The so-called "end-replication" problem applies primarily to somatic cells and is a direct consequence of DNA polymerase's biochemical properties (Figure 1.3). DNA polymerase requires short RNA primers to initiate replication, and it then extends the primers in a 5'- to-3'-direction. Thus, as the replication fork moves along the chromosome, one of the two daughter strands is synthesized continuously. The other daughter strand, known as the lagging strand, is synthesized discontinuously in short fragments known as Okazaki fragments, each of which has its own RNA primer. The RNA primers are subsequently degraded, and the gaps between the Okazaki fragments are then filled in by the DNA repair machinery. A problem arises at the end of the chromosome, however, because the DNA repair machinery is unable to repair the gap left by the terminal RNA primer.
Consequently, the new DNA molecule is shorter than the parent DNA molecule by at least the length of one RNA primer.
The ends of telomeres in germline and immortal cell populations are replicated by the enzyme telomerase, a specialised ribonucleoprotein. Here, it functions to maintain a constant telomere length. In human cells, shortening of telomeres is fundamental to replicative senescence and is considered to be an anti-neoplastic mechanism (150). Indeed, unrestrained telomere attrition can expose chromosome ends, trigger cell cycle checkpoints and lead to a senescent state (151) (M1 in Figure 1.4). Cells that are driven to continue dividing by abnormal stimuli develop massive genomic instability or crisis (M2 in Figure 1.4). Germline cells and immortal cell populations like most cancer cell lines possess mechanisms (telomerase activation or an alternative mechanism) to preserve their telomere length indefinitely despite cell division, thus protecting their genome (152).
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Figure 1.3 The “End Replication Problem”
The 3'–5' leading strand (above) is copied continuously to the end of the DNA molecule using DNA polymerase; the 5'–3' lagging parental strand (below) is copied in discontinuous Okazaki fragments initiated by labile RNA fragments (black boxes). The RNA primers are degraded, the internal gaps are filled, and the Okazaki fragments ligated. The terminal gap is not filled, leaving an unreplicated terminal region varying between the size of the RNA primer and the Okazaki fragment. The function of telomerase is to fill the terminal gap in the telomere. If telomerase is not present, as is generally true for human cells in vitro, the 5' end of the progeny strand is shortened every time the cell divides and DNA is replicated, eventually resulting in cessation of division.
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Figure 1.4 Telomeres, Hayflick limit and Crisis
Telomerase is active in germline cells, maintaining long stable telomeres, but is repressed in most normal somatic cells, resulting in telomere loss in dividing cells. At M1, the Hayflick limit, there is a presumed critical telomere loss in one or perhaps a few chromosomes signalling irreversible cell cycle arrest. This corresponds to the phenotype of replicative senescence. Transformation events may allow somatic cells to bypass M1 without activating telomerase. When chromosomes become critically short on a large number of telomeres, cells are genomically unstable and enter crisis (M2). Rare clones that activate telomerase escape M2, stabilize their genome, and acquire indefinite growth capacity. (Figure adapted from: Melk and Halloran – Cell Senescence and its implications for nephrology, J Am Soc Nephrol 12: 386, 2001)
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The term cellular senescence was coined by Hayflick and Moorhead who described the phenomenon that human diploid fibroblasts have a limited proliferative capacity in culture (41). After 50–70 generations, human fibroblasts show a permanent and irreversible growth arrest change in cell morphology. Subsequently, it was shown that replicative senescence occurs as soon as telomeres become critically shortened (46). Bodnar et al showed how telomerase, an enzyme that maintains telomere length, is able to rescue fibroblasts from replicative senescence (45). As the majority of human cells do not express telomerase, their ability to divide is therefore limited to a certain threshold (Hayflick number). If telomeres become critically short, they have the potential to unfold from their presumed closed structure, this may precipitate chromosomal fusions. The cell may detect this uncapping as DNA damage and then either stop growing (entering senescence), or begin programmed self-destruction (apoptosis). Alternatively, the cell may enter a state of immortality, depending on the cell's genetic background/p53 status (153) (Figure 1.5).
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Figure 1.5 Critical telomere shortening and p53
Telomere shortening activates p53 and drives formation of epithelial cancers through gene amplification and deletion. Telomeres shorten progressively with cell division due to the end- replication problem in cells with no telomerase. Critical telomere shortening compromises the telomere cap and results in a DNA damage response that activates the p53 tumour suppressor protein. This activation of p53 induces replicative senescence in cultured human fibroblasts, impairs stem cell self renewal, induces apoptosis in tissue progenitor cells, causes premature ageing and strongly suppresses tumour formation. If p53 is mutated or deleted, these responses to telomere dysfunction are mitigated and chromosomal fusions are tolerated. Chromosome breakage subsequently occurs predisposing to translocations, deletions and amplifications with resultant carcinogenesis. (Adapted from: Artandi and DePinho – Telomeres and telomerase in cancer, Carcinogenesis Vol.31 no.1 pg 10, 2010)
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Paradoxically therefore, an age-related decline in telomere length may promote genetic instability and increase the risk of malignancy (154). This is associated with an increase in oxidative damage accruing in biologically ageing tissues. Organs deteriorate as more and more of their cells die off or enter cellular senescence. A wide range of different diseases exhibit accelerated telomere attrition including psychological, cardiovascular, neurodegenerative, renal, osteo- and hepatic diseases (1;4;155-160). With respect to patients in end stage renal failure (ESRF), Carrero and colleagues, first showed that shortened telomere length is associated with higher levels of DNA damage (8-OH-dG) and increased mortality in haemodialysis patients (161). These findings were independent of age and gender which may be considered strong confounders for telomere length in humans (162). Such data proves invaluable as a means of progression to studies in enhancing the quantity of kidneys available for transplantation. Interestingly, the latter study also confirmed observations by Nawrot et al that females show less age related telomere attrition. They hypothesize that oestrogen may directly or indirectly exert protective effects on telomere length due to its anti inflammatory and anti oxidant properties (155;163;164).
Replicative senescenceand critical telomere attrition result in the activation of a number of cyclin dependent kinase inhibitors. Typically, p21 expression is elevated following acute oxidant insult followed by elevation of CDKN2A (p16INK4a) expression, necessary for the maintenance of the senescent state (165).