Introduction

Telomeres are protein structures located at the ends of each eukaryotic DNA chromosomal arm. These chromosomal caps are one 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] In humans, telomeres play a significant role in cellular senescence with 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 the course of 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 conformation called a T-loop. Of note, the T-loop biochemical structure is thermodynamically unfavorable. As such, proteins are required in manufacturing and maintaining the T-loop.[4]

The telomere is associated with six 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 repeat 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 to 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 that comprises two parts: a functional RNA component and a catalytic reverse transcriptase component. The RNA component houses a template for the synthesis of telomeric DNA. In humans, the functional RNA component 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 two main components, hTERC and hTERT, essential supportive proteins exist for the proper functioning of the entire telomerase structure. Tcab1, Gar1, Nhp2, Reptin, and Pontin, are proteins that are 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 ends of the chromosome from DNA end-joining to one another or each other, from 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] Due to these reasons, telomeres and their necessary maintenance are essential to eukaryotic genomic stability and the longevity of cellular information.

Mechanism

DNA replication is facilitated by DNA polymerase. This enzyme only has the ability to 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 will begin. 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 one 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, then subsequently elongated, then 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 replication is complete.[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 presents numerous challenges. Repetitive tandem repeats of DNA predisposes DNA polymerase slippage during DNA replication. Frequent slippage of the enzyme may cause insertion or deletion of nucleotide bases as well as 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 amount of hydrogen bonds. The G-quadruplexes, which require specific helicases for proper disassembly, may induce replication fork stalling if the specialized helicase is unable to 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 will be unable to 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 (DDR). The purpose of this phenomenon is to prevent abnormal fusion of exposed chromosomal ends as well as chromosomal instability. Without telomere elongation, which is characteristic of most somatic cells, the telomeres will shorten. Telomerase has the ability to elongate telomere structures; however, with persistent telomeric DNA damage response activation, in addition to DNA damage, a senescence-initiating signal will be elicited. Cellular senescence, or replicative senescence, will also initiate when the telomere shortens to below a critical length.[25][26] DDR 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 (ROS). 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 highly associated with chronic inflammation and increased ROS levels in adipose tissue. These patients with a higher body mass index (BMI) are associated with a higher blood volume, leading to greater proliferation of blood cells- all of which are related to the shortening of the telomere [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 the majority of 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 by way of hTERT involvement may also occur by hTERT promoter methylation. Methylation prevents the binding of transcriptional repressors from blocking transcription machinery. TERT promoter mutations have been implicated in cancer cells as well, 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 are able to inhibit telomerase, thereby inducing telomere shortening and forced 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]