Telomeres The Guardian Of Chromosome Ends Biology
Telomeres are repeated nucleoprotein structures, sited at the chromosome termini, which help to preserve the integrity and stability of the genome (Bhattacharyya & Lustig, 2006). The protective function owes much to their unusual architecture and composition. Human telomeres consist of tandem repeats of the sequence 5'-TTAGGG-3' (Moyzis et al., 1988) and terminate with a single-stranded G-rich overhang (Makarov et al., 1997). The ends of telomeres are 'capped' by telomere-binding proteins (collectively called shelterin) (de Lange, 2005), which promote invasion of the telomeric arrays by the 3' overhang to form a higher-order structure known as a T-loop (Figure 1) (Griffith et al., 1999; de Lange, 2004). This is thought to prevent the chromosome ends from being mistaken for DNA breaks, thus avoiding unnecessary activation of repair systems and subsequent chromosome fusions or degradation (de Lange, 2002). Telomeres also play important role in chromosome segregation during mitosis (Kirk et al.; 1997).
Figure 1: Electron Micrograph of Human T-loop (Griffith et al., 1999)
Immediately adjacent to telomeres are highly variable repeat regions called subtelomeres, (Allshire et al, 1989; Riethman, 2005). Currently, there is no means of separating the telomere repeats from the subtelomeric DNA, thus telomere length cannot be measured directly. Digestion of DNA with restriction endonucleases liberates terminal restriction fragments (TRFs), which are composed of both regions. In humans, telomere length is estimated to be between 0.5 and 15 kilobase pairs (kbp) long (Aubert & Lansdorp, 2008).
The End-Replication Problem
Telomeres provide a buffer of disposable DNA, which solves the 'end-replication problem' (i.e. inability of DNA polymerase to completely replicate the ends of linear chromosomes) (Watson, 1972; Olovnikov, 1973)
DNA polymerase can only replicate DNA in the 5'â†’3' direction and requires a RNA primer to initiate replication. The leading strand, which has the 3' to 5' parent strand, undergoes continuous DNA replication whilst the lagging (discontinuous) strand is replicated as a series of short Okazaki fragments. Removal of the terminal RNA primer at the end of the lagging strand leaves a gap that cannot be filled by the enzyme due the absence of a free 3'-OH to prime synthesis (Figure 1). This results in incomplete replication of telomeres.
The end-replication problem has always been regarded as a 'lagging strand problem' (Levy et al., 1992). However, it could also stem from the inability of leading strand synthesis to produce a 3' overhang (Lingner, 1995).
Figure 2: End-Replication Problem.
Regardless, this phenomenon shortens telomeres by approximately 120 base pairs per cell doubling (Vaziri et al., 1993) and serves as a 'mitotic clock' that defines the proliferative potential of the cells (Harley, 1991). The loss of telomeric repeats ultimately leads to permanent cellular growth arrest called replicative senescence (or M1 stage) though initiation of DNA damage signals and activation of p53 and/or p16-Rb tumour suppressor pathways (Hara et al., 1991; Shay et al., 1991; Vaziri & Benchimo, 1996; Lin et al., 1998; d'Adda di Fagagna et al., 2003). By restricting the proliferative capacity of cells, including those with deleterious mutations, telomere shortening and replicative senescence form a major barrier to tumourigenesis.
Replicative Senescence & Beyond
Although, there is universal agreement that telomere shortening causes the onset of senescence there remains some doubt about the specific molecular trigger. Some studies suggest that a single short telomere or a group of the chromosomes with the shortest telomeres may induce senescence (Henman et al., 2001; Zou et al., 2004). Other researchers argue that an altered telomeric state (function) rather than an exact length triggers senescence (Karlseder et al., 2002). Telomeres are able to repress the transcription of neighbouring genes by a length-dependent mechanism known as the telomere position effect (TPE) (Baur et al., 2001), thus it has been proposed that telomere shortening may lead to reactivation of genes implicated in cell cycle arrest. However, this is unlikely since specific genes controlled by telomere length are yet to be identified.
In the absence of senescence pathways (e.g. due to loss of p53 function), cells continue to divide and telomeres shorten further, destabilising chromosome ends (Duncan & Reddel, 1997). The resulting telomeric fusions can initiate repeated breakage-fusion-bridge (BFB) cycles that lead to extensive chromosome rearrangements and aneuploidy (Lundblad, 2001; Shay & Roninson, 2004). This genomic instability invariably leads to a period of crisis (or M2 stage), which is characterised by widespread apoptotic cell death (Shay & Wright, 1992). Nonetheless, selected rare cells (10-7) may emerge from crisis with a mechanism for telomere maintenance and often exhibit genomic abnormalities (Shay et al., 1993; Artandi & DePinho, 2000; Murnane, 2006). In these cases, telomere dysfunction serves as a mutational mechanism that drives cancer cell formation.
Telomerase: The Mediator of Immortality
Telomere shortening can be counteracted by activation of a specialised enzyme named telomerase, which was first identified in the protozoan Tetrahymena (Greider & Blackburn, 1985). Telomerase is a ribonucleoprotein composed of a catalytic subunit called telomerase reverse transcriptase (TERT) and an RNA moiety known as telomerase RNA (TERC), which serves as the template for de novo synthesis of telomeric repeats (Feng et al., 1995; Shippen-Lentz & Blackburn, 1990; Nakamura et al., 1997; Autexier & Lue, 2006).
Figure 3: Telomere Extension by Telomerase
The enzyme elongates the 3' end of the DNA, thus enabling other polymerases to synthesise the complementary strand (Autexier & Lue, 2006). A short segment within the TERC is repeatedly used to extend the telomere (Figure 3). Firstly, the DNA is recognised by telomerase and hybrid structure is formed between the 3' end of the DNA and the integral RNA template. DNA sequences upstream of this region interact with an anchor site. Next, nucleotides are sequentially added onto the 3' end of the DNA until the 5' end of the template is reached. The enzyme then translocates to 3' end of the newly synthesised DNA sequence and another reaction cycle is initiated. It is remains unclear how the process is halted, but telomere structure could be involved since folding of DNA into a special G-quartet configuration has been shown to prevent elongation (Figure 4) (Zahler et al., 1991).
Figure 4: Model of G-Quartet Configuration
Telomerase is active in about 80% of cancer cells (Kim et al., 1994, Shay & Bacchetti, 1997), indicating that its acquisition is critical for tumour progression. A characteristic of these cells is that they have very short telomeres (Counter et al., 1994; Schmitt et al., 1994). Most somatic cells do not display telomerase activity (Forsyth et al., 2002). However forced expression of human TERT (hTERT) triggers enzyme activity and extends the replicative lifespan of the cells (Bodnar et al., 1998; Counter et al., 1998; Vaziri et al., 1998). The cells do not undergo changes associated with a malignant phenotype (Jiang et al., 1999), suggesting that telomerase expression alone does not cause tumours.
How telomerase is activated during crisis remains unknown, though amplification of the hTERT gene has been observed in some tumours (Takuma et al., 2004; Nowak et al., 2006). Also, the promoter of the hTERT gene is a regulatory target for several tumour suppressors and oncogenes (Janknecht, 2004), which could acquire mutations during genomic instability.
A small minority of immortalised cells maintain telomere length by a telomerase-independent process termed alternative lengthening of telomeres (ALT) (Bryan et al., 1995; Bryan et al., 1997). ALT appears to be a recombination-based mechanism, in which one telomere is used as a template to extend another telomere (Dunham et al., 2000; Muntoni & Reddel, 2005). The cells usually exhibit long, heterogonous telomeres (Bryan et al., 1995).
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