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Back to the future: The intimate and evolving connection between telomere-related factors and genotoxic stress

Open AccessPublished:August 21, 2019DOI:https://doi.org/10.1074/jbc.AW119.008145
      The conversion of circular genomes to linear chromosomes during molecular evolution required the invention of telomeres. This entailed the acquisition of factors necessary to fulfill two new requirements: the need to fully replicate terminal DNA sequences and the ability to distinguish chromosome ends from damaged DNA. Here we consider the multifaceted functions of factors recruited to perpetuate and stabilize telomeres. We discuss recent theories for how telomere factors evolved from existing cellular machineries and examine their engagement in nontelomeric functions such as DNA repair, replication, and transcriptional regulation. We highlight the remarkable versatility of protection of telomeres 1 (POT1) proteins that was fueled by gene duplication and divergence events that occurred independently across several eukaryotic lineages. Finally, we consider the relationship between oxidative stress and telomeres and the enigmatic role of telomere-associated proteins in mitochondria. These findings point to an evolving and intimate connection between telomeres and cellular physiology and the strong drive to maintain chromosome integrity.

      Introduction

      Molecular evolution is opportunistic, enabling novel cellular mechanisms to arise in response to biological challenges. One such challenge was conversion of the circular prokaryotic genome into the multiple linear DNA forms that comprise the eukaryotic genome (
      • Cavalier-Smith T.
      Origin of the cell nucleus, mitosis and sex: roles of intracellular coevolution.
      ). This challenge necessitated the invention of telomeres. Here we discuss the origin and evolution of telomere-related functions. Although the factors associated with chromosome ends were initially thought to be specific for this locale, in-depth analysis has revealed many such factors having noncanonical, so-called “moonlighting” roles in other transactions within the nucleus and the cytoplasm. We now appreciate that some of the moonlighting contributions may reflect ancestral functions preserved from the dawn of genome linearization, whereas others may be newly emergent.
      There are several theories for how linear chromosomes evolved from their circular progenitors (
      • Ishikawa F.
      • Naito T.
      Why do we have linear chromosomes? A matter of Adam and Eve.
      ,
      • Volff J.-N.
      • Altenbuchner J.
      A new beginning with new ends: linearisation of circular chromosomes during bacterial evolution.
      ), but one of the more intriguing proposals is that invasion of circular genomes by group II introns (
      • Cavalier-Smith T.
      Origin of the cell nucleus, mitosis and sex: roles of intracellular coevolution.
      ), via reverse splicing and reverse transcription, led to DNA linearization (
      • Eickbush T.H.
      Molecular biology: telomerase and retrotransposons: which came first?.
      ,
      • de Lange T.
      A loopy view of telomere evolution.
      ) (Fig. 1). Specifically, it is posited that non-LTR
      The abbreviations used are: LTR
      long terminal repeat
      DSB
      double-strand break
      NHEJ
      nonhomologous DNA end joining
      OB-fold
      oligonucleotide/oligosaccharide-binding fold
      ROS
      reactive oxygen species
      8-oxoG
      8-oxo-guanine
      Tg
      thymine glycol
      BER
      base excision repair
      MTS
      mitochondrial targeting sequence.
      retrotransposons targeted to double-strand breaks (DSBs) served as “proto-telomeres” (
      • Garavís M.
      • González C.
      • Villasante A.
      On the origin of the eukaryotic chromosome: The role of noncanonical DNA structures in telomere evolution.
      ). The nascent chromosome ends presented two immediate challenges: the “end replication” problem and need for “end protection” (
      • Lingner J.
      • Cooper J.P.
      • Cech T.R.
      Telomerase and DNA end replication: no longer a lagging strand problem?.
      ,
      • de Lange T.
      How telomeres solve the end-protection problem.
      ). The end replication problem occurs because the DNA replication machinery cannot fully replicate the extreme terminus of the lagging strand, which would lead to the gradual depletion of terminal DNA sequences when the genome is duplicated (
      • Watson J.D.
      Origin of concatemeric T7 DNA.
      ,
      • Olovnikov A.M.
      A theory of marginotomy: the incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon.
      ). The chromosome ends may also be perceived as a DSB and must therefore be sequestered to prevent activation of the DNA damage response. Such end protection is also crucial for the avoidance of end-to-end fusions of chromosomes, which would cause improper chromosome segregation during mitosis, cell cycle arrest, genome instability, senescence, and cell death (
      • de Lange T.
      How telomeres solve the end-protection problem.
      ,
      • Sfeir A.
      • de Lange T.
      Removal of shelterin reveals the telomere end-protection problem.
      ). Most eukaryotes cope with these problems by 1) adding long arrays of noncoding DNA repeats to serve as a physical buffer to protect coding regions from attrition and 2) formation of higher-order DNA architecture that helps distinguish chromosome ends from a DSB (i.e. fold-back structures in yeast (
      • de Bruin D.
      • Kantrow S.M.
      • Liberatore R.A.
      • Zakian V.A.
      Telomere folding is required for the stable maintenance of telomere position effects in yeast.
      ) and t-loops in other species (
      • de Lange T.
      T-loops and the origin of telomeres.
      ,
      • Griffith J.D.
      • Comeau L.
      • Rosenfield S.
      • Stansel R.M.
      • Bianchi A.
      • Moss H.
      • de Lange T.
      Mammalian telomeres end in a large duplex loop.
      )).
      Figure thumbnail gr1
      Figure 1Model for evolution of telomerase and telomere-associated components from group II retrotransposons and DNA repair proteins. After chromosome linearization by the insertion of group II retrotransposons, telomerase and DNA repair proteins evolved roles in telomere maintenance and end protection. Telomere-associated factors also participate genome-wide in transcription, replication, and repair. Other factors function in mitochondria to modulate the response to oxidative stress. Whether the mitochondrial functions of telomere proteins reflect an ancient or newly evolved function is unknown (see text for details).

      Emergence of telomerase

      To help overcome the telomere end replication problem, a group II intron likely gained the ability to use the 3′ end of linearized chromosomes as a template for reverse transcription (
      • de Lange T.
      A loopy view of telomere evolution.
      ). There is strong evidence that the telomerase catalytic subunit TERT evolved from a non-LTR class 2 retrotransposon (
      • Nakamura T.M.
      • Cech T.R.
      Reversing time: origin of telomerase.
      ,
      • Belfort M.
      • Curcio M.J.
      • Lue N.F.
      Telomerase and retrotransposons: reverse transcriptases that shaped genomes.
      • Pardue M.-L.
      • DeBaryshe P.G.
      Retrotransposons that maintain chromosome ends.
      ) (Fig. 1). Fruit flies and silkworms maintain their chromosome ends through a telomerase-independent mechanism that employs a different class of retrotransposons (
      • Tatsuke T.
      • Sakashita K.
      • Masaki Y.
      • Lee J.M.
      • Kawaguchi Y.
      • Kusakabe T.
      The telomere-specific non-LTR retrotransposons SART1 and TRAS1 are suppressed by Piwi subfamily proteins in the silkworm, Bombyx mori.
      ,
      • Silva-Sousa R.
      • López-Panadès E.
      • Casacuberta E.
      Drosophila telomeres: an example of co-evolution with transposable elements.
      ), supporting the idea that retrotransposons played an early and critical role in establishing and maintaining telomere architecture (
      • Luan D.D.
      • Korman M.H.
      • Jakubczak J.L.
      • Eickbush T.H.
      Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition.
      ,
      • Han J.S.
      Non-long terminal repeat (non-LTR) retrotransposons: mechanisms, recent developments, and unanswered questions.
      ).
      The modern-day enzyme that helps solve the end replication problem is telomerase, a reverse transcriptase that compensates for incomplete replication by continually replenishing terminal DNA using a long noncoding RNA, TER, as template (
      • Greider C.W.
      • Blackburn E.H.
      A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis.
      ). It is possible that TER arose from a transcript derived from the progenitor group II intron (
      • de Lange T.
      A loopy view of telomere evolution.
      ) (Fig. 1), but TER and TERT are now encoded from separate loci in the genome. TERT can interact with a large array of RNAs in vivo (
      • Maida Y.
      • Yasukawa M.
      • Furuuchi M.
      • Lassmann T.
      • Possemato R.
      • Okamoto N.
      • Kasim V.
      • Hayashizaki Y.
      • Hahn W.C.
      • Masutomi K.
      An RNA-dependent RNA polymerase formed by TERT and the RMRP RNA.
      ). Ultimately, an RNA emerged with a higher affinity for TERT, a short C-rich repeat that could serve as a telomere sequence template, and a stem-loop element abutting the template that could form a functional template boundary element to allow fidelity of telomere repeat addition by TERT (
      • Podlevsky J.D.
      • Chen J.J.-L.
      Evolutionary perspectives of telomerase RNA structure and function.
      ). Unlike TERT, TER is constrained by structure and not sequence (
      • Bhattacharyya A.
      • Blackburn E.H.
      Architecture of telomerase RNA.
      ,
      • Webb C.J.
      • Zakian V.A.
      Telomerase RNA is more than a DNA template.
      ). Consequently, TER sequences diverged and expanded to give accessory proteins a foothold in the RNP complex. These new telomerase proteins enabled RNP maturation and both positive and negative regulation of the enzyme (
      • Zappulla D.C.
      • Cech T.R.
      Yeast telomerase RNA: a flexible scaffold for protein subunits.
      ,
      • Zhang Q.
      • Kim N.-K.
      • Feigon J.
      Architecture of human telomerase RNA.
      ).

      Telomere-associated proteins: Origins and their role in telomere end protection

      In vertebrates and fission yeast, telomere end protection is mediated by shelterin (
      • de Lange T.
      Shelterin: the protein complex that shapes and safeguards human telomeres.
      ,
      • Moser B.A.
      • Nakamura T.M.
      Protection and replication of telomeres in fission yeast.
      ) (Fig. 2 and Table 1). Shelterin physically caps the telomere ends, preventing the termini from being recognized as DNA damage and suffering DNA attrition via nucleolytic processing and DNA damage checkpoint activation. Shelterin is composed of TRF1/TRF2 (SpTAZ1), which binds the duplex DNA, and POT1-TPP1/SpPot1-SpTpz1, which binds the 3′ single-strand extension on the extreme terminus (termed the G-overhang). Additional proteins bridge the two DNA-binding complexes (TIN2(SpPOZ1) and RAP1). In addition to end protection, shelterin controls telomerase access and therefore contributes to telomere length regulation (
      • de Lange T.
      Shelterin: the protein complex that shapes and safeguards human telomeres.
      ).
      Figure thumbnail gr2
      Figure 2Models for chromosome end protection. Human telomeres are protected by the shelterin complex. CST transiently associates with the telomeric G-overhang during S phase to facilitate replication of the C-rich telomeric strand. In budding yeast, CST provides a stable, protective cap on the G-overhang, and RAP1, a shelterin component ortholog, binds the duplex region of telomeric DNA. Arabidopsis telomeres are asymmetrical. Ku maintains a blunt end on one chromosome terminus, whereas the other end harbors a conventional G-overhang that is bound by CST. There are two functional POT1 paralogs in A. thaliana. AtPOT1a is a component of the telomerase RNP, whereas AtPOT1b promotes genome stability and is proposed to reside in the cytoplasm.
      Table 1Localization and functions of telomere-related components
      Figure thumbnail gr5
      In budding yeast, instead of chromosome end protection by shelterin, the G-overhang is stably bound by the CST complex, comprised of Cdc13(CTC1), STN1, and TEN1 proteins (
      • Price C.M.
      • Boltz K.A.
      • Chaiken M.F.
      • Stewart J.A.
      • Beilstein M.A.
      • Shippen D.E.
      Evolution of CST function in telomere maintenance.
      ) (Fig. 2 and Table 1). Notably, vertebrates also possess CST, but this complex only transiently associates with telomeres during S phase to promote telomeric DNA replication. CST is structurally related to the single-strand DNA-binding complex RPA and had likely evolved from the latter (
      • Sun J.
      • Yu E.Y.
      • Yang Y.
      • Confer L.A.
      • Sun S.H.
      • Wan K.
      • Lue N.F.
      • Lei M.
      Stn1-Ten1 is an Rpa2-Rpa3-like complex at telomeres.
      ,
      • Bryan C.
      • Rice C.
      • Harkisheimer M.
      • Schultz D.C.
      • Skordalakes E.
      Structure of the human telomeric Stn1-Ten1 capping complex.
      ). Interestingly, Drosophila lacks canonical telomere repeat arrays at its chromosome termini and yet encodes one or more proteins related to CST subunits (
      • Raffa G.D.
      • Raimondo D.
      • Sorino C.
      • Cugusi S.
      • Cenci G.
      • Cacchione S.
      • Gatti M.
      • Ciapponi L.
      Verrocchio, a Drosophila OB fold-containing protein, is a component of the terminin telomere-capping complex.
      ,
      • Biessmann H.
      • Mason J.M.
      Telomere maintenance without telomerase.
      ). Flowering plants, including Arabidopsis, present yet another twist on the telomere protection apparatus wherein one half of the chromosome ends harbor a G-overhang bound by CST, whereas the other half are blunt-ended and bound by Ku, which functions in the nonhomologous DNA end joining (NHEJ) pathway of DSB repair and has high affinity for DNA ends (
      • Kazda A.
      • Zellinger B.
      • Rössler M.
      • Derboven E.
      • Kusenda B.
      • Riha K.
      Chromosome end protection by blunt-ended telomeres.
      ) (Fig. 2). The asymmetry of plant telomeres may reflect the absence of a 5′ exonuclease (e.g. Apollo) (
      • Wu P.
      • van Overbeek M.
      • Rooney S.
      • de Lange T.
      Apollo contributes to G-overhang maintenance and protects leading-end telomeres.
      ) that normally converts blunt-end telomeres created from leading-strand synthesis into termini with the typical 3′ G-overhang (
      • Nelson A.D.L.
      • Shippen D.E.
      Blunt-ended telomeres: an alternative ending to the replication and end protection stories.
      ).
      The shelterin and CST proteins employ one of two DNA binding motifs: the MYB domain for duplex DNA binding and the oligonucleotide/oligosaccharide-binding fold (OB-fold) for interaction with single-strand DNA. The MYB motif is common in transcription factors and may have been predisposed to function at telomeres as it is capable of binding tandemly repeated sequences (
      • Du H.
      • Wang Y.-B.
      • Xie Y.
      • Liang Z.
      • Jiang S.-J.
      • Zhang S.-S.
      • Huang Y.-B.
      • Tang Y.-X.
      Genome-wide identification and evolutionary and expression analyses of MYB-related genes in land plants.
      ,
      • Horvath M.P.
      Evolution of telomere binding proteins. In Madame Curie Bioscience Database [Internet].
      ). OB-folds, on the other hand, function in a vast array of nucleic acid transactions and are found in proteins ranging from t-RNA synthetases to nucleases and RPA (
      • Theobald D.L.
      • Mitton-Fry R.M.
      • Wuttke D.S.
      Nucleic acid recognition by OB-fold proteins.
      ). Thus, the single-strand telomere-binding proteins have likely diverged from a common OB-fold ancestor with a role in DNA repair and/or replication (
      • Kerr I.D.
      • Wadsworth R.I.M.
      • Cubeddu L.
      • Blankenfeldt W.
      • Naismith J.H.
      • White M.F.
      Insights into ssDNA recognition by the OB fold from a structural and thermodynamic study of Sulfolobus SSB protein.
      ).

      Telomere protection and DNA repair

      DNA damage repair pathways and telomere-associated proteins act collaboratively to promote genome integrity. Both TERT and TER have been linked to the DNA damage response (Table 1). In the presence of a DSB, human TERT relocalizes to the nucleolus (
      • Wong J.M.Y.
      • Kusdra L.
      • Collins K.
      Subnuclear shuttling of human telomerase induced by transformation and DNA damage.
      ), an outcome that would decrease the probability of de novo telomere formation at sites of DNA damage. In addition, human cells lacking TERT fail to mount an effective DNA damage response to ionizing radiation (
      • Masutomi K.
      • Possemato R.
      • Wong J.M.Y.
      • Currier J.L.
      • Tothova Z.
      • Manola J.B.
      • Ganesan S.
      • Lansdorp P.M.
      • Collins K.
      • Hahn W.C.
      The telomerase reverse transcriptase regulates chromatin state and DNA damage responses.
      ). Intriguingly, these cells also display altered chromatin structure and fragmented chromosomes, suggesting that TERT plays a role in chromatin reorganization (
      • Park J.-I.
      • Venteicher A.S.
      • Hong J.Y.
      • Choi J.
      • Jun S.
      • Shkreli M.
      • Chang W.
      • Meng Z.
      • Cheung P.
      • Ji H.
      • McLaughlin M.
      • Veenstra T.D.
      • Nusse R.
      • McCrea P.D.
      • Artandi S.E.
      Telomerase modulates Wnt signalling by association with target gene chromatin.
      ). Human TER (hTR) has been proposed to play a TERT-independent role in the response to DNA damage. Inhibition of hTR causes rapid arrest of cell growth, whereas increased hTR, which occurs in response to DNA damage induced by UV light, inhibits the DNA damage checkpoint kinase ATR (
      • Kedde M.
      • le Sage C.
      • Duursma A.
      • Zlotorynski E.
      • van Leeuwen B.
      • Nijkamp W.
      • Beijersbergen R.
      • Agami R.
      Telomerase-independent regulation of ATR by human telomerase RNA.
      ). In contrast, loss of TR in mice does not trigger phenotypes distinct from those of mTERT mutants, suggesting that the core RNA and protein components of telomerase act in the same pathways (
      • Blasco M.A.
      • Lee H.W.
      • Hande M.P.
      • Samper E.
      • Lansdorp P.M.
      • DePinho R.A.
      • Greider C.W.
      Telomere shortening and tumor formation by mouse cells lacking telomerase RNA.
      ,
      • Strong M.A.
      • Vidal-Cardenas S.L.
      • Karim B.
      • Yu H.
      • Guo N.
      • Greider C.W.
      Phenotypes in mTERT+/− and mTERT−/− mice are due to short telomeres, not telomere-independent functions of telomerase reverse transcriptase.
      ).
      Shelterin proteins also modulate the DNA damage response (Table 1). TRF2, for instance, prevents ATM-mediated DNA damage signaling at telomeres (
      • Karlseder J.
      • Hoke K.
      • Mirzoeva O.K.
      • Bakkenist C.
      • Kastan M.B.
      • Petrini J.H.J.
      • de Lange T.
      The telomeric protein TRF2 binds the ATM kinase and can inhibit the ATM-dependent DNA damage response.
      ) and also helps recruit various DNA damage response and repair factors, such as ERCC1, Apollo, the MRE11-RAD50-NBS1 complex, helicases BLM and WRN, Ku, and PARP1/2 (
      • Xin H.
      • Liu D.
      • Songyang Z.
      The telosome/shelterin complex and its functions.
      ) (Table 1). The recruitment of these factors facilitates telomeric DNA replication, promotes the formation of a single-strand overhang on the chromosome terminus, and ensures that telomeres are properly sequestered to prevent inappropriate recombination or activation of a DNA damage response (
      • Arnoult N.
      • Karlseder J.
      Complex interactions between the DNA-damage response and mammalian telomeres.
      ,
      • Vannier J.-B.
      • Depeiges A.
      • White C.
      • Gallego M.E.
      ERCC1/XPF protects short telomeres from homologous recombination in Arabidopsis thaliana.
      ). TRF2 can also associate with DSBs within the body of the chromosome as part of the early response to DNA damage (
      • Bradshaw P.S.
      • Stavropoulos D.J.
      • Meyn M.S.
      Human telomeric protein TRF2 associates with genomic double-strand breaks as an early response to DNA damage.
      ,
      • Williams E.S.
      • Stap J.
      • Essers J.
      • Ponnaiya B.
      • Luijsterburg M.S.
      • Krawczyk P.M.
      • Ullrich R.L.
      • Aten J.A.
      • Bailey S.M.
      DNA double-strand breaks are not sufficient to initiate recruitment of TRF2.
      ). As such, the ability of TRF2 to engage the machineries concerned with the DNA damage response and DNA repair likely promotes genome stability on a global scale. Interestingly, both TRF1 and TRF2 are modified by MMS21, a SUMO ligase involved in DNA repair and recombination. This modification is associated with alternative lengthening of telomeres (ALT) (
      • Potts P.R.
      • Yu H.
      The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins.
      ), a mechanism germane for telomere maintenance in cancer cells that lack telomerase (
      • Bryan T.M.
      • Englezou A.
      • Gupta J.
      • Bacchetti S.
      • Reddel R.R.
      Telomere elongation in immortal human cells without detectable telomerase activity.
      ).
      Like TRF2, TIN2 and RAP1 associate with chromosome locales other than the telomeres. TIN2 accumulates at nontelomeric regions (
      • Kaminker P.
      • Plachot C.
      • Kim S.-H.
      • Chung P.
      • Crippen D.
      • Petersen O.W.
      • Bissell M.J.
      • Campisi J.
      • Lelièvre S.A.
      Higher-order nuclear organization in growth arrest of human mammary epithelial cells: a novel role for telomere-associated protein TIN2.
      ) associated with HP1 (
      • Bártová E.
      • Malyšková B.
      • Komůrková D.
      • Legartová S.
      • Suchánková J.
      • Krejčí J.
      • Kozubek S.
      Function of heterochromatin protein 1 during DNA repair.
      ), a heterochromatin mark that has been implicated in the DNA damage response (
      • Dinant C.
      • Luijsterburg M.S.
      The emerging role of HP1 in the DNA damage response.
      ). Moreover, in human cells, RAP1 interacts with noncoding interstitial TTTAGGG repeats present on some chromosomes, raising the possibility that RAP1 helps prevent fragility and recombination at these sites (
      • Martinez P.
      • Thanasoula M.
      • Carlos A.R.
      • Gómez-López G.
      • Tejera A.M.
      • Schoeftner S.
      • Dominguez O.
      • Pisano D.G.
      • Tarsounas M.
      • Blasco M.A.
      Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites.
      ).
      POT1 has also been implicated in the DNA damage response (Table 1) (Fig. 3). The association of POT1 with the telomeric G-overhang prevents activation of an ATR-mediated DNA damage response (
      • Denchi E.L.
      • de Lange T.
      Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1.
      ), and recent studies indicate that human POT1 increases the fidelity of NHEJ at nontelomeric sites (
      • Yu Y.
      • Tan R.
      • Ren Q.
      • Gao B.
      • Sheng Z.
      • Zhang J.
      • Zheng X.
      • Jiang Y.
      • Lan L.
      • Mao Z.
      POT1 inhibits the efficiency but promotes the fidelity of nonhomologous end joining at non-telomeric DNA regions.
      ). Intriguingly, the C terminus of hPOT1 bears structural similarity to a Holliday junction resolvase domain (
      • Rice C.
      • Shastrula P.K.
      • Kossenkov A.V.
      • Hills R.
      • Baird D.M.
      • Showe L.C.
      • Doukov T.
      • Janicki S.
      • Skordalakes E.
      Structural and functional analysis of the human POT1-TPP1 telomeric complex.
      ), supporting the notion that POT1 affects other facets of DNA metabolism beyond telomere biology.
      Figure thumbnail gr3
      Figure 3Diverse functions of POT1. Many POT1 orthologs bind single-stranded G-rich telomeric DNA, serving to control telomere length and to protect chromosome ends from eliciting the DNA damage response. Other POT1 proteins are tailored to engage the telomeric C-strand and its replication machinery. There are also examples of POT1 proteins that do not stably engage the chromosome terminus, but rather function to stimulate telomerase activity or to facilitate DNA repair. In addition, several POT1 proteins have been shown to accumulate in the cytoplasm or are predicted to reside here. Cytoplasmic mouse POT1b (mPOT1b) is proposed to promote an innate immunity response. Shown are the A. thaliana POT1a (AtPOT1a) and POT1b (AtPOT1b); C. elegans POT1 proteins CeOB1, CeOB2, and MRT1; human POT1 (hPOT1), mouse POT1a (mPOT1a), and POT1b (mPOT1b); P. patens POT1 (PpPOT1); and T. thermophila POT1 (TtPOT1) and POT2 (TtPOT2).
      Ku harbors two subunits (Ku70 and Ku80) and is a core component of the NHEJ pathway (
      • Bertuch A.A.
      • Lundblad V.
      Which end: dissecting Ku's function at telomeres and double-strand breaks.
      ). Within the context of telomere biology, Ku facilitates telomere protection and telomeric DNA replication (
      • Kazda A.
      • Zellinger B.
      • Rössler M.
      • Derboven E.
      • Kusenda B.
      • Riha K.
      Chromosome end protection by blunt-ended telomeres.
      ,
      • Baumann P.
      • Cech T.R.
      Protection of telomeres by the Ku protein in fission yeast.
      ,
      • Gravel S.
      • Wellinger R.J.
      Maintenance of double-stranded telomeric repeats as the critical determinant for cell viability in yeast cells lacking Ku.
      ). Recent studies in budding yeast provide clues for how the DNA repair and telomere protection functions of Ku might be parsed at chromosome termini. Ku harbors two solvent-exposed α-helices on opposite sides of the heterodimer. The surface facing the telomere end is necessary for NHEJ, whereas the inward facing helix is required for telomeric heterochromatin formation (
      • Ribes-Zamora A.
      • Mihalek I.
      • Lichtarge O.
      • Bertuch A.A.
      Distinct faces of the Ku heterodimer mediate DNA repair and telomeric functions.
      ). In addition to discrete structural boundaries, separation of function can be influenced by cell cycle regulation. For example, the cell cycle regulator CYREN was recently shown to interact with Ku and block NHEJ at telomeres during the S and G2 cell cycle phases (
      • Arnoult N.
      • Correia A.
      • Ma J.
      • Merlo A.
      • Garcia-Gomez S.
      • Maric M.
      • Tognetti M.
      • Benner C.W.
      • Boulton S.J.
      • Saghatelian A.
      • Karlseder J.
      Regulation of DNA repair pathway choice in S and G2 phases by the NHEJ inhibitor CYREN.
      ). Ebrahimi and Cooper (
      • Ebrahimi H.
      • Cooper J.P.
      Finding a place in the SUN: telomere maintenance in a diverse nuclear landscape.
      ) have postulated that localization of telomeres within different regions of the nucleus influences a broad range of cellular processes, including meiotic recombination, chromosome segregation, and gene expression. Hence, in a broader sense, both temporal and spatial regulation of telomeres impact cellular physiology.

      A role for telomere-associated proteins in DNA replication and transcription

      Given that telomere accessory factors have likely evolved from factors that function in DNA repair, DNA replication, and transcription, it is not surprising that some of the telomere-associated factors also function in the aforementioned processes. Because of the highly repetitive nature of G-rich telomeric DNA and its propensity to form higher-order structures, such as the G-quartet, auxiliary factors are needed to ensure timely and proper replication through telomeric tracts. Notably, both POT1 and TRF2 stimulate the helicase activity of WRN (
      • Opresko P.L.
      • von Kobbe C.
      • Laine J.-P.
      • Harrigan J.
      • Hickson I.D.
      • Bohr V.A.
      Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases.
      ,
      • Machwe A.
      • Xiao L.
      • Orren D.K.
      TRF2 recruits the Werner syndrome (WRN) exonuclease for processing of telomeric DNA.
      ), and POT1 has been found to promote G-quartet unwinding by the WRN and BLM helicases (
      • Opresko P.L.
      • Mason P.A.
      • Podell E.R.
      • Lei M.
      • Hickson I.D.
      • Cech T.R.
      • Bohr V.A.
      POT1 stimulates RecQ helicases WRN and BLM to unwind telomeric DNA substrates.
      ) (Table 1). TRF2 has been proposed to assist in telomeric replication, and it does so by inducing positive supercoiling in DNA that favors enhanced access by DNA topoisomerases and the Apollo nuclease, enzymes critical for replication (
      • Amiard S.
      • Doudeau M.
      • Pinte S.
      • Poulet A.
      • Lenain C.
      • Faivre-Moskalenko C.
      • Angelov D.
      • Hug N.
      • Vindigni A.
      • Bouvet P.
      • Paoletti J.
      • Gilson E.
      • Giraud-Panis M.-J.
      A topological mechanism for TRF2-enhanced strand invasion.
      ,
      • Ye J.
      • Lenain C.
      • Bauwens S.
      • Rizzo A.
      • Saint-Léger A.
      • Poulet A.
      • Benarroch D.
      • Magdinier F.
      • Morere J.
      • Amiard S.
      • Verhoeyen E.
      • Britton S.
      • Calsou P.
      • Salles B.
      • Bizard A.
      • et al.
      TRF2 and Apollo cooperate with topoisomerase 2α to protect human telomeres from replicative damage.
      ). Furthermore, TRF2 is also hypothesized to assist in the assembly of the prereplication complex during telomere replication (
      • Deng Z.
      • Dheekollu J.
      • Broccoli D.
      • Dutta A.
      • Lieberman P.M.
      The origin recognition complex localizes to telomere repeats and prevents telomere-circle formation.
      ,
      • Tatsumi Y.
      • Ezura K.
      • Yoshida K.
      • Yugawa T.
      • Narisawa-Saito M.
      • Kiyono T.
      • Ohta S.
      • Obuse C.
      • Fujita M.
      Involvement of human ORC and TRF2 in pre-replication complex assembly at telomeres.
      ).
      The primary function of the CST heterotrimer appears to be in telomere replication (Table 1). Originally identified as a DNA Pol α accessory factor (
      • Casteel D.E.
      • Zhuang S.
      • Zeng Y.
      • Perrino F.W.
      • Boss G.R.
      • Goulian M.
      • Pilz R.B.
      A DNA polymerase α primase cofactor with homology to replication protein A-32 regulates DNA replication in mammalian cells.
      ), the vertebrate CST complex was subsequently shown to stimulate synthesis of the telomeric C-strand after telomerase extends the G-strand (
      • Chen L.-Y.
      • Redon S.
      • Lingner J.
      The human CST complex is a terminator of telomerase activity.
      ,
      • Nakaoka H.
      • Nishiyama A.
      • Saito M.
      • Ishikawa F.
      Xenopus laevis Ctc1-Stn1-Ten1 (xCST) protein complex is involved in priming DNA synthesis on single-stranded DNA template in Xenopus egg extract.
      • Feng X.
      • Hsu S.-J.
      • Bhattacharjee A.
      • Wang Y.
      • Diao J.
      • Price C.M.
      CTC1-STN1 terminates telomerase while STN1-TEN1 enables C-strand synthesis during telomere replication in colon cancer cells.
      ). CST plays a crucial role in the restart of stalled replication forks at nontelomeric sites (
      • Stewart J.A.
      • Wang F.
      • Chaiken M.F.
      • Kasbek C.
      • Chastain 2nd, P.D.
      • Wright W.E.
      • Price C.M.
      Human CST promotes telomere duplex replication and general replication restart after fork stalling.
      ), and CST mutations lead to genome-wide instability (
      • Surovtseva Y.V.
      • Churikov D.
      • Boltz K.A.
      • Song X.
      • Lamb J.C.
      • Warrington R.
      • Leehy K.
      • Heacock M.
      • Price C.M.
      • Shippen D.E.
      Conserved telomere maintenance component 1 interacts with STN1 and maintains chromosome ends in higher eukaryotes.
      ,
      • Miyake Y.
      • Nakamura M.
      • Nabetani A.
      • Shimamura S.
      • Tamura M.
      • Yonehara S.
      • Saito M.
      • Ishikawa F.
      RPA-like mammalian Ctc1-Stn1-Ten1 complex binds to single-stranded DNA and protects telomeres independently of the Pot1 pathway.
      ). Vertebrate CST only transiently engages telomeres (
      • Renfrew K.B.
      • Song X.
      • Lee J.R.
      • Arora A.
      • Shippen D.E.
      POT1a and components of CST engage telomerase and regulate its activity in Arabidopsis.
      ), but in budding yeast and in Arabidopsis thaliana, CST is a constitutive component of telomeres that facilitates both replication of the C-rich telomere strand by Pol α/primase and the G-rich strand by stimulating telomerase activity (
      • Surovtseva Y.V.
      • Churikov D.
      • Boltz K.A.
      • Song X.
      • Lamb J.C.
      • Warrington R.
      • Leehy K.
      • Heacock M.
      • Price C.M.
      • Shippen D.E.
      Conserved telomere maintenance component 1 interacts with STN1 and maintains chromosome ends in higher eukaryotes.
      ,
      • Renfrew K.B.
      • Song X.
      • Lee J.R.
      • Arora A.
      • Shippen D.E.
      POT1a and components of CST engage telomerase and regulate its activity in Arabidopsis.
      ,
      • Grandin N.
      • Damon C.
      • Charbonneau M.
      Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13.
      ). Hence, some of the POT1-TPP1 functions within the context of shelterin (
      • Nandakumar J.
      • Bell C.F.
      • Weidenfeld I.
      • Zaug A.J.
      • Leinwand L.A.
      • Cech T.R.
      The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity.
      ) may be fulfilled by CST. Indeed, Lue (
      • Lue N.F.
      Evolving linear chromosomes and telomeres: a C-strand-centric view.
      ) has provided a compelling argument that POT1-TPP1 evolved from CST. The multifunctional nature of CST is further evidenced by the involvement of components of the yeast complex in transcriptional regulation through interactions with RNA polymerase II and the elongation factor Spt5. The interactions of CST with the transcription machinery are thought to help mitigate the consequences of RNA polymerase II collision with replication forks (
      • Calvo O.
      • Grandin N.
      • Jordán-Pla A.
      • Miñambres E.
      • González-Polo N.
      • Pérez-Ortín J.E.
      • Charbonneau M.
      The telomeric Cdc13–Stn1–Ten1 complex regulates RNA polymerase II transcription.
      ). In addition, studies in Arabidopsis have revealed that the CST component TEN1 possesses protein chaperone activity that is activated in response to heat stress (
      • Lee J.R.
      • Xie X.
      • Yang K.
      • Zhang J.
      • Lee S.Y.
      • Shippen D.E.
      Dynamic interactions of Arabidopsis TEN1: stabilizing telomeres in response to heat stress.
      ) (Table 1).
      Besides CST, other telomere-associated proteins also influence transcriptional regulation (Table 1). Yeast RAP1 was originally described as a transcriptional regulator at many promoters (
      • Shore D.
      • Nasmyth K.
      Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements.
      ,
      • Shore D.
      RAP1: a protean regulator in yeast.
      ). Human RAP1 modulates NF-κB expression (
      • Teo H.
      • Ghosh S.
      • Luesch H.
      • Ghosh A.
      • Wong E.T.
      • Malik N.
      • Orth A.
      • de Jesus P.
      • Perry A.S.
      • Oliver J.D.
      • Tran N.L.
      • Speiser L.J.
      • Wong M.
      • Saez E.
      • Schultz P.
      • Chanda S.K.
      • Verma I.M.
      • Tergaonkar V.
      Telomere-independent Rap1 is an IKK adaptor and regulates NF-κB-dependent gene expression.
      ), whereas interaction of TRF2 with the promoter of the cyclin-dependent kinase CDKN1a affects its expression (
      • Hussain T.
      • Saha D.
      • Purohit G.
      • Kar A.
      • Kishore Mukherjee A.
      • Sharma S.
      • Sengupta S.
      • Dhapola P.
      • Maji B.
      • Vedagopuram S.
      • Horikoshi N.T.
      • Horikoshi N.
      • Pandita R.K.
      • Bhattacharya S.
      • Bajaj A.
      • et al.
      Transcription regulation of CDKN1A (p21/CIP1/WAF1) by TRF2 is epigenetically controlled through the REST repressor complex.
      ). TERT has also been reported to enhance the expression of genes such as cyclin D1 (
      • Hong J.
      • Lee J.H.
      • Chung I.K.
      Telomerase activates transcription of cyclin D1 gene through an interaction with NOL1.
      ) and NF-κB (
      • Ghosh A.
      • Saginc G.
      • Leow S.C.
      • Khattar E.
      • Shin E.M.
      • Yan T.D.
      • Wong M.
      • Zhang Z.
      • Li G.
      • Sung W.-K.
      • Zhou J.
      • Chng W.J.
      • Li S.
      • Liu E.
      • Tergaonkar V.
      Telomerase directly regulates NF-κB-dependent transcription.
      ).

      Gene duplication: Refining the landscape of telomere protein function

      Gene duplication has fueled protein evolution, including telomere proteins. The duplication event giving rise to vertebrate TRF1 and TRF2 dates back 540 million years ago (
      • Poulet A.
      • Pisano S.
      • Faivre-Moskalenko C.
      • Pei B.
      • Tauran Y.
      • Haftek-Terreau Z.
      • Brunet F.
      • Le Bihan Y.-V.
      • Ledu M.-H.
      • Montel F.
      • Hugo N.
      • Amiard S.
      • Argoul F.
      • Chaboud A.
      • Gilson E.
      • Giraud-Panis M.-J.
      The N-terminal domains of TRF1 and TRF2 regulate their ability to condense telomeric DNA.
      ), at the beginning of the Chordate lineage (
      • Dehal P.
      • Boore J.L.
      Two rounds of whole genome duplication in the ancestral vertebrate.
      ). The conserved C-terminal MYB domain of TRF1/2 facilitates telomeric DNA engagement, whereas divergent N-terminal domains (
      • Broccoli D.
      • Smogorzewska A.
      • Chong L.
      • de Lange T.
      Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2.
      ) are important for telomeric DNA length regulation (primarily accomplished by TRF1) (
      • Smogorzewska A.
      • van Steensel B.
      • Bianchi A.
      • Oelmann S.
      • Schaefer M.R.
      • Schnapp G.
      • de Lange T.
      Control of human telomere length by TRF1 and TRF2.
      ) or chromosome end protection (TRF2) (
      • van Steensel B.
      • Smogorzewska A.
      • de Lange T.
      TRF2 protects human telomeres from end-to-end fusions.
      ) (Table 1). The Candida clade possesses two copies of the gene that encodes the Cdc13 component of CST (
      • Lue N.F.
      • Chan J.
      Duplication and functional specialization of the telomere-capping protein Cdc13 in Candida species.
      ). The two paralogous proteins, Cdc13A and Cdc13B, are significantly smaller than their counterparts in budding yeast and have overlapping but nonredundant functions in telomere length regulation.
      One of the most fascinating outcomes of gene duplication is seen with POT1 (Fig. 3) (Table 1). Here, independent gene duplication events occurred repeatedly throughout evolution. Although humans have a single POT1 protein, mice possess two POT1 paralogs, mPOT1a and mPOT1b, that share 72% sequence similarity (
      • Hockemeyer D.
      • Daniels J.-P.
      • Takai H.
      • de Lange T.
      Recent expansion of the telomeric complex in rodents: two distinct POT1 proteins protect mouse telomeres.
      ). Recent studies suggest that both mPOT1a and mPOT1b attenuate ATR signaling at chromosome ends (
      • Kratz K.
      • de Lange T.
      ATR repression by POT1a and POT1b 1 Both protection of telomeres 1 proteins POT1a and POT1b can repress ATR signaling by RPA exclusion but binding to CST limits ATR repression by POT1b.
      ). However, mPOT1b uniquely contributes to the regulation of 5′ end resection to form the 3′ G-overhang (
      • Kratz K.
      • de Lange T.
      ATR repression by POT1a and POT1b 1 Both protection of telomeres 1 proteins POT1a and POT1b can repress ATR signaling by RPA exclusion but binding to CST limits ATR repression by POT1b.
      ) and may also play a cytosolic role in the innate immunity response (
      • Hagiwara M.
      • Komatsu T.
      • Sugiura S.S.
      • Isoda R.
      • Tada H.
      • Tanigawa N.
      • Kato Y.
      • Ishida N.
      • Kobayashi K.
      • Matsushita K.
      POT1b regulates phagocytosis and NO production by modulating activity of the small GTPase Rab5.
      ).
      The POT1 isoforms in worms and ciliated protozoa exhibit more profound functional divergence. Caenorhabditis elegans encodes four single OB-fold proteins with structural similarity to the OB-folds of mammalian POT1 (
      • Raices M.
      • Verdun R.E.
      • Compton S.A.
      • Haggblom C.I.
      • Griffith J.D.
      • Dillin A.
      • Karlseder J.
      C. elegans telomeres contain G-strand and C-strand overhangs that are bound by distinct proteins.
      ). CeOB1 binds the telomeric G-rich strand, whereas CeOB2 engages the complementary C-rich strand. Mutation in either of these CeOB genes leads to telomere elongation, providing evidence that their encoded proteins serve as a negative regulator of telomerase (
      • Cheng C.
      • Shtessel L.
      • Brady M.M.
      • Ahmed S.
      Caenorhabditis elegans POT-2 telomere protein represses a mode of alternative lengthening of telomeres with normal telomere lengths.
      ,
      • Shtessel L.
      • Lowden M.R.
      • Cheng C.
      • Simon M.
      • Wang K.
      • Ahmed S.
      Caenorhabditis elegans POT-1 and POT-2 repress telomere maintenance pathways.
      ). The function for CeOB3 is unknown; however, CeOB4 (MRT1) was originally identified in a screen for genes required for germ line mortality as a result of telomere shortening (
      • Meier B.
      • Barber L.J.
      • Liu Y.
      • Shtessel L.
      • Boulton S.J.
      • Gartner A.
      • Ahmed S.
      The MRT-1 nuclease is required for DNA crosslink repair and telomerase activity in vivo in Caenorhabditis elegans.
      ). CeOB4 is required for telomerase activity in vivo. Intriguingly, CeOB4 also bears a SNM1 family nuclease domain and has been implicated in both DNA cross-link and nucleotide excision repair (
      • Meier B.
      • Barber L.J.
      • Liu Y.
      • Shtessel L.
      • Boulton S.J.
      • Gartner A.
      • Ahmed S.
      The MRT-1 nuclease is required for DNA crosslink repair and telomerase activity in vivo in Caenorhabditis elegans.
      ).
      In the ciliates Euplotes crassus and Tetrahymena thermophila, there are two POT1 paralogs (
      • Wang W.
      • Skopp R.
      • Scofield M.
      • Price C.
      Euplotes crassus has genes encoding telomere-binding proteins and telomere-binding protein homologs.
      ,
      • Jacob N.K.
      • Lescasse R.
      • Linger B.R.
      • Price C.M.
      Tetrahymena POT1a regulates telomere length and prevents activation of a cell cycle checkpoint.
      ). The Tetrahymena TtPOT1-encoded protein is essential for telomere length maintenance and prevents checkpoint activation much like the vertebrate POT1 proteins (
      • Jacob N.K.
      • Lescasse R.
      • Linger B.R.
      • Price C.M.
      Tetrahymena POT1a regulates telomere length and prevents activation of a cell cycle checkpoint.
      ). However, TtPOT2 protein does not associate with chromosome ends, but instead localizes to internal sites in macronuclear chromosomes that are destined for developmentally programmed cleavage and de novo telomere formation (
      • Cranert S.
      • Heyse S.
      • Linger B.R.
      • Lescasse R.
      • Price C.
      Tetrahymena Pot2 is a developmentally regulated paralog of Pot1 that localizes to chromosome breakage sites but not to telomeres.
      ). In E. crassus, the telomere end-binding protein caps chromosome ends (
      • Wang W.
      • Skopp R.
      • Scofield M.
      • Price C.
      Euplotes crassus has genes encoding telomere-binding proteins and telomere-binding protein homologs.
      ). Replication telomere protein, the other POT1-like protein, is not associated with telomeres, but rather co-localizes with the replication apparatus as it moves through the macronuclear genome (
      • Skopp R.
      • Wang W.
      • Price C.
      rTP: a candidate telomere protein that is associated with DNA replication.
      ). This remarkable observation underscores the strong connection between telomere proteins and the DNA replication machinery.
      The plant kingdom is replete with large gene families, arising from both localized gene duplication and whole-genome duplication. It is therefore noteworthy that most POT1 genes in plants are not duplicated. The POT1 gene in the early diverging land plant Physcomitrella patens retains the ancestral functions of binding single-stranded G-rich telomeric DNA and protecting chromosome ends from fusion (
      • Shakirov E.V.
      • Perroud P.-F.
      • Nelson A.D.
      • Cannell M.E.
      • Quatrano R.S.
      • Shippen D.E.
      Protection of telomeres 1 is required for telomere integrity in the moss Physcomitrella patens.
      ). However, at least two independent POT1 duplications occurred in higher plants, one in the grasses and the other in the Brassicaceae family to which A. thaliana belongs (
      • Beilstein M.A.
      • Renfrew K.B.
      • Song X.
      • Shakirov E.V.
      • Zanis M.J.
      • Shippen D.E.
      Evolution of the telomere-associated protein POT1a in Arabidopsis thaliana is characterized by positive selection to reinforce protein–protein interaction.
      ). There are three POT1 paralogs in A. thaliana, AtPOT1a, AtPOT1b, and AtPOT1c (
      • Beilstein M.A.
      • Renfrew K.B.
      • Song X.
      • Shakirov E.V.
      • Zanis M.J.
      • Shippen D.E.
      Evolution of the telomere-associated protein POT1a in Arabidopsis thaliana is characterized by positive selection to reinforce protein–protein interaction.
      ,
      • Rossignol P.
      • Collier S.
      • Bush M.
      • Shaw P.
      • Doonan J.H.
      Arabidopsis POT1A interacts with TERT-V(I8), an N-terminal splicing variant of telomerase.
      ). AtPOT1a and AtPOT1b exhibit only 52% sequence similarity. AtPOT1a resembles the mammalian shelterin component TPP1 (
      • Nandakumar J.
      • Bell C.F.
      • Weidenfeld I.
      • Zaug A.J.
      • Leinwand L.A.
      • Cech T.R.
      The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity.
      ,
      • Wang F.
      • Podell E.R.
      • Zaug A.J.
      • Yang Y.
      • Baciu P.
      • Cech T.R.
      • Lei M.
      The POT1–TPP1 telomere complex is a telomerase processivity factor.
      ) in that it physically associates with the telomerase RNP and stimulates its repeat addition processivity (
      • Renfrew K.B.
      • Song X.
      • Lee J.R.
      • Arora A.
      • Shippen D.E.
      POT1a and components of CST engage telomerase and regulate its activity in Arabidopsis.
      ,
      • Surovtseva Y.V.
      • Shakirov E.V.
      • Vespa L.
      • Osbun N.
      • Song X.
      • Shippen D.E.
      Arabidopsis POT1 associates with the telomerase RNP and is required for telomere maintenance.
      ). However, unlike TPP1 (
      • Latrick C.M.
      • Cech T.R.
      POT1–TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation.
      ), AtPOT1a accumulates at telomeres only in S phase (
      • Surovtseva Y.V.
      • Shakirov E.V.
      • Vespa L.
      • Osbun N.
      • Song X.
      • Shippen D.E.
      Arabidopsis POT1 associates with the telomerase RNP and is required for telomere maintenance.
      ), indicating that it is not a stable component of the end protection complex. Initially, AtPOT1a was not thought to bind telomeric DNA (
      • Shakirov E.V.
      • McKnight T.D.
      • Shippen D.E.
      POT1-independent single-strand telomeric DNA binding activities in Brassicaceae.
      ), but a recent study showed that the first OB-fold of AtPOT1a has single-strand telomeric DNA-binding activity (
      • Arora A.
      • Beilstein M.A.
      • Shippen D.E.
      Evolution of Arabidopsis protection of telomeres 1 alters nucleic acid recognition and telomerase regulation.
      ). Strikingly, the AtPOT1a lineage, but not AtPOT1b, has been subjected to positive selection from an ancestral POT1 protein, leading to enhanced interaction with CST (
      • Beilstein M.A.
      • Renfrew K.B.
      • Song X.
      • Shakirov E.V.
      • Zanis M.J.
      • Shippen D.E.
      Evolution of the telomere-associated protein POT1a in Arabidopsis thaliana is characterized by positive selection to reinforce protein–protein interaction.
      ). Hence, AtPOT1a appears to have been evolved to be specialized for telomere maintenance through CST interaction. A role for AtPOT1b in telomere biology is not clear. It cannot complement the pot1a mutant (
      • Beilstein M.A.
      • Renfrew K.B.
      • Song X.
      • Shakirov E.V.
      • Zanis M.J.
      • Shippen D.E.
      Evolution of the telomere-associated protein POT1a in Arabidopsis thaliana is characterized by positive selection to reinforce protein–protein interaction.
      ) and cannot bind telomeric DNA in vitro (
      • Arora A.
      • Beilstein M.A.
      • Shippen D.E.
      Evolution of Arabidopsis protection of telomeres 1 alters nucleic acid recognition and telomerase regulation.
      ). However, overexpression of the AtPOT1b C-terminal domain leads to massive chromosome fusion (
      • Shakirov E.V.
      • Surovtseva Y.V.
      • Osbun N.
      • Shippen D.E.
      The Arabidopsis Pot1 and Pot2 proteins function in telomere length homeostasis and chromosome end protection.
      ). Whereas this finding implicates AtPOT1b in chromosome end protection, AtPOT1b probably does so in a manner distinct from the single-copy POT1 proteins from vertebrates and fission yeast. The third POT1 gene in A. thaliana, AtPOT1c, arose only 5 million years ago as a partial duplication of the AtPOT1a locus. The insertion of a transposon into the promoter of AtPOT1c rendered this gene silent almost immediately after its genesis (
      • Kobayashi C.R.
      • Castillo-González C.
      • Survotseva Y.
      • Canal E.
      • Nelson A.D.L.
      • Shippen D.E.
      Recent emergence and extinction of the protection of telomeres 1c gene in Arabidopsis thaliana.
      ). This finding, coupled with the remarkable functional divergence associated with POT1 paralogs across eukarya, argues that POT1 dosage affects the fitness of organisms, and one or more of the duplicated copies must diverge quickly or be silenced.

      Telomere proteins and their role in the genome-wide response to oxidative stress

      The majority of DNA lesions in mammalian and plant cells can be attributed to oxidative damage (
      • Sharma P.
      • Jha A.B.
      • Dubey R.S.
      • Pessarakli M.
      Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions.
      ,
      • Liguori I.
      • Russo G.
      • Curcio F.
      • Bulli G.
      • Aran L.
      • Della-Morte D.
      • Gargiulo G.
      • Testa G.
      • Cacciatore F.
      • Bonaduce D.
      • Abete P.
      Oxidative stress, aging, and diseases.
      ), and recent data indicate that several shelterin components safeguard the genome against this assault (Fig. 4). Reactive oxygen species (ROS) modifies DNA bases, most commonly resulting in 8-oxo-guanine (8-oxoG) and thymine glycol (Tg) (
      • Lee H.-T.
      • Bose A.
      • Lee C.-Y.
      • Opresko P.L.
      • Myong S.
      Molecular mechanisms by which oxidative DNA damage promotes telomerase activity.
      ). If not repaired, 8-oxoG induces GC-TA transversion mutations as well as single-strand or double-strand breaks, leading to genomic instability (
      • Fouquerel E.
      • Barnes R.P.
      • Uttam S.
      • Watkins S.C.
      • Bruchez M.P.
      • Opresko P.L.
      Targeted and persistent 8-oxoguanine base damage at telomeres promotes telomere loss and crisis.
      ). Tg is the most prevalent oxidative product of thymine, responsible for 10–20% of ionizing radiation-induced genomic damage (
      • Frenkel K.
      • Goldstein M.S.
      • Duker N.J.
      • Teebor G.W.
      Identification of the cis-thymine glycol moiety in oxidized deoxyribonucleic acid.
      ). Due to their high G-T content, telomeres are a hot spot for oxidative damage (
      • Coluzzi E.
      • Leone S.
      • Sgura A.
      Oxidative stress induces telomere dysfunction and senescence by replication fork arrest.
      ,
      • Suram A.
      • Herbig U.
      The replicometer is broken: telomeres activate cellular senescence in response to genotoxic stresses.
      • Tian R.
      • Zhang L.-N.
      • Zhang T.-T.
      • Pang H.-Y.
      • Chen L.-F.
      • Shen Z.-J.
      • Liu Z.
      • Fang Q.
      • Zhang S.-Y.
      Association between oxidative stress and peripheral leukocyte telomere length in patients with premature coronary artery disease.
      ).
      Figure thumbnail gr4
      Figure 4Impact of oxidative stress on telomeres and telomere-associated proteins in mammals. Telomeres are a hot spot for oxidative damage causing base modifications including thymine to thymine glycol and guanine to 8-oxoG. These lesions interfere with DNA binding by TRF1, TRF2, and POT1. These same proteins stimulate BER at telomeres and perhaps elsewhere in the genome, enabling the removal of damaged bases from the DNA. Oxidative DNA damage decreases the abundance of both cytoplasmic and nuclear RAP1, which in turn triggers apoptosis. Conversely, oxidative stress leads to the accumulation in mitochondria of TERT and TIN2, which promote mitochondrial functions that protect against apoptosis.
      Base excision repair (BER) is the most important pathway for removing 8-oxoG and Tg lesions (
      • Bohr V.A.
      Repair of oxidative DNA damage in nuclear and mitochondrial DNA, and some changes with aging in mammalian cells.
      ). Mice lacking the glycosylase NTH1, which removes Tg via BER, exhibit increased telomere fragility (
      • Vallabhaneni H.
      • O'Callaghan N.
      • Sidorova J.
      • Liu Y.
      Defective repair of oxidative base lesions by the DNA glycosylase Nth1 associates with multiple telomere defects.
      ). Intriguingly, TRF1, TRF2, and POT1 stimulate BER after oxidative damage (
      • Miller A.S.
      • Balakrishnan L.
      • Buncher N.A.
      • Opresko P.L.
      • Bambara R.A.
      Telomere proteins POT1, TRF1 and TRF2 augment long-patch base excision repair in vitro.
      ). Interestingly, 8-OxoG and Tg modifications inhibit telomeric DNA binding by TRF1, TRF2, and POT1 in vitro (
      • Opresko P.L.
      • Fan J.
      • Danzy S.
      • Wilson 3rd, D.M.
      • Bohr V.A.
      • Bohr V.A.
      Oxidative damage in telomeric DNA disrupts recognition by TRF1 and TRF2.
      ). These observations suggest a feedback loop wherein oxidative damage at telomeres leads to the expulsion of the aforementioned telomere proteins, which then become available to assist in the BER-mediated repair of damaged telomeric bases, so as to enable the re-engagement of shelterin at the chromosome terminus (
      • Miller A.S.
      • Balakrishnan L.
      • Buncher N.A.
      • Opresko P.L.
      • Bambara R.A.
      Telomere proteins POT1, TRF1 and TRF2 augment long-patch base excision repair in vitro.
      ) (Fig. 4). Because TRF1 and TRF2 can associate with other genomic locales, they may exert a broader impact in the response to oxidative stress.
      Recent data reveal an intriguing response of RAP1 to oxidative stress and other types of DNA damage (
      • Swanson M.J.
      • Baribault M.E.
      • Israel J.N.
      • Bae N.S.
      Telomere protein RAP1 levels are affected by cellular aging and oxidative stress.
      ). RAP1 levels decrease in the nucleus and the cytoplasm in response to ROS. Diminished levels of cytoplasmic RAP1 appear to promote apoptosis in aging cells (
      • Teo H.
      • Ghosh S.
      • Luesch H.
      • Ghosh A.
      • Wong E.T.
      • Malik N.
      • Orth A.
      • de Jesus P.
      • Perry A.S.
      • Oliver J.D.
      • Tran N.L.
      • Speiser L.J.
      • Wong M.
      • Saez E.
      • Schultz P.
      • Chanda S.K.
      • Verma I.M.
      • Tergaonkar V.
      Telomere-independent Rap1 is an IKK adaptor and regulates NF-κB-dependent gene expression.
      ) (Fig. 4). Notably, in yeast, shortening of telomeres due to senescence releases RAP1, which then becomes associated with extratelomeric sites. Release of RAP1 from telomeres correlates with the down-regulation of genes encoding core histones and the translational apparatus and up-regulation of genes responsive to senescence (
      • Platt J.M.
      • Ryvkin P.
      • Wanat J.J.
      • Donahue G.
      • Ricketts M.D.
      • Barrett S.P.
      • Waters H.J.
      • Song S.
      • Chavez A.
      • Abdallah K.O.
      • Master S.R.
      • Wang L.-S.
      • Johnson F.B.
      Rap1 relocalization contributes to the chromatin-mediated gene expression profile and pace of cell senescence.
      ) (Table 1).

      Genome protection from a distance: The role of telomere proteins outside the nucleus

      The role of telomere proteins in the response to oxidative stress correlates with cytoplasmic activities, but the molecular mechanisms that govern telomere protein function outside the nucleus are largely unexplored. In addition to RAP1, several other telomere-related proteins accumulate in the cytoplasm (Table 1). POT1, TPP1, and trace amounts of TIN2 shuttle in and out of the nucleus and can be detected as subcomplexes in the cytoplasm (
      • Chen L.-Y.
      • Liu D.
      • Songyang Z.
      Telomere maintenance through spatial control of telomeric proteins.
      ). TTP1 bears a nuclear export signal that is crucial for modulating the levels of the TTP1-POT1 complex within the nucleus. Abrogation of TPP1 nuclear export causes overelongation of telomeres and activates the DNA damage response (
      • Chen L.-Y.
      • Liu D.
      • Songyang Z.
      Telomere maintenance through spatial control of telomeric proteins.
      ).
      TIN2 possesses a mitochondrial targeting sequence (MTS) that enables its transport into the mitochondria, where it is post-translationally modified (
      • Chen L.-Y.
      • Zhang Y.
      • Zhang Q.
      • Li H.
      • Luo Z.
      • Fang H.
      • Kim S.H.
      • Qin L.
      • Yotnda P.
      • Xu J.
      • Tu B.P.
      • Bai Y.
      • Songyang Z.
      Mitochondrial localization of telomeric protein TIN2 links telomere regulation to metabolic control.
      ) (Fig. 4). Interestingly, in cells lacking TIN2, glycolysis is inhibited, and ROS production is elevated along with ATP and oxygen consumption. Strikingly, these phenotypes do not correlate with telomeric abnormalities (
      • Chen L.-Y.
      • Zhang Y.
      • Zhang Q.
      • Li H.
      • Luo Z.
      • Fang H.
      • Kim S.H.
      • Qin L.
      • Yotnda P.
      • Xu J.
      • Tu B.P.
      • Bai Y.
      • Songyang Z.
      Mitochondrial localization of telomeric protein TIN2 links telomere regulation to metabolic control.
      ), indicating that TIN2's mitochondria-related functions are distinct from its role at telomeres.
      TERT proteins from vertebrates and plants also harbor a MTS (Table 1). Extracts prepared from mitochondria are enriched in telomerase activity (
      • Santos J.H.
      • Meyer J.N.
      • Skorvaga M.
      • Annab L.A.
      • Van Houten B.
      Mitochondrial hTERT exacerbates free-radical-mediated mtDNA damage.
      ). In addition, TERT is associated with the outer mitochondrial membrane translocators TOM20 and TOM40 (
      • Gabriel K.
      • Egan B.
      • Lithgow T.
      Tom40, the import channel of the mitochondrial outer membrane, plays an active role in sorting imported proteins.
      ,
      • Haendeler J.
      • Dröse S.
      • Büchner N.
      • Jakob S.
      • Altschmied J.
      • Goy C.
      • Spyridopoulos I.
      • Zeiher A.M.
      • Brandt U.
      • Dimmeler S.
      Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage.
      ) as well as tFAM, HSP60, tim23, and a variety of mitochondrial RNAs (
      • Maida Y.
      • Yasukawa M.
      • Furuuchi M.
      • Lassmann T.
      • Possemato R.
      • Okamoto N.
      • Kasim V.
      • Hayashizaki Y.
      • Hahn W.C.
      • Masutomi K.
      An RNA-dependent RNA polymerase formed by TERT and the RMRP RNA.
      ,
      • Sharma N.K.
      • Reyes A.
      • Green P.
      • Caron M.J.
      • Bonini M.G.
      • Gordon D.M.
      • Holt I.J.
      • Santos J.H.
      Human telomerase acts as a hTR-independent reverse transcriptase in mitochondria.
      ). Notably, oxidative stress triggers hTERT export from the nucleus to mitochondria, and elevated levels of hTERT in this compartment correlate with stabilization of mitochondrial DNA, reduced ROS, increased mitochondrial membrane potential, and enhanced mitochondrial function (
      • Passos J.F.
      • Saretzki G.
      • von Zglinicki T.
      DNA damage in telomeres and mitochondria during cellular senescence: is there a connection?.
      ,
      • Kovalenko O.A.
      • Caron M.J.
      • Ulema P.
      • Medrano C.
      • Thomas A.P.
      • Kimura M.
      • Bonini M.G.
      • Herbig U.
      • Santos J.H.
      A mutant telomerase defective in nuclear-cytoplasmic shuttling fails to immortalize cells and is associated with mitochondrial dysfunction.
      ,
      • Monaghan R.M.
      • Whitmarsh A.J.
      Mitochondrial proteins moonlighting in the nucleus.
      ) (Fig. 4). There are also reports that mitochondrial TERT not only associates with non-TER RNAs but also possesses noncanonical enzyme activities. These include an RNA-dependent RNA polymerase activity that is implicated in the production of siRNA (
      • Maida Y.
      • Yasukawa M.
      • Furuuchi M.
      • Lassmann T.
      • Possemato R.
      • Okamoto N.
      • Kasim V.
      • Hayashizaki Y.
      • Hahn W.C.
      • Masutomi K.
      An RNA-dependent RNA polymerase formed by TERT and the RMRP RNA.
      ) and reverse transcriptase activity using mitochondrial tRNA as a template (
      • Sharma N.K.
      • Reyes A.
      • Green P.
      • Caron M.J.
      • Bonini M.G.
      • Gordon D.M.
      • Holt I.J.
      • Santos J.H.
      Human telomerase acts as a hTR-independent reverse transcriptase in mitochondria.
      ). The biological relevance of this latter activity is unknown.
      TERT in the mitochondria has been proposed to stimulate mitochondrial DNA replication and repair (
      • Sharma N.K.
      • Reyes A.
      • Green P.
      • Caron M.J.
      • Bonini M.G.
      • Gordon D.M.
      • Holt I.J.
      • Santos J.H.
      Human telomerase acts as a hTR-independent reverse transcriptase in mitochondria.
      ). Compared with WT mice, the RNA expression profiles of tert mutants monitored for four consecutive generations (G1–G4) reveal statistically significant changes in the expression of both mitochondrial and nuclear encoded genes required for oxidative phosphorylation, mitochondrial function, and antioxidant defense (
      • Sahin E.
      • Colla S.
      • Liesa M.
      • Moslehi J.
      • Müller F.L.
      • Guo M.
      • Cooper M.
      • Kotton D.
      • Fabian A.J.
      • Walkey C.
      • Maser R.S.
      • Tonon G.
      • Foerster F.
      • Xiong R.
      • Wang Y.A.
      • et al.
      Telomere dysfunction induces metabolic and mitochondrial compromise.
      ). Similar results were obtained A. thaliana tert mutants of generations G2 and G7 (
      • Amiard S.
      • Da Ines O.
      • Gallego M.E.
      • White C.I.
      Responses to telomere erosion in plants.
      ). Interestingly, yeast and ciliate TERT proteins lack an MTS (
      • Santos J.H.
      • Meyer J.N.
      • Skorvaga M.
      • Annab L.A.
      • Van Houten B.
      Mitochondrial hTERT exacerbates free-radical-mediated mtDNA damage.
      ), raising the possibility that the mitochondrial function of TERT is not conserved in these species or that these TERT proteins are transported into mitochondria via a different mechanism.

      Conclusions and future directions

      With the advent of linear chromosomes, factors involved in different facets of DNA metabolism were coopted to solve the telomere end protection and end replication problems. Some of these factors retain their functions in DNA replication, DNA repair, and transcriptional regulation. Telomeric DNA is a magnet for oxidative damage, and hence in the drive to maintain genome integrity, telomere proteins may have gained the capacity to protect chromosome ends from this assault by promoting BER proximally, or at a distance by affecting mitochondrial function. Alternatively, some noncanonical functions of telomere proteins may have an older origin. Mitochondria, which possess group II introns (
      • Novikova O.
      • Belfort M.
      Mobile group II introns as ancestral eukaryotic elements.
      ) and proteins structurally similar to the ancestral OB-folds of RPA (
      • Webster G.
      • Genschel J.
      • Curth U.
      • Urbanke C.
      • Kang C.
      • Hilgenfeld R.
      A common core for binding single-stranded DNA: structural comparison of the single-stranded DNA-binding proteins (SSB) from E. coli and human mitochondria.
      ), emerged 1.45 billion years ago (
      • Martin W.F.
      • Tielens A.G.M.
      • Mentel M.
      • Garg S.G.
      • Gould S.B.
      The physiology of phagocytosis in the context of mitochondrial origin.
      ). Thus, the building blocks for some of the modern-day telomere proteins and their functions in the oxidative stress response may reflect a mitochondrial ancestry. Finally, the ancient and emerging functions of telomere proteins have been linked to gene duplication events. In particular, POT1 gene duplications that occurred across evolution have given rise to multifaceted roles of telomere proteins in chromosome biology and their integration into the broader context of cellular physiology.

      Acknowledgments

      We apologize to colleagues whose work we were unable to cite due to space limitations. We thank members of the Shippen laboratory for insightful comments on the manuscript and the American Society for Biochemistry and Molecular Biology for highlighting our research. Components of all of the figures were created using BioRender software.

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