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Telomeres are regions of repetitive nucleotide sequences located at the end of each sister chromatid. Telomeres function to prevent gene deterioration during DNA replication, cap the chromosomes to deter chromatid or chromosome fusion, and are involved in important cellular processes including aging (senescence), cell immortality and the formation of cancers. This essay will discuss the role of telomeres and associated proteins in each of these critical processes.
At birth, human telomeres are around 10-15 kilobases in length, although there is considerable variation in length between individuals (Palm and de Lange, 2008). Human telomeres contain a specific sequence (5'-TTAGGG-3') repeated from about 500 to 5000 times, depending on the chromosome, and the telomere length gradually shortens during DNA replication and cell division. The polymerase enzyme requires the presence of 3' hydroxyl ends to initiate DNA replication; these are provided by RNA primers. At the 5' end of the lagging strand there is no free 3' hydroxyl group, therefore this strand shortens (by the length of the primer) during each DNA replication step (Figure 1A). If left unchecked, telomeres shorten with each mitotic division, which may lead, in extremis, to genes situated at the 3' end of chromosomes not being replicated. In turn, this leads to genomic instability, loss of cell division and cellular senescence (Aubert and Lansdorp, 2008). Chromosome shortening is ameliorated via a dedicated ribonucleoprotein, telomerase (reviewed in detail by Zvereva et al., 2010).
The telomerase core enzyme consists of a reverse transcriptase protein made up of 1,132 amino acids, encoded by the hTERT gene (Harrington et al., 1997). The telomerase RNA component contains 451 nucleotides, including a CAAUCCCAAUC telomere template, encoded by the telomerase RNA gene hTERC (Feng et al., 1995). Telomerase recognises guanine-rich telomere repeat sequences and elongates the copied DNA strand in the 5'→3'direction.Telomerase then adds repeat sequences and the lagging strand is subsequently completed by DNA polymerase alpha catalytic subunit (Pol-α), which contains a DNA primase that synthesises a cytosine-rich RNA primer close to the 3' end (Figure 1B).
Figure 1: The end-replication problem and the role of telomerase. (A) Stages of DNA replication showing the newly-synthesised strands (red), containing short RNA primers (green) at the 5' end (step 1). These primers are removed in step 2, leaving shorter DNA strands, which are processed (step 3) leaving overhangs. (B) Action of telomerase enzyme, containing the RNA primer, to synthesise the telomere ends. Figure adapted from (Karp, 2010, p.494).
Telomeres and chromosome stability
Telomeres 'cap' the ends of DNA strands, thereby preventing homologous recombination between chromosomes, which can cause the formation of double-stranded DNA breaks. Mammalian telomeres contain a protein complex, termed shelterin, which functions to protect chromosome ends from the cellular DNA damage response and to regulate telomere maintenance by telomerase (reviewed in detail by Palm and de Lange, 2008). Shelterin consists of six fixed proteins (TRF1, TRF2, POT1, TIN2, TPP1, and Rap1), and a plethora of transiently-associated proteins (e.g. tankyrases, helicases, Rif1 and Apollo), which 'shelter' the chromosome ends to avoid them being detected by cellular DNA damage surveillance mechanisms and subsequently processed inappropriately by DNA repair pathways (de Lange, 2005). Cells that do not possess a functioning shelterin complex undergo chromosomal fusions despite the presence of the telomere repeat sequences. For example, deficiencies in TRF2 results in double-strand break repair at telomeres and subsequent non-homologous end joining at the site of the breaks (Takai et al., 2003), leading to extensive chromosome fusion (Smogorzewska et al., 2002). TRF2 also functions to inhibit the protein ATM, which responds specifically to double-strand breaks in DNA (Karlseder et al., 2004). When TRF2 is absent or inactive, chromosome fusion has been shown to be dependent on ATM (Denchi and de Lange, 2007), despite the fact that ATM performs double-strand break-induced non-homologous end joining in only ten percent of cases (Cornforth and Bedford, 1985).
The importance of telomere capping during mitosis to prevent the formation of chromosome fusions, was first demonstrated more than 70 years ago. In cases where telomeres are absent, sister chromatids or entire chromosomes can fuse together, forming a bridge during anaphase of mitosis. As the cells divide and the chromatids pull apart, the chromosomes break at locations other than the site of fusion. This results in the unregulated acquisition of DNA on one chromosome and concomitant loss on the second chromosome. Furthermore, because the broken chromosomes still do not contain telomeres, they are liable to fuse again during the next cell division, continuing the breakage-fusion-bridge cycle (McClintock, 1941). The effect of this genomic instability depends on the extent of the breakage and whether the fusions occur between sister chromatids or between different chromosomes, with the latter fusion likely to cause chromosomal disorders, similar to those resulting from homologous recombination (Murnane, 2006).
Broken chromosomes in mammalian and yeast cells can have additional telomere repeat sequences added to the ends; a process termed chromosome healing (Melek and Shippen, 1996, Pennaneach et al., 2006). Pennaneach et al. (2006) identified that chromosome healing in Saccharomyces cerevisiae required a functioning telomerase, however chromosome healing has not been observed in response to ionizing radiation-induced DNA damage at interstitial sites, even with the presence of telomerase (Latre et al., 2004). In the EJ-30 human tumour cell line and in mouse embryonic stem (ES) cells chromosome healing has been detected in response to double-strand breaks near to telomeres. In EJ-30 cells, chromosome healing only occurs in about one percent of cases of telomere loss (Zschenker et al., 2009), however this frequency was markedly increased in mouse ES cells, occurring in approximately one-third of the total events (Gao et al., 2008, Sprung et al., 1999). Chromosomal healing in mouse ES cells required telomerase and nearly always occurred at the site of the double-strand break. Interestingly, the sequence of the de novo 'healed' telomere can bear little resemblance to the lost telomere repeat sequence, with as little as a single base-pair homology (Harrington and Greider, 1991). Although chromosome healing re-stabilises chromosomes that have lost their telomeres, the mechanism underpinning the process remains to be fully elucidated, and an explanation for the differing frequencies of chromosome healing between EJ-30 tumour and mouse ES cells has still to be found (Murnane, 2012).
Telomeres: senescence, cell immortality and cancer
The high G-C content of telomeres has been hypothesised to make these regions more susceptible to oxidative damage compared to non-telomeric regions. Single strand breaks in telomeric regions are not efficiently repaired, and a gradual loss of telomeres is thought to contribute to cellular senescence. This hypothesis has been supported by experimental evidence which demonstrated that antioxidants decrease telomere shortening rates in vitro (von Zglinicki, 2002). Environmental factors such as smoking and obesity have been associated with shorter telomeres in patients (Valdes et al., 2005); conversely, a healthy lifestyle has been proposed to maintain more stable telomere length in individuals from diverse ethnic backgrounds (Nettleton et al., 2008). Therefore, telomere length has been proposed as an indicator of 'somatic fitness' rather than chronological age (Aviv, 2006).
Telomeres in germ cells and embryonic stem cells are maintained by telomerase however these regions decrease in length during somatic cell division due to insufficient telomerase activity (Murnane, 2012). Stem cells are thought to undergo fewer than 100 cell divisions in a human lifetime (Lansdorp, 1997), therefore there appears to be a correlation between DNA replication, cell division and senescence. However, it is not clear whether telomere shortening is the cause or the result of senescence (Hornsby, 2006). Advances in understanding links between senescence, telomere length and telomerase expression have allowed the rescue of human primary cell lines from cellular senescence, usually through the ectopic expression of hTERT (Aubert and Lansdorp, 2008).
In contrast to cellular senescence, maintenance of telomere length by increase telomerase activity is critical for cell immortality, a key feature of cancer cells (Caino et al., 2009, Shay and Roninson, 2004). In vitro cancer models have identified that unregulated cell division is due in part to upregulation of the hTERT gene, encoding the telomerase core enzyme, resulting in a delay of cellular senescence (Gonzalez-Suarez et al., 2005). Interestingly, sustained expression (or over-expression) of telomerase has been shown to cause genomic instability (Roth et al., 2005), possibly due to an accumulation of DNA mutations, resulting from continued proliferation and replication of cancer cells. This genomic instability is likely to fuel further cancer progression, as observed in a case of uterine cervical dysplasia progressing to invasive cancer, in response to expression of multiple copies of hTERC (Hopman et al., 2006).
The fact that the majority of tumour cells express telomerase has made targeting this enzyme the basis of novel therapeutic approaches (reviewed by Mergny et al., 2002). Success to date has been limited, due to issues with toxicity, modes of delivery and the long lag time between treatment and cancer cell death. Despite this, a telomerase-targeting small molecule (GRN163L) is now undergoing clinical trials to determine the treatment efficacy (Dikmen et al., 2005).
Telomeres are distinct and essential components of chromosomes in eukaryotic cells, preventing structural DNA damage at the gene and chromosomal level. Decreased telomere length is a hallmark of senescence and aging, although the exact mechanism remains unclear. Crucially, telomere length is maintained in immortal cell lines and increased in cancer cells, through elevated telomerase levels. Although the mechanisms underpinning telomere maintenance in human cells are not fully understood, it is hoped that targeting telomeres of cancer cells, through the supporting protein machinery will form the basis of future anticancer treatments.