1) What is the problem with completing DNA replication in linear chromosomes? DNA polymerases are known to be dependent on a preformed RNA or DNA primer to initiate elongation for DNA replication from the 3' end of the DNA strand. The replication process occur simultaneously but in opposite directions and while the leading strand (3' - 5') goes all the way to the end of the parental template without losing any deoxyribonucleotides, the lagging strand (5' - 3') replicates in a stepwise fashion dependent on an RNA primer that is subsequently degraded and the gaps filled in with DNA polymerase and ligated by DNA ligase. But at the very end, the ribonucleotides cannot be served as a template for replicative DNA polymerases; the DNA polymerases therefore cannot fill in the gaps at the end of the lagging strand, resulting in the lagging strand successively becoming shorter and shorter upon each round of replication.
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2) How have eukaryotes solved this problem?
This problem is solved by the addition of an enzyme that adds telomeric (TEL) sequences to the ends of each chromosome, a protein-RNA complex called telomere terminal transferase or telomerase. The sequence of telomerase-associated RNA acts as the template for addition of deoxyribonucleotides to the ends of the telomeres and using the extended template strand, the okazaki fragments can then be added to the template strand and continue the replication process.
∴ The shortening of the lagging strand prevented.
3) How was the existence of the telomeres demonstrated?
The trials to create DNA ends for a stabilized in vitro DNA replication showed that a very specialized piece of DNA is required for function as a stable DNA terminus in yeast. DNA ends originated by gamma irradiation and DNA ends of plasmids generated by restrictive enzymes are unstable as they are very recombinogenic, they ligate to other free ends and are susceptible to exonucleolytic degradation. Artificially constructed hairpin terminus too was unable to stabilize a linear plasmid in yeast.
A study was then conducted by constructing a linear yeast plasmid by joining fragments from the termini of Tetrahymena ribosomal DNA (rDNA) to a yeast vector.
During a comparison of Tetrahymena rDNA terminus and yeast, it was found that there are cluster of repeats of the hexonucleotides C-C-C-C-A-A (C4A2) at or near the DNA termini; the clusters are of variable size and with specific gaps or nicks, and there are present at the 5' end of the Tetrahymena rDNA strands, 3-4 repeats apart in yeast, thus suggesting that the enzymatic process responsible for these lesions to be similar in the two organisms. The fact that yeast can recognize and use DNA ends from organism Tetrahymena suggests a high evolutional conservation for the structural features required for telomere replication and resolution.
One Tetrahymena end was removed by restriction enzyme, and yeast fragments that can function as an end on a linear plasmid were selected. As the unusual structure of Tetrahymena end is maintained in yeast, restriction fragment of the tetrahymena rDNA plasmid can function as a telomere on a linear plasmid in yeast. The linear plasmid is then used as vector to clone chromosomal telomeres from yeast. Restriction mapping and hybridization analysis demonstrated that these fragments were yeast telomeres and suggested that all yeast chromosomes might have a common telomere sequence.
(Szostak and Blackburn 1982)
4) What is the mechanism of action of telomerase?
The telomerase complex contains a telomerase-associated RNA template and telomerase catalytic site. Telomerase basically functions by reverse transcription and translocation hybridization steps.
After recognition of the TTGGGG sequence, the telomerase-associated RNA template base pairs with the 3' end of the lagging strand template.
The telomerase catalytic site then uses reverse transcription and adds deoxyribonucleotides on the lagging strand using the RNA molecule as template, thus elongating the lagging strand.
The DNA-RNA duplex are then thought to be slip relative to each other, leading to translocation of a single-stranded region of the telomeric DNA strand and the 3' most TTG nucleotides of the lagging strand are hybridized to the telomerase-associated RNA template.
The lagging strand is elongated again as in step (ii). This mechanism explains how oligonucleotides with 3' ends terminating at any nucleotide with TTGGGG sequence are correctly elongated to yield perfect tandem repeats of (TTGGGG)n. (Or any sequence for that matter).
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Figure 4. Mechanism of action of telomerase using a Tetrahymena model.
5) How does a telomerase assay work?
A telomeric sequence of G-rich DNA is added de novo onto the preexisting telomeric end, and once the strand is formed, it serves as the template for the synthesis of the complementary C-rich strand by conventional primase and DNA polymerase activities.
Using TTGGGG sequence as primer, the telomere elongation activity is assayed in the Tetrahymena cell extracts by adding various combinations of α32P-labeled deoxynucleoside triphosphates (α32P-dNTPs) and unlabeled nucleoside triphosphates (dNTPs). The bands are observed on the electrophoresis and this assay also shows that the bases sequence is not due to sequence-specific nuclease activity.
The assay also comprises of several tests to eliminate other possible factors. For one, the whole cell extracts were separated into S100 supernatant and pellet fractions, pretreated with micrococcal nuclease, in order to test whether the repeat addition was independent of Tetrahymena DNA. Different endogenous oligomers (CCCAA) (TTGGGG) are primed in repeat synthesis to test the possibility of their hybridization as the input synthetic oligomer. The involvement of endogenous Tetrahymena α-type DNA polymerase in the repeat synthesis is also tested using the inhibitor drug aphidicolin. These experiments indicate that the addition activity is independent of both endogenous DNA and endogenous DNA polymerase CI.
The oligomer sequence is further verified using the Sanger sequencing method with dideoxynucleotides (Sanger et al., 1977), where each dideoxynucleoside triphosphate (ddNTP) - that was added in turn to the in vitro reactions in the presence of labeled dGTP and the other unlabeled dNTPs - terminates the chain at the site of addition, making sequencing deduction possible with the addition products.
The extent of α32P-dGTP incorporation was also measured as a function of increasing input oligomer concentration with a high concentration suggesting a high affinity for the oligomer primer hence a high efficiency at micromolar concentrations and a high affinity for triphosphate substrates, providing evidence that the addition activity in the Tetrahymena extracts has the properties expected of an enzyme. The label incorporation is then stopped by heating the extracts or by treatment with proteinase K.
6) What are the roles of telomeres and telomerase in the following:
Genetic evidence showed that short telomeres induce senescence; shortening telomere is heavily associated with the reduction of replicative life span of cultured human cells. Conversely, introduction of telomerase into these cultured human cells proved to extend their life spans in the absence of other cellular alterations. However the link between ageing and telomere length is not absolute and compromised telomere function can cause tissue degeneration even in the absence of telomere shortening. Also, it's now known that human telomerase, TERT, has several other activities aside telomere maintenance that could be potentially contributing to increased cell proliferation. Maintenance of telomere length and protection are undoubtedly important factors in the control of cellular life span but ageing in itself is a complex process influenced by many different factors, therefore, further research needs to be done before one can affirm the roles of telomeres and telomerase in ageing.
Absence of telomeres from the end of a chromosome triggers breakage-fusion-bridge (BFB) cycles that consequently inflict major damage to chromosome structures. Absence of telomerase causes fusion (bridge) of sister chromatids during mitosis and during anaphase, this subsequently leads to amplication or deletion of terminal genes wherein the sister cells separate unevenly (refer to figure 6.2).
Transformation of cultured human cells is known to be in three steps (Hahn et al., 1999, 2000): activation proliferation e.g., induced by expression of a mutant ras ocogene; inactivation of tumor suppressors p53 and Rb; and activation of telomerase by expression of hTERT.
Extensive shortening of telomeres is recognized as a type of DNA damage and at the loss of telomeres, consequent stabilization and activation of p53 protein by the cells leads to p53 triggered apoptosis and eventual cell death. Loss of telomeres limits the number of rounds of cell division, therefore, in order to survive and avoid eventual cell death, cancer cells strive to maintain the telomere length as to ensure infinite cell division. About 85%-90% of human cancer cases showed increased telomerase activity reactivation while the rest maintain telomeres by ALT (alternative lengthening of telomeres) which occurs by exchange of sequences between telomeres (Dunham et al., 2000).
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Antisense RNA against telomerase production causes HeLa cells (tumour cells) to cease growth in four weeks. Dominant negative (DN) telomerase e.g., the modified RNA templates, can interfere with cancer cell growth and lead to apoptosis (Hahn et al., 1999; Zhang et al., 1999). Telomerase-null mice are also found to develop tumours less readily than normal mice and in another case, high levels of telomerase predict a poor response to therapy and vice versa. Level of N-myc protein, a transcription factor that regulates expression of telomerase too, is predictive for tumour outcomes. Other mutations are also found to be reduced if mice lack telomerase.
The loss of telomeres in cancer cells coupled with multiple mechanisms can trigger the BFB cycles.
∴Telomerase expression is essential for cancer cells to survive, therefore inhibiting telomerase can be a useful therapeutic treatment for cancer cells.
There is, however, a setback as a recent observation in mouse models showed that tumours can still develop in the absence of telomerase activity and that short telomeres may in some situations enhance genomic instability and accelerate tumour development, particularly in organisms with familial disease syndromes characterized by defects in telomerase functions.
It is found that induced pluripotent stem cells (iPS) show telomere characteristics similar to embryonic stem cells with respect to epigenetic marks. Telomerase reintroductions are also found to be decreasing reprogramming efficiency of cells in mice with telomerase deficiency. However, a recent discovery showed that dividing primary fibroblasts have low levels of hTERT expression and telomerase activity and that disruption of this activity by ectopic expression e.g by catalytically inactive mutant of hTERT (DN-hTERT) or by RNA interference (RNAi) leads to premature senescence. Conclusively, more evidence is needed to emphasize the roles of telomeres and telomerase in stem cells.