Introduction

Telomeres are protein structures located at the ends of each eukaryotic DNA chromosomal arm. These chromosomal caps are 1 of the most important structures that preserve the structural integrity of linear DNA during each cycle of replication.[1] Functions of telomeres include protecting the ends of the DNA from binding to one another and to itself, allowing for complete chromosomal replication, and serving as a molecular timer by controlling the lifespan of a eukaryotic cell. Telomeres also prevent the free ends of the chromosome from appearing as DNA double-stranded breaks, which in turn safeguards the ends from accidental DNA repair.[2] Telomeres play a significant role in cellular senescence in humans and have made major contributions to human aging. Pathologically, dysregulated expression of the telomere synthesis mechanism causes cellular immortality, leading to potential oncogenesis and tumorigenesis.[3]

Molecular Level

Telomere

A telomere structure consists of repeats of non-coding nitrogenous bases (5'-TTAGGG-3'). In mammals, telomeres are highly conserved, indicating that this nuclear sequence remains relatively unchanged throughout evolutionary biology.[1] The hexameric segments of DNA are located in tandem with one another. The 3' G-rich end of the chromosome is longer than the 5' C-rich end.[4][5] In humans, the length of the telomere segment is between 5,000 to 15,000 base pairs long.[6] This long stretch of repetitive DNA sequences is characterized by a 3' end single-stranded overhang, which tucks itself into the end of the chromosome, creating a T-loop conformation. Of note, the T-loop biochemical structure is thermodynamically unfavorable. As such, proteins are required to manufacture and maintain the T-loop.[4]

The telomere is associated with 6 proteins that collectively create the Shelterin complex. This complex helps to create the final end cap structure of the chromosome. The associated proteins are described as follows: telomere repeats binding factor 1 (TERF1 or TRF1) regulates the telomere length. Telomere repeat binding factor 2 (TERF2 or TRF2) stabilizes the T-loop. Protection of telomeres 1 (POT1) inhibits DNA damage response at the single-stranded telomere overhang.[1][7] Telomerase recruitment factor (ACD or TPP1) facilitates POT1 binding to single-stranded telomere DNA. TERF1 interacting nuclear factor 2 (TIN2 or TINF2) tethers POT1 and ACD to TERF1 and TERF2. TIN2 is also responsible for stabilizing TERF2 in the telomere. TERF interacting protein 2 (TERF2 or RAP1), in addition to the proteins mentioned above, are all responsible for the regulation of telomere length.[8][9]

Telomerase

The synthesis of a telomere involves a reverse transcriptase telomerase, which functions as an RNA-dependent DNA polymerase. Telomerase is present in germline and stem cells and has enhanced activity in cancer cells. This enzyme is responsible for elongating telomeres by de novo addition of TTAGGG sequences onto 3' chromosome ends to prevent replicative cellular senescence.[10] Telomerase is a ribonucleoprotein structure comprising a functional RNA component and a catalytic reverse transcriptase component. The RNA component houses a template for the synthesis of telomeric DNA. The functional RNA component in humans is called hTERC or hTR [11]. It is encoded by the TERC gene located at the 3q26 region of the chromosome. The reverse transcriptase component is called hTERT and is encoded by the TERT gene located at chromosome 5p13.33.[12] While the telomerase core complex mainly consists of the 2 main components, hTERC and hTERT, essential supportive proteins exist to properly function the entire telomerase structure. Tcab1, Gar1, Nhp2, Reptin, and Pontin are proteins required for telomerase assembly and the proper recruitment of chromosomes.[13][14] Next, the proteins responsible for stabilizing the telomerase structure are TEP1 and dyskerin. Lastly, the additional protein subunits, Es1p, and Es3p, aid in the assembly and maturation of the catalytic complex.[15][16]

Function

The main functions of a telomere are to maintain chromosomal stability and prevent chromosomal degradation. Additionally, telomeres protect the ends of the chromosome from DNA end-joining to one another, damage response to DNA, and accidental DNA recombination.[6] The longer 3' G-rich end overhang that creates the T-loop protects the end of that chromosome from appearing as a double-stranded break in the DNA strand, thus preventing unwanted DNA repair.[17] For these reasons, telomeres and their maintenance are essential to eukaryotic genomic stability and the longevity of cellular information.

Mechanism

DNA replication is facilitated by DNA polymerase. This enzyme can only synthesize DNA in the 5' to 3' direction. DNA replication begins with an RNA primer, which is synthesized by primase. The RNA primer allows the DNA to locate the area of the chromosome where replication begins. The RNA primer anneals to the template DNA to provide a free 3'-OH group where new nucleotides are added. During the synthesis of the leading strand, which runs from the 5' to 3' direction, only 1 primer is needed for synthesis at this location to be continuous. This is due to the addition of new nucleotides in the direction of the replication fork.[18] Simultaneously, the synthesis of the DNA strand occurs in a lagging fashion in the 3' to 5' direction. Multiple RNA primers are necessary for the lagging strand, which is then replaced by DNA nucleotides via DNA polymerase, elongated, and ligated to create the new DNA strand.[19] The challenge arises at the 5' end of the lagging strand, where a stretch of DNA the size of the RNA primer is lost. This "end replication problem" occurs when the final RNA primer is removed after complete replication.[20] DNA polymerase cannot synthesize the end of the lagging strand due to the lack of a 3'-OH group after removing the RNA primer. Thus, due to the inherent properties of DNA polymerase, after each S phase of cell division, telomeres shorten 50-150 base pairs.[21][22]

Telomere replication and maintenance present numerous challenges. Repetitive tandem repeats of DNA predispose DNA polymerase slippage during DNA replication. Frequent slippage of the enzyme may cause insertion or deletion of nucleotide bases and strand mispairing. The next challenge is the G-rich structure of the telomere. A higher number of guanine nucleotides can cause G-quadruplexes to form. Tethered G-rich tetrads are highly stable due to their increased hydrogen bonds. The G-quadruplexes, which require specific helicases for proper disassembly, may induce replication fork stalling if the specialized helicase cannot function.[23] Additionally, the final step of telomere replication involves unwinding the T-loops to facilitate the passage of the replisome. With the considerably large structural make-up of the telomere, inadequate unwinding may cause failure for timely disassembly. Thus, replication machinery cannot copy the end of the chromosome, leading to a significant loss of telomere sequences. To combat the challenges mentioned above, telomeric proteins such as homology-dependent recombination factors, specialized helicases, and nucleases exist to promote smooth replication of the telomere.[17][24]

Pathophysiology

Cellular Senescence 

Telomeres play a crucial role in cellular senescence and, thus, biological aging. Cellular senescence refers to the irreversible loss of cellular division capability. The end replication problem, which describes the loss of base pairs during each S phase of cellular synthesis, can expose the ends of the DNA of a somatic cell, activating a process called DNA damage response. The purpose of this phenomenon is to prevent abnormal fusion of exposed chromosomal ends as well as chromosomal instability. The telomeres shorten without telomere elongation, characteristic of most somatic cells. Telomerase can elongate telomere structures; however, with persistent telomeric DNA damage response activation, a senescence-initiating signal can be elicited in addition to DNA damage. Cellular or replicative senescence also initiates when the telomere shortens to below a critical length.[25][26] DNA damage response involves multiple cellular signaling pathways that activate cell cycle checkpoints to prevent the formation of potentially pathophysiologic mutations.[27] In cancer cells, as described later under clinical significance, unlimited self-renewal capacity is acquired through uninhibited telomerase activation.[13]

Clinical Significance

Telomeres and Oxidative Stress

DNA stressors include numerous endogenous and exogenous factors such as mitochondrial dysfunction, cigarette smoking, alcohol consumption, inflammation, a high-fat diet, and other lifestyle and environmental factors.[28][29] Iatrogenically, inducers of cell senescence include chemotherapy and radiation.[30][31] Most importantly, the relationship between these inducers and cellular senescence is the production of reactive oxygen species. Researchers believe that G-rich telomeres are especially susceptible to oxidative stress.[32] Additionally, telomeres have a repressed DNA damage response, leading to inefficient DNA repair if exposed to oxidative damage.[6] Obesity is associated with chronic inflammation and increased reactive oxygen species levels in adipose tissue. These patients with a higher body mass index are associated with a higher blood volume, leading to greater proliferation of blood cells- all related to the telomere's shortening [29] [33]. It has also been reported that telomere length is inversely correlated with patients who suffer from psychosocial stress and major depressive disorder due to increased oxidative stress and inflammatory factors.[33][34] Notably, it has been reported that those who participate in increased physical activity levels have longer telomeres.[35][36]

Cancer

While the synthesis of telomeres by the reverse transcriptase, telomerase, is absent in most human somatic cells, it is found in greater than 90% of tumorigenic cells and in-vitro immortalized cells.[37] Telomerase gains oncogenic function when its expression is deregulated in human somatic cells.[2][12] hTERT gene amplification, which results from a breakage at DNA sites or abnormal chromosomal fusions, causes a pathologic upregulation of telomerase activity. Cancer cell immortalization through hTERT involvement may also occur by hTERT promoter methylation. Methylation prevents the binding of transcriptional repressors from blocking transcription machinery. TERT promoter mutations have also been implicated in cancer cells, including uroepithelial, bladder, thyroid, cutaneous melanoma, basal cell carcinomas, squamous cell carcinomas, and glioblastoma.[13][38]

Telomerase-targeted Cancer Immunotherapy

Because upregulated telomerase activity is significant in tumor cells, hTERT makes an attractive tumor antigen for telomerase-targeted cancer immunotherapy. Several approaches exist, including oligonucleotide inhibitors, immunotherapeutic approaches, and telomerase-directed gene therapy. Oligonucleotide inhibitors are modified nucleic acids that inhibit telomerase, inducing telomere shortening and forcing cellular senescence and apoptosis. Immunotherapeutic approaches use high-avidity T lymphocytes that are reactive against the catalytic enzyme. Finally, telomerase-directed gene therapy involves the selective killing of tumor cells by targeting telomerase promoters.[39]

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References

1.

Turner KJ, Vasu V, Griffin DK. Telomere Biology and Human Phenotype. Cells. 2019 Jan 19;8(1) [PMC free article: PMC6356320] [PubMed: 30669451]

2.

Cong YS, Wright WE, Shay JW. Human telomerase and its regulation. Microbiol Mol Biol Rev. 2002 Sep;66(3):407-25, table of contents. [PMC free article: PMC120798] [PubMed: 12208997]

3.

Bejarano L, Bosso G, Louzame J, Serrano R, Gómez-Casero E, Martínez-Torrecuadrada J, Martínez S, Blanco-Aparicio C, Pastor J, Blasco MA. Multiple cancer pathways regulate telomere protection. EMBO Mol Med. 2019 Jul;11(7):e10292. [PMC free article: PMC6609915] [PubMed: 31273934]

4.

Giardini MA, Segatto M, da Silva MS, Nunes VS, Cano MI. Telomere and telomerase biology. Prog Mol Biol Transl Sci. 2014;125:1-40. [PubMed: 24993696]

5.

Wright WE, Tesmer VM, Huffman KE, Levene SD, Shay JW. Normal human chromosomes have long G-rich telomeric overhangs at one end. Genes Dev. 1997 Nov 01;11(21):2801-9. [PMC free article: PMC316649] [PubMed: 9353250]

6.

Ahmed W, Lingner J. Impact of oxidative stress on telomere biology. Differentiation. 2018 Jan-Feb;99:21-27. [PubMed: 29274896]

7.

de Lange T. Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev. 2005 Sep 15;19(18):2100-10. [PubMed: 16166375]

8.

Xin H, Liu D, Songyang Z. The telosome/shelterin complex and its functions. Genome Biol. 2008;9(9):232. [PMC free article: PMC2592706] [PubMed: 18828880]

9.

Lazzerini-Denchi E, Sfeir A. Stop pulling my strings - what telomeres taught us about the DNA damage response. Nat Rev Mol Cell Biol. 2016 Jun;17(6):364-78. [PMC free article: PMC5385261] [PubMed: 27165790]

10.

Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell. 1985 Dec;43(2 Pt 1):405-13. [PubMed: 3907856]

11.

Feng J, Funk WD, Wang SS, Weinrich SL, Avilion AA, Chiu CP, Adams RR, Chang E, Allsopp RC, Yu J. The RNA component of human telomerase. Science. 1995 Sep 01;269(5228):1236-41. [PubMed: 7544491]

12.

Zhang Y, Toh L, Lau P, Wang X. Human telomerase reverse transcriptase (hTERT) is a novel target of the Wnt/β-catenin pathway in human cancer. J Biol Chem. 2012 Sep 21;287(39):32494-511. [PMC free article: PMC3463325] [PubMed: 22854964]

13.

Leão R, Apolónio JD, Lee D, Figueiredo A, Tabori U, Castelo-Branco P. Mechanisms of human telomerase reverse transcriptase (hTERT) regulation: clinical impacts in cancer. J Biomed Sci. 2018 Mar 12;25(1):22. [PMC free article: PMC5846307] [PubMed: 29526163]

14.

Venteicher AS, Meng Z, Mason PJ, Veenstra TD, Artandi SE. Identification of ATPases pontin and reptin as telomerase components essential for holoenzyme assembly. Cell. 2008 Mar 21;132(6):945-57. [PMC free article: PMC2291539] [PubMed: 18358808]

15.

Cohen SB, Graham ME, Lovrecz GO, Bache N, Robinson PJ, Reddel RR. Protein composition of catalytically active human telomerase from immortal cells. Science. 2007 Mar 30;315(5820):1850-3. [PubMed: 17395830]

16.

Liu L, Lai S, Andrews LG, Tollefsbol TO. Genetic and epigenetic modulation of telomerase activity in development and disease. Gene. 2004 Sep 29;340(1):1-10. [PubMed: 15556289]

17.

Stroik S, Hendrickson EA. Telomere replication-When the going gets tough. DNA Repair (Amst). 2020 Oct;94:102875. [PubMed: 32650286]

18.

Ohki R, Tsurimoto T, Ishikawa F. In vitro reconstitution of the end replication problem. Mol Cell Biol. 2001 Sep;21(17):5753-66. [PMC free article: PMC87295] [PubMed: 11486015]

19.

Okazaki R, Okazaki T, Sakabe K, Sugimoto K, Sugino A. Mechanism of DNA chain growth. I. Possible discontinuity and unusual secondary structure of newly synthesized chains. Proc Natl Acad Sci U S A. 1968 Feb;59(2):598-605. [PMC free article: PMC224714] [PubMed: 4967086]

20.

Watson JD. Origin of concatemeric T7 DNA. Nat New Biol. 1972 Oct 18;239(94):197-201. [PubMed: 4507727]

21.

Maestroni L, Matmati S, Coulon S. Solving the Telomere Replication Problem. Genes (Basel). 2017 Jan 31;8(2) [PMC free article: PMC5333044] [PubMed: 28146113]

22.

Griffith JD, Comeau L, Rosenfield S, Stansel RM, Bianchi A, Moss H, de Lange T. Mammalian telomeres end in a large duplex loop. Cell. 1999 May 14;97(4):503-14. [PubMed: 10338214]

23.

Vannier JB, Pavicic-Kaltenbrunner V, Petalcorin MI, Ding H, Boulton SJ. RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity. Cell. 2012 May 11;149(4):795-806. [PubMed: 22579284]

24.

Stroik S, Kurtz K, Hendrickson EA. CtIP is essential for telomere replication. Nucleic Acids Res. 2019 Sep 26;47(17):8927-8940. [PMC free article: PMC6755089] [PubMed: 31378812]

25.

d'Adda di Fagagna F, Reaper PM, Clay-Farrace L, Fiegler H, Carr P, Von Zglinicki T, Saretzki G, Carter NP, Jackson SP. A DNA damage checkpoint response in telomere-initiated senescence. Nature. 2003 Nov 13;426(6963):194-8. [PubMed: 14608368]

26.

Aguado J, d'Adda di Fagagna F, Wolvetang E. Telomere transcription in ageing. Ageing Res Rev. 2020 Sep;62:101115. [PubMed: 32565330]

27.

Rossiello F, Aguado J, Sepe S, Iannelli F, Nguyen Q, Pitchiaya S, Carninci P, d'Adda di Fagagna F. Corrigendum: DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs. Nat Commun. 2017 Apr 13;8:15344. [PMC free article: PMC5399297] [PubMed: 28406145]

28.

Wiley CD, Velarde MC, Lecot P, Liu S, Sarnoski EA, Freund A, Shirakawa K, Lim HW, Davis SS, Ramanathan A, Gerencser AA, Verdin E, Campisi J. Mitochondrial Dysfunction Induces Senescence with a Distinct Secretory Phenotype. Cell Metab. 2016 Feb 09;23(2):303-14. [PMC free article: PMC4749409] [PubMed: 26686024]

29.

McGrath M, Wong JY, Michaud D, Hunter DJ, De Vivo I. Telomere length, cigarette smoking, and bladder cancer risk in men and women. Cancer Epidemiol Biomarkers Prev. 2007 Apr;16(4):815-9. [PubMed: 17416776]

30.

Childs BG, Durik M, Baker DJ, van Deursen JM. Cellular senescence in aging and age-related disease: from mechanisms to therapy. Nat Med. 2015 Dec;21(12):1424-35. [PMC free article: PMC4748967] [PubMed: 26646499]

31.

Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature. 2000 Nov 09;408(6809):239-47. [PubMed: 11089981]

32.

Oikawa S, Kawanishi S. Site-specific DNA damage at GGG sequence by oxidative stress may accelerate telomere shortening. FEBS Lett. 1999 Jun 25;453(3):365-8. [PubMed: 10405177]

33.

Epel ES. Psychological and metabolic stress: a recipe for accelerated cellular aging? Hormones (Athens). 2009 Jan-Mar;8(1):7-22. [PubMed: 19269917]

34.

Wolkowitz OM, Mellon SH, Epel ES, Lin J, Dhabhar FS, Su Y, Reus VI, Rosser R, Burke HM, Kupferman E, Compagnone M, Nelson JC, Blackburn EH. Leukocyte telomere length in major depression: correlations with chronicity, inflammation and oxidative stress--preliminary findings. PLoS One. 2011 Mar 23;6(3):e17837. [PMC free article: PMC3063175] [PubMed: 21448457]

35.

Puterman E, Lin J, Blackburn E, O'Donovan A, Adler N, Epel E. The power of exercise: buffering the effect of chronic stress on telomere length. PLoS One. 2010 May 26;5(5):e10837. [PMC free article: PMC2877102] [PubMed: 20520771]

36.

Ludlow AT, Zimmerman JB, Witkowski S, Hearn JW, Hatfield BD, Roth SM. Relationship between physical activity level, telomere length, and telomerase activity. Med Sci Sports Exerc. 2008 Oct;40(10):1764-71. [PMC free article: PMC2581416] [PubMed: 18799986]

37.

Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW. Specific association of human telomerase activity with immortal cells and cancer. Science. 1994 Dec 23;266(5193):2011-5. [PubMed: 7605428]

38.

Azouz A, Wu YL, Hillion J, Tarkanyi I, Karniguian A, Aradi J, Lanotte M, Chen GQ, Chehna M, Ségal-Bendirdjian E. Epigenetic plasticity of hTERT gene promoter determines retinoid capacity to repress telomerase in maturation-resistant acute promyelocytic leukemia cells. Leukemia. 2010 Mar;24(3):613-22. [PubMed: 20072159]

39.

Jäger K, Walter M. Therapeutic Targeting of Telomerase. Genes (Basel). 2016 Jul 21;7(7) [PMC free article: PMC4962009] [PubMed: 27455328]

Disclosure: Jenna Lee declares no relevant financial relationships with ineligible companies.

Disclosure: Mark Pellegrini declares no relevant financial relationships with ineligible companies.