Telomerase Substrate Research Articles

Inhibition of Human Telomerase by 7-Deaza-2′-deoxyguanosine Nucleoside Triphosphate Analogs: Potent Inhibition by 6-Thio-7-deaza-2′-deoxyguanosine 5′-Triphosphate

We have examined analogs of the previously reported 7-deaza-2′-deoxypurine nucleoside triphosphate series of human telomerase inhibitors. Two new telomerase-inhibiting nucleotides are reported: 6-methoxy-7-deaza-2′-deoxyguanosine 5′-triphosphate (OMDG-TP) and 6-thio-7-deaza-2′-deoxyguanosine 5′-triphosphate (TDG-TP). In particular, TDG-TP is a very potent inhibitor of human telomerase with an IC50 of 60 nM. TDG-TP can substitute for dGTP as a substrate for telomerase, but only at relatively high concentrations. Under conditions in which TDG-TP is the only available guanosine substrate, telomerase becomes nonprocessive, synthesizing short products that appear to contain only one to three TDG residues. Similarly, the less potent telomerase inhibitor OMDG-TP gives rise to short telomerase products, but less efficiently than TDG-TP. We show here that TDG-TP, and to a lesser extent OMDG-TP, can serve as substrates for both templated (Klenow exo) and nontemplated (terminal transferase) DNA polymerases. For either polymerase, the products arising from TDG-TP are relatively short, and give rise to bands of unusual mobility under PAGE conditions. These anomalous bands revert, under treatment with DTT, to normal mobility bands, indicating that these products may contain thiol-labile disulfide linkages involving the incorporated TDG residues. This observation of potential TDG-crosslinks may have bearing on the mechanism of telomerase inhibition by this nucleotide analog.

Telomeric repeat amplification, without shortening or lengthening of the telomerase products: a method to analyze the processivity of telomerase enzyme.

The Telomeric Repeat Amplification Protocol (TRAP) and its modified versions (including ours, TP-TRAP) change the size and/or the ratio of the telomerase products in the amplification stage of the assay. Based on our recently published method we developed a new TRAP. This method ensures that the number of telomeric repeats present in the original telomerase products does not change on PCR amplification. The usefulness of the method was proved with amplification of chemically synthesized telomerase products and a newly designed telomerase substrate oligonucleotide. This is the first report in which the PCR products directly reflect the size distribution of telomerase products generated by the enzyme.

The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase α and the telomerase-associated Est1 protein

Saccharomyces telomeres consist of approximately 350 bp of C(1-3)A/TG(1-3) DNA. Most of this approximately 350 bp is replicated by standard, semiconservative DNA replication. After conventional replication, the C(1-3)A strand is degraded to generate a long single strand TG(1-3) tail that can serve as a substrate for telomerase. Cdc13p is a single strand TG(1-3) DNA-binding protein that localizes to telomeres in vivo. Genetic data suggest that the Cdc13p has multiple roles in telomere replication. We used two hybrid analysis to demonstrate that Cdc13p interacted with both the catalytic subunit of DNA polymerase alpha, Pol1p, and the telomerase RNA-associated protein, Est1p. The association of these proteins was confirmed by biochemical analysis using full-length or nearly full-length proteins. Point mutations in either CDC13 or POL1 that reduced the Cdc13p-Pol1p interaction resulted in telomerase mediated telomere lengthening. Over-expression of the carboxyl terminus of Est1p partially suppressed the temperature sensitive lethality of a cdc13-1 strain. We propose that Cdc13p's interaction with Est1p promotes TG(1-3) strand lengthening by telomerase and its interaction with Pol1p promotes C(1-3)A strand resynthesis by DNA polymerase alpha.

Inhibition of human telomerase by rubromycins: implication of spiroketal system of the compounds as an active moiety.

We found that a group of rubromycins and their analogues, a class of quinone antibiotics that possesses benzofuran and benzodipyran rings to form a spiroketal system, strongly inhibited human telomerase as assessed with a modified telomeric repeat amplification protocol. beta- and gamma-Rubromycins and purpuromycin appeared to be the most potent telomerase inhibitors, with 50% inhibitory concentrations (IC(50)) of about 3 microM, and griseorhodins A and C also showed comparable potencies for the inhibition (IC(50) = 6-12 microM). In contrast, opening of the spiroketal system of beta-rubromycin, giving rise to alpha-rubromycin, substantially decreased its inhibitory potency toward telomerase (IC(50) > 200 microM), indicating the essential role of the spiroketal system in telomerase inhibition. A kinetic study of the inhibition by beta-rubromycin revealed a competitive interaction with respect to the telomerase substrate primer, with a K(i) of 0.74 microM, whereas a mixed type inhibition was observed with respect to the nucleotide substrate. beta-Rubromycin was also potent in inhibiting retroviral reverse transcriptases but had virtually no effect on other DNA/RNA-modifying enzymes including DNA and RNA polymerases, deoxyribonuclease, and topoisomerase. Although beta-rubromycin showed nonspecific cytotoxicities, reducing proliferation of cancer cells (IC(50) approximately 20 microM), we conclude that beta-rubromycin appears to be a lead structure for the development of more potent and selective inhibitors of human telomerase.

Control of human telomere length by TRF1 and TRF2.

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.

Molecular interactions between telomerase and the tumor suppressor protein p53 in vitro.

The telomere DNA polymerase (telomerase) and the tumor suppressor protein p53 are frequently associated with human cancers, and activation of telomerase and inactivation of p53 involved in cancer cell immortalization. In this report, we demonstrate a direct interaction of telomerase with p53 in the nuclear lysates of human breast cancer cells, and with recombinant human p53, by affinity chromatography and immunoprecipitation. On activity criteria, the interaction is between the carboxyl-terminal region of p53 and a region close to the amino-terminus of human telomerase-associated protein 1 (hTEP1). Incubation of recombinant p53 with nuclear telomerase extracts results in inhibition of telomerase activity, with the C-terminal region of p53 being essential for inhibition. This effect is not mediated by binding to telomerase substrate DNA, but requires the region near the N-terminus of hTEP1, in that a synthetic peptide derived from this region of hTEP1 similarly inhibits telomerase activity. Together, these in vitro interactions between telomerase and p53 suggest that the activity of telomerase may be regulated by p53, down-regulation of which in turn would favor up-regulation of telomerase activity in cancer cell development.

Ciliate telomerase biochemistry.

Telomerase is a cellular reverse transcriptase specialized for use of a template carried within the RNA component of the enzyme ribonucleoprotein complex. Substrates for telomerase are single-stranded oligonucleotides in vitro and chromosome ends in vivo. In vitro, a bound substrate is extended by an initial round of DNA synthesis on the internal RNA template and in some cases by multiple rounds of template copying before product dissociation. In vivo, de novo synthesis of one strand of a telomeric repeat sequence by telomerase balances the sequence loss resulting from incomplete replication of linear chromosome ends by RNA primer-requiring DNA polymerases. Telomerase biochemistry has been studied extensively by using partially purified cell extracts. Telomerase components are being identified and beginning to be produced in recombinant form. This review focuses on the enzyme mechanism of telomerases from ciliate species, thus far the most intensively studied systems.

Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha.

Telomere proteins protect the chromosomal terminus from nucleolytic degradation and end-to-end fusion, and they may contribute to telomere length control and the regulation of telomerase. The current studies investigate the effect of Oxytricha single-stranded telomere DNA-binding protein subunits alpha and beta on telomerase elongation of telomeric DNA. A native agarose gel system was used to evaluate telomere DNA-binding protein complex composition, and the ability of telomerase to use these complexes as substrates was characterized. Efficient elongation occurred in the presence of the alpha subunit. Moreover, the alpha-DNA cross-linked complex was a substrate for telomerase. At higher alpha concentrations, two alpha subunits bound to the 16-nucleotide single-stranded DNA substrate and rendered it inaccessible to telomerase. The formation of this alpha . DNA . alpha complex may contribute to regulation of telomere length. The alpha . beta . DNA ternary complex was not a substrate for telomerase. Even when telomerase was prebound to telomeric DNA, the addition of alpha and beta inhibited elongation, suggesting that these telomere protein subunits have a greater affinity for the DNA and are able to displace telomerase. In addition, the ternary complex was not a substrate for terminal deoxynucleotidyltransferase. We conclude that the telomere protein inhibits telomerase by rendering the telomeric DNA inaccessible, thereby helping to maintain telomere length.

Interaction of recombinant Tetrahymena telomerase proteins p80 and p95 with telomerase RNA and telomeric DNA substrates.

Telomerase is a specialized reverse transcriptase that catalyzes telomeric repeat addition at the ends of existing telomeres or fragmented chromosomes. Two telomerase proteins from Tetrahymena thermophila, p80 and p95, were identified on the basis of their association with telomerase activity and telomerase RNA. Here we have produced recombinant versions of these proteins to characterize their functions in the ribonucleoprotein. Our findings indicate that the two proteins can form a complex whose association is independent of RNA. Each protein interacts directly with telomerase RNA, but the p80/p95 complex binds RNA with an affinity substantially greater than either protein alone. We have also characterized the DNA binding properties of p95 and show that it interacts with telomeric substrate DNAs with a specificity characteristic of the functionally defined Tetrahymena telomerase substrate anchor site. Together, these findings suggest a model in which protein-nucleic acid interactions separable from the active site contribute to positioning the template and primer, rather than exclusively the direct nucleic acid-active site interaction typical of other polymerases.

Variable telomeric repeat synthesis in Paramecium tetraurelia is consistent with misincorporation by telomerase

Telomeric DNA at the ends of chromosomes consist of short, tandem repeat sequences. The telomeres of Paramecium tetraurelia are made up of variable repeats, whereas Paramecium caudatum telomeric repeats are largely invariant. To investigate variable repeat synthesis in P. tetraurelia, mutated telomerase RNA genes were expressed in vivo. We demonstrate that the P. caudatum telomerase RNA can participate in telomere synthesis when expressed in the P. tetraurelia macronucleus, despite 24% primary sequence divergence of the RNAs between the two species. De novo telomeric repeats from transformants indicate that P. tetraurelia telomerase fidelity is dramatically affected by template substitutions and that misincorporation at a single templating position is likely to account for the majority of P. tetraurelia telomeric DNA variability. Furthermore, we show that fidelity is not solely a function of the RNA moiety, as the P. caudatum telomerase RNA does not impart high fidelity to the chimeric enzyme.

Telomere structure and telomerase expression during mouse development and tumorigenesis

Mouse telomeres are on average longer than those of man, raising questions regarding the link between telomere loss and replicative senescence in mice and the requirement for telomerase activity for mouse cell immortalisation. However, the emerging data on telomerase activity during tumorigenesis in the mouse must be interpreted in the context of the very different structure of mouse telomeres. It will be argued here that the evidence for a casual link between telomere loss and replicative senescence is weak in the mouse, with the observed upregulation of telomerase activity in mouse tumours perhaps instead reflecting co-ordinated regulatory changes in tumour cells. Its absence would be consistent with evolutionary considerations, which hypothesise that such a link is an additional layer of control against tumour formation that has evolved in man. The very different genomic substrates for telomerase in humans and mice mean that the initial phenotype of a telomerase knock-out mouse does not necessarily critically address the existence of a link between telomerase and tumorigenesis in man.

Cell cycle-regulated generation of single-stranded G-rich DNA in the absence of telomerase.

Current models of telomere replication predict that due to the properties of the polymerases implicated in semiconservative replication of linear DNA, the two daughter molecules have one end that is blunt and one end with a short 3' overhang. Telomerase is thought to extend the short 3' overhang to produce long single-stranded overhangs. Recently, such overhangs, or TG1-3 tails, were shown to occur on both telomeres of replicated linear plasmids in yeast. Moreover, indirect evidence suggested that the TG1-3 tails also occurred in a yeast strain lacking telomerase. We report herein a novel in-gel hybridization technique to probe telomeres for single-stranded DNA. Using this method, it is shown directly that in yeast strains lacking the TLC1 gene encoding the yeast telomerase RNA, TG1-3 single-stranded DNA was generated on chromosomal and plasmid telomeres. The single-stranded DNA only appeared in S phase and was sensitive to digestion with a single-strand-specific exonuclease. These data demonstrate that during replication of telomeres, TG1-3 tails can be generated in a way that is independent of telomerase-mediated strand elongation. In wild-type strains, these TG1-3 tails could subsequently serve as substrates for telomerase and telomere binding proteins on all telomeres.

Evidence for a New Step in Telomere Maintenance

The strand of telomeric DNA that runs 5′–3′ toward a chromosome end is typically G rich. Telomerase-generated G tails are expected at one end of individual DNA molecules. Saccharomyces telomeres acquire TG 1–3 tails late in S phase. Moreover, the telomeres of linear plasmids can interact when the TG 1–3 tails are present. Molecules that mimic the structures predicted for telomere replication intermediates were generated in vitro. These in vitro generated molecules formed telomere–telomere interactions similar to those on molecules isolated from yeast, but only if both ends that interacted had a TG 1–3 tail. Moreover, TG 1–3 tails were generated in vivo in cells lacking telomerase. These data suggest a new step in telomere maintenance, cell cycle–regulated degradation of the C 1–3A strand, which can generate a potential substrate for telomerase and telomere-binding proteins at every telomere.