the 30 end of the chromosome. in humans and other organisms, allowing the Cloning and sequencing of telomeres from oth- study of telomerase during development and er eukaryotes revealed that the repetitive nature in cancer. Significantly, it was shown that ectop- of telomeric repeats and the presence of a G-rich ic expression of human Tert in various pri- strand are common features of nearly all eu- mary human cells is sufficient to reconstitute karyotes. Furthermore, it has been established telomerase, rendering them immortal (Bodnar that the 30-end-containing strand protrudes at et al. 1998). both ends of the chromosome. However,notable Ciliated protozoa were also instrumental in exceptions do exist. The roundworm Caeno- purifying and cloning the first eukaryotic telo- rhabditis elegans contains both 30 G-overhangs mere-binding proteins (Gottschling and Zakian as well as 50 C-overhangs (Raices et al. 2008). 1986; Hicke et al. 1990; Gray et al. 1991). How- Several genera of insects and plants do contain ever, orthologs of these proteins were at first long DNA repeats (Martinez et al. 2001). Droso- not recognized in other eukaryotes, and inde- phila contains, instead of short telomeric re- pendent approaches were undertaken to identify peats, retrotransposons at chromosome ends the first telomeric proteins invarious eukaryotes that have overtaken telomere functions (Pardue including yeasts and humans. Only much later and Debaryshe 2011). was it realized that the ciliate telomere-binding The formulation of the DNA end-replica- proteins contain counterparts in a broad range tion problem by Watson and Olovnikov in- of eukaryotes (Baumann and Cech 2001). ferred that specialized mechanisms must exist In this review, we introduce known basic to maintain telomere length (Olovnikov 1971; components of telomeres and review the mech- Watson 1972). Greider and Blackburn discov- anisms of semiconservative DNA replication of ered telomerase activity in extracts from Tet ra- telomeric DNA and the regulation of telome- hymena (Greider and Blackburn 1985). They rase. For the discussion of telomere protection also identified the telomerase RNA moiety in and the three-dimensional structures of telo- this organism, which contains a sequence com- mere components, the reader is referred to ex- plementary to Tetrahymena telomeric repeats cellent recent reviews (de Lange 2009; Jain and (Greider and Blackburn 1989). Mutation of Cooper 2010; Lewis and Wuttke 2012). this sequence proved that the telomerase RNA moiety provides the template for DNA repeat TELOMERE COMPONENTS synthesis and that the telomerase functions as reverse transcriptase (Yu et al. 1990). Around In the following section, the stable constituents the same time, Lundblad and Szostak were the of telomeres that are implicated in regulating first to discover an essential telomere mainte- telomere replication are discussed. Abundant nance protein gene in Saccharomyces cerevisiae. telomere-binding proteins as well as telomerase Dysfunction of the identified gene gave rise to subunits from S. cerevisiae, Schizosaccharomyces an ever shorter telomeres (est) phenotype cul- pombe, and Homo sapiens described in detail in minating in cellular senescence (Lundblad and the text are summarized in Table 1. Szostak 1989). The discovery of EST1 was fol- lowed by the identification of EST2, EST3, and S. cerevisiae EST4 in the Lundblad laboratory (Lendvay et al. 1996). Est2 turned out to be orthologous with The telomeric DNA repeat consensus sequence the p123 telomerase reverse transcriptase (Tert) for the 30-end-containing strand from S. cerevi- 0 0 protein subunit (Lingner et al. 1997) that was siae is 5 -(TG)0–6TGGGTGTG(G)-3 (Forste- identified in the ciliate Euplotes aediculatus mann and Lingner 2001). The telomere length upon biochemical purification of the telome- is around 300 bp. Native chromosome ends in rase enzyme (Lingner and Cech 1996). The S. cerevisiae have a number of subtelomeric 2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a010405 Downloaded from Advanced Online Article. Cite this article as http://cshperspectives.cshlp.org/ Table 1. Abundant telomere-binding proteins and telomerase proteins in S. cerevisiae, S. pombe, and H. sapiens Organism Subcomplex Protein Interaction partner Ortholog Function S. cerevisiae Rap1 Sir3, Sir4, or Rif1, Rap1 (S. p., H. s.) Essential; major double-stranded telomere-binding Rif2 protein; transcriptional regulation of telomeres and protein-coding genes; protection from NHEJ; negative regulator of telomere length Sirtuins Sir2 Sir3, Sir4 NADþ-dependent histone deacetylase; telomeric silencing Sir3 Sir2, Sir4, Rap1 Telomeric silencing Sir4 Sir2, Sir3, Rap1 Telomeric silencing onOctober6,2021-PublishedbyColdSpringHarborLaboratoryPress Rifs Rif1 Rif2, Rap1 Negative regulator of telomere length Cold Spring Harb Perspect Biol Rif2 Rif1, Rap1 Negative regulator of telomere length CST Cdc13 Ten1, Stn1, Est1, Ctc1 (H. s.; limited Essential G-strand single-stranded telomere-binding Pol1 sequence protein; protects from C-strand loss; recruits similarity) telomerase through interaction with Est1; essential for telomerase activity in vivo but not in vitro; interacts with Pol1, the catalytic subunit of DNA polymerase a-primase, promoting fill-in synthesis of telomerase-elongated telomeres doi: 10.1101 Stn1 Ten1, Cdc13, Pol12 Stn1 (S. p., H. s.) Essential; protects from C-strand loss; negative regulator of telomere length; interacts with Pol12, the B subunit of DNA polymerase a-primase, promoting fill-in synthesis of telomerase-elongated / cshperspect.a010405 telomeres Ten1 Stn1, Cdc13 Ten1 (S. p., H. s.) Essential; protects from C-strand loss; negative regulator of telomere length Telomerase Est1 Tlc1, Est2, Cdc13 Est1 (S. p., H. s. est phenotype; binds Tlc1 and G-strand single- Est1A has limited stranded telomeric DNA; recruits telomerase Telomere Replication sequence through interaction with Cdc13; essential for similarity) telomerase activity in vivo but not in vitro Est2 Tlc1,