The structure of Telomerase and its function Introduction: Telomerase is an essential ribonucleoprotein enzyme for the stability of eukaryotic chromosome termini, which is recognized as telomere. Telomeres are specialized nucleoprotein complexes, consists of tandem DNA tracts (TTAGGG for all vertebrates) with specific telomeric protein bind to it. As the replication of eukaryotic chromosome can not performed all the way to the end of chromosome, telomere acts as a disposable buffer in replication. They are consumed during the replication of chromosome instead of the meaningful chromosome.
Telomerase replenish the consuming of telomere by adding the specific DNA sequence to the termini. This process is regulated by the proteins bind to the telemetric DNA (like POT1 in humans). Besides of this, some other protein binds along (TRF1, TIN2, tankyrase in human) are part of the negative feedback of the elongation of the chromosome. Telomerase is a reverse transcriptase as it carries its RNA template inside. However, it possesses an unusual character that its RNA template seems also acting in its enzyme function. The detail of the mechanism is still unclarified.
In this review, the structure, reaction and function of the telomerase will be discussed. Structure: Telomerase ribonucleoprotein (RNP) was first purified from Euplotes aediculatus (1). The purification process gives out that telomerase complex had a molecular mass of about 230 kD, which RNA subunit counts for 66kD, and a pair of 43kD and 123kd of proteins counts for 166kD. (1) Through the experiment of photocross-linking, we could specify 123kD part binds to telomeric DNA part. Through the SDS-polyacrylamide gels process, we could separates the polypeptides of the telomerase p123 subunits.
Then place trypsin to digest telomerase p123, and further take nanoelectrospray tandem mass spectrometry to determine the amino acid sequences(2). Thus a miniatuized form of electrospray that allows mass spectrometric interrogation of minute analyte volumes. (3) After these process, 14 peptides of p123 would be sequenced: Fig 1. Telomerase p123 subunit’s sequencing under tandem mass nanoelectrospray spectrometry. Part A is the unseparated peptide mixture under mass spectrum and part B is the tandem mass spectrum of the doubly charged precursor as the mass to charge ratio. 4) The peptide sequences were assigned: T1, YLLFQR; T2, DYNEEDFQVL (VK,AR); T3, (L, N)WDVLMK; T4, SFLMNNLTHYFR; T5, TLYSWLQK; T6, ETLAEVQEK; T7, AMLGNELFR;T8, LQTSFPLSPSK; T9,(A,Q) TLFTNLFR; T10, (L,T) ALMPNLNLR; T11, LFAQTNLVATPR; T12, LESWMQVETSAK; T13, QYFFQDEWNQVR; T14, (E,D) SMNPENPNVNLLMR. (5) Take 2 of these peptide sequences to design degenerate polymerase chain reaction (PCR) primers to amplify a part of p123’s gene, thus a gene library was prepared from it, then screen the fragment the integrate gene could be acquired. 4) It was found that p123 gene is 3279 base pair long, containing an continuous 1031-amino acid open reading frame. (4) The open reading frame predicts the protein is 122,562 daltons, matched the estimated size from SDS-polyacrylamide gel electrophoresis. The amino acids also matched well. These validate the experiment data. Telomerase reaction: Different species may bear the identical telomere sequence with each other but one species must have only one characteristic sequence. The repeating subunit of telomere sequence may be a perfect repeating one but may also an irregular one with variations on repeat unit. 6) But one thing must be unchanged, that one strand of DNA must contain clusters of G and another clusters of C. And the strand is rich of G is always at the 3’ end of the chromosome DNA strand. The telomerase synthesizes the G-rich strand DNA sequence. The elongation of telomere needs DNA primer to assist the telomerase so the telomeric repeats are added by polymerization in the 5’-3’ direction. Deoxynucleoside triphosphates (dNTPs) are used as substrates. Both a few repeats of a single stranded G-rich telomeric or telomere-like DNA sequence could act as perfect primers.
Here take telomerase in Tetrahymena thermophila as an example, in which the primer is the DNA oligonucleotde GGGGTTGGGGTT , it’s repeat subunit is (GGGGTT), GGGGTTGGGGTT+ 4n dGTP+ 2n TTp GGGGTTGGGGTT(GGGGTT)n +6n pyrophosphate. In the reaction, no other deoxynucleoside triphosphates, or nucleotide cofactors are required for the reaction. (7) And as the telomerase replenish the 3’end of the telomere, the 3’ end of the primer is always included in a perfect repeat subunit. (8, 9) In order to maintain the activity of telomerase, its RNA part is also essential.
This fact is first indicated that partially purified telomerase activity in tetrahymena extracts is sensitive to nuclease. When pretreated with nuclease or RNase A, the enzyme is inactivated. (10, 11) The RNA moiety was identified also from the Tetrahymena telomerase by its co-chromatographyic functions. The telomerase RNAs from different species have their species- specific telomeric repeat sequence. (12). Now only Tetrahymena spp and some related ciliate Glaucoma were identified by cross-hybridization of their single-copy genes to the Tetrahymena thermophila gene.
Human telomerase RNA has suggestion template sequence but this RNA has not yet been identified. The Tetrahymena telomerase RNA is RNA polymerase III transcript and their RNA polymerase III- mediated transcription is regulated by upstream cis- acting elements (11, 12). The regulation of telomerase RNA transcription has not been well studied. But when overexpressed in vivo, the stable-state level of accumulated telomerase RNA almost the same to the normal situation, though the transcription rate of the overexpressed genes is very high.
These facts indicated that excess telomerase RNA transcribed but not assembled into the telomerase RNA complex was degraded. The telomerase RNA acts a template function. This assumption is approved by the experiment designed to test the effect on telomerase activity of treatment with RNase H in the presence of DNA oligonucleotides complementary to the putative telomerase RNA sequence by the Tetrahymena telomerase RNA. (9) When treated with RNase H on RNA where it is base-paired to a complementary DNA sequence, the telomerase loss its activity. This indicated the corresponding RNA sequence was required for telomerase.
A DNA oligonucleotide which 3’end was complementary to the sequence in telomerase RNA directing RNase H cleavage would specifically inactivated telomerase. And this oligonucleotide could further block the access of the primer to the telomerase activity site. A DNA oligonucleotide whose 3’end was complementary to the sequence near, but ending one nucleotide from the 3’ end of the putative template sequence was also extend by GGGGTT repeat addition, suggesting that base- pairing of a DNA oligonucleotide to the telomerase RNA in the vicinity of the template would allow the oligonucleotide to be utilized as primer.
Base on this fact, it was proposed that the telomerase RNA sequence is the template sequence. (9) To verify this assumption, the prediction that if the RNA template sequence is altered, the corresponding DNA telomeric DNA will also be altered must be proved. And this prediction was confirmed by experiments done in vivo (13) Fig 2 Telomerase from Tetrahymena synthesis telomeric DNA. (9, 12) The mechanism of the telomeric DNA synthesis by telomerase is the model introduced above; the model is supported by results obtained with telomerase activities from oxytricha and human cells. 14, 15) In the model for synthesis of telomeric DNA: 1. The 3’ terminal chromosome with G-rich overhang in vivo or a single- stranded DNA primer oligonucleotide in vitro, base-pair with the telomere-complementary sequence in the telomerase RNA. 2. The chromosomal end is extended by polymerization of dGTP and dTTP using the RNA as template, resulting in the addition of repeat subunit. 3. The extended DNA strand unwinds from the RNA template and then is placed back to the 3’ terminal of the template, available for a new round of elongation.
In the phase 2, the DNA polymerization, is the process of the six nucleotide polymerization (for Tetrahymena) cycle. In order to make the polymerization move along, the telomerase’s conformation should has flexibility, as the polymerization active site and the template region of the RNA must move, as each template position is copied. (16) Actually, the telomerases have characteristic patterns of pausing points along the template, which can be different for different telomerases. ( 9, 15,16) Function
The regulation of telomerase function at the end of the telomere: One would expect the major site of telomerase regulation should happen at the telomere terminus, where it exerts its catalytic activity. The regulation of telomerase function is conducted in three steps- the recruitment of telomerase to its action site, the initiation of its catalytic reaction, and the elongation rate and processivity. First, is the recruitment process. The major regulator of the telomerase activity, at least in S.
Cerevisiae, is a telomere terminus specific factor Cdc13. Cdc13 is a single-stranded DNA binding protein that also function in the DNA repair pathway and DNA recombination. This factor was first identified by a cell division cycle mutant: CDC13 knock-out cells accumulate single-stranded DNA at chromosome ends, which finally leads to RAD-9 dependent cell cycle arrest (17). The most accepted model so far of how the Cdc 13 recruits the telomerase is that it interacts with one of the components of the telomerase complex: Est1.
This suspicion is substantiated by the rescue of telomere maintenance deficiency, and hence the restoration of normal cell cycle regulation, of Cdc13 mutant (at the suspected site of interacting with Est1) cell line, by introducing yet another mutation on corresponding site on the Est13 gene. This kind of allele specific suppression is most conveniently interpreted by the restoration of intact physical interaction between the two molecules.
This model is further supported by several fusion experiments, in which the Cdc13 protein (or part of it) is genetically designed to fuse to essential telomerase components including Est1, and other proteins. Interestingly, the fusion of Cdc13 with telomerase components other than Est1 liviated the requirement of intact Est1 in telomere maintenance, leading to the conclusion that the recruitment of telomerase to the action site, rather than precisely positioning the telomerase or other more complex activation processes, are the mechanism of Cdc13 maintaining the intact structure of telomeres. 18) Chromatin immunoprecipitation experiments did by Taggart and his colleagues (2002) has also shown that the recruitment of telomerase to the ends of telomeres is also facilitated by interactions of other telomere proteins and telomerase complex components. Est2 could binds to the telomeres and this association is tested to be independent of Cdc13. this interaction may further help to position the telomerase in the right gesture relating to the telomere end. Further evidences have shown that this interaction is possibly mediated by yet another protein TLC1.
On the contrary of Cdc13’s role in recruitment of telomerase, it also potentially limits the elongation rate of TERT. To maintain the telomere homeostasis, which means the stable yet dynamic length of telomeres by balancing the prolonging of telomere by telomerase and the erosion by the end replication problem intrinsic to eukaryotic DNA replication mechanism, the activity and abundance of telomerase should be strictly regulated. This is postulated to be controlled by negative feedback loop involving the RAP1/RIF1/RIF2 complex in yeast and TRF1 complex in mammalian cells.
Interestingly there is a drastic change in the telomere homeostasis control complex from yeast to mammals. In this review, we only deal with mammalian complex and detailed mechanism. TRF1 binds to the tandem repeat in telomeres (TTAGGG repeat binding factor 1). Long telomeres are preferable. The role of TRF1 in telomere maintenance was first found when observing the fact that over-expression of TRF1 would set the average length of telomeres to a shorter equilibrium length.
More importantly, this did not happen in telomerase negative cells, in which, the telomere shorten at the same rate in spite of the level of TRF1. So TRF1 is postulated to control the length of telomeres by interfering the function of telomerase at end of telomeres. In accordance with this model, the short telomeres are preferable lengthened and the long telomeres are more likely to be eroded, simply because short telomeres tends to have less TRF1 bound, leaving full capacity of telomerase (17). Telomerase in cancer and aging:
The knowledge of telomerase in aging is mainly derived from experiments with human primary fibroblasts. The over-expression of TERT subunit of telomerase was observed to have unlimited proliferate potential in the absence of malignancy. Human population studies had also correlated relatively shorter telomere length in peripheral blood cells with high mortality rate and shorter life expectancy. Human diseases caused by genetic mutations in either TERT or TERC components of telomerase results in obvious accelerated or premature aging phenotypes.
TERT or TERC knockout mice seemed have normal phenotypes and unaffected aging process in the first generation, but there were significant reduction of life expectancy and obvious premature aging syndromes observed in the G2 and especially G3 generation, which signified the process of telomeres being worn off in a couple of generations, and indicates that telomerase mainly affects aging by maintaining the telomeres length (19). However there is also growing evidences showing that telomerase has adverse effects on aging independent of it function with telomere maintenance.
The detailed mechanism behind it remains largely unknown, but the potential candidate pathways involving those mechanisms encompasses a lot of important cellular functions including DNA damage repair, cell cycle control and others(20). As telomere shortening was shown to suppress cancer and keep control of cell populations in regenerative tissues, one would expect that expression of telomerase would be positively related to tumorigenesis. Although some observations supports this conclusion, this is not true by large. (19).
Actually the shorting of telomeres in the absence of telomerase would lead to the instabilities chromosome; this is one of the forms of genetic instability, which increases the chance of oncogenetic mutation. However those mutations were suppressed by other mechanisms, and those the increases of carcinogenesis are only signified in certain genetic backgrounds like p53 heterozygous mics (21, 22). Discussion: Telomerase research has its great potential in a variety of aspects. The most promising application in the future is introducing exogenous telomerase genes into normal somatic cells by gene therapy.
As the telomerase helps to maintain the homeostasis of telomeres which are speculated to be the clock of cellular aging, and the level of telomerase expression (especially the gene for TERT component) is far lower than counteracting the effect of telomere shortening caused by endogenous DNA replication drawback, the introduction of telomerase gene under stronger promoters would probably prolong the replicative life of somatic cells in our body and hopefully, prolong the life expectancy of the whole organism.
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