SUMOylation- and GAR1-Dependent Regulation of Dyskerin Nuclear and Subnuclear Localization.

The nuclear and subnuclear compartmentalization of the telomerase-associated protein and H/ACA ribonucleoprotein component dyskerin is an important although incompletely understood aspect of H/ACA ribonucleoprotein function. Four SUMOylation sites were previously identified in the C-terminal nuclear/nucleolar localization signal (N/NoLS) of dyskerin. We found that a cytoplasmic localized C-terminal truncation variant of dyskerin lacking most of the C-terminal N/NoLS represents an under-SUMOylated variant of dyskerin compared to wild-type dyskerin. We demonstrate that mimicking constitutive SUMOylation of dyskerin using a SUMO3 fusion construct can drive nuclear accumulation of this variant and that the SUMO site K467 in this N/NoLS is particularly important for the subnuclear localization of dyskerin to the nucleolus in a mature H/ACA complex assembly- and SUMO-dependent manner. We also characterize a novel SUMO-interacting motif in the mature H/ACA complex component GAR1 that mediates the interaction between dyskerin and GAR1. Mislocalization of dyskerin, either in the cytoplasm or excluded from the nucleolus, disrupts dyskerin function and leads to reduced interaction of dyskerin with the telomerase RNA. These data indicate a role for dyskerin C-terminal N/NoLS SUMOylation in regulating the nuclear and subnuclear localization of dyskerin, which is essential for dyskerin function as both a telomerase-associated protein and as an H/ACA ribonucleoprotein.

Mutated telomeres sensitize tumor cells to anticancer drugs independently of telomere shortening and mechanisms of telomere maintenance.

Telomerase is a ribonucleoprotein complex that maintains the stability of chromosome ends and regulates replicative potential. Telomerase is upregulated in over 85% of human tumors, but not in adjacent normal tissues and represents a promising target for anticancer therapy. Most telomerase-based therapies rely on the inhibition of telomerase activity and require extensive telomere shortening before inducing any antiproliferative effect. Disturbances of telomere structure rather than length may be more effective in inducing cell death. Telomerase RNA subunits (hTRs) with mutations in the template region reconstitute active holoenzymes that incorporate mutated telomeric sequences. Here, we analysed the feasibility of an anticancer approach based on the combination of telomere destabilization and conventional chemotherapeutic drugs. We show that a mutant template hTR dictates the synthesis of mutated telomeric repeats in telomerase-positive cancer cells, without significantly affecting their viability and proliferative ability. Nevertheless, the mutant hTR increased sensitivity to anticancer drugs in cells with different initial telomere lengths and mechanisms of telomere maintenance and without requiring overall telomere shortening. This report is the first to show that interfering with telomere structure maintenance in a telomerase-dependent manner may be used to increase the susceptibility of tumor cells to anticancer drugs and may lead to the development of a general therapy for the treatment of human cancers.

Rapid upregulation of telomerase activity in human leukemia HL-60 cells treated with clinical doses of the DNA-damaging drug etoposide.

The enzyme telomerase is implicated in cellular resistance to apoptosis, but the mechanism for this resistance remains to be elucidated. The ability of telomerase to synthesize new DNA at telomeres suggests that this enzyme might function in the repair of double-stranded DNA breaks. To distinguish the effects of double-stranded DNA break damage and apoptosis on human telomerase activity, we treated the HL-60 human hematopoietic cancer cell line with clinical doses of the chemotherapeutic drug etoposide (0.5 to 5 microM), which allowed us to distinguish between events associated with DNA damage-induced cell cycle arrest, and events associated with apoptosis. Large (three- to seven-fold) upregulation of telomerase activity occurred soon after etoposide treatment (3 h) in S/G2/M-arresting populations; this upregulation was abolished at onset of apoptotic cell death. No upregulation of telomerase activity was observed in cells treated with a larger dose of etoposide (5 microM) that caused cells to undergo rapid apoptosis without intervening cell cycle arrests. These observations are consistent with a possible role for telomerase upregulation during the DNA damage response.

Tetrahymena proteins p80 and p95 are not core telomerase components.

Telomeres provide stability to eukaryotic chromosomes and consist of tandem DNA repeat sequences. Telomeric repeats are synthesized and maintained by a specialized reverse transcriptase, termed telomerase. Tetrahymena thermophila telomerase contains two essential components: Tetrahymena telomerase reverse transcriptase (tTERT), the catalytic protein component, and telomerase RNA that provides the template for telomere repeat synthesis. In addition to these two components, two proteins, p80 and p95, were previously found to copurify with telomerase activity and to interact with tTERT and telomerase RNA. To investigate the role of p80 and p95 in the telomerase enzyme, we tested the interaction of p80, p95, and tTERT in several different recombinant expression systems and in Tetrahymena extracts. Immunoprecipitation of recombinant proteins showed that although p80 and p95 associated with each other, they did not associate with tTERT. In in vitro transcription and translation lysates, tTERT was associated with telomerase activity, but p80 and p95 were not. p80 bound telomerase RNA as well as several other unrelated RNAs, suggesting p80 has a general affinity for RNA. Immunoprecipitations from Tetrahymena extracts also showed no evidence for an interaction between the core tTERT/telomerase RNA complex and the p80 and p95 proteins. These data suggest that p80 and p95 are not associated with the bulk of active telomerase in Tetrahymena.

Human telomerase RNA-protein interactions.

Telomere length is maintained in most eukaryotic cells by telomerase. The core components of this ribonucleoprotein (RNP) enzyme include a protein catalytic subunit, composed of motifs conserved among reverse transcriptases (RT), and an RNA subunit that contains a short template sequence essential for the synthesis of telomeric repeats. We developed an electrophoretic mobility shift assay using active telomerase partially purified from 293 cells and radiolabeled, in vitro-transcribed human telomerase RNA (hTR) to investigate the molecular interactions of the human telomerase RT (hTERT) and telomerase-associated proteins with hTR. A specific hTR-protein complex was identified and shown to contain hTERT and human Staufen by antibody supershift assays. Variants of hTR altered in distinct structural elements were analyzed for their ability to competitively inhibit complex formation. Human telomerase RNAs lacking the CR4-CR5 domain were poor inhibitors of hTR-protein complex formation, suggesting that the CR4-CR5 domain of hTR is a potential protein-binding site. Furthermore, alterations in the telomerase RNA pseudoknot's P3 helix, the CR7 domain, or the H/ACA box efficiently inhibited formation of the complex, indicating that these domains are dispensable for the assembly of a telomerase RNP in vitro. Potential telomerase-associated proteins that bind hTR were also identified using a UV cross-linking assay.

Functional regions of human telomerase reverse transcriptase and human telomerase RNA required for telomerase activity and RNA-protein interactions.

Telomerase is a specialized reverse transcriptase (RT) that is minimally composed of a protein catalytic subunit and an RNA component. The RNA subunit contains a short template sequence that directs the synthesis of DNA repeats at the ends of chromosomes. Human telomerase activity can be reconstituted in vitro by the expression of the human telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. We analyzed telomerase activity and binding of hTR to hTERT in RRL by expressing different hTERT and hTR variants. hTRs containing nucleotide substitutions that are predicted to disrupt base pairing in the P3 helix of the pseudoknot weakly reconstituted human telomerase activity yet retained their ability to bind hTERT. Our results also identified two distinct regions of hTR that can independently bind hTERT in vitro. Furthermore, sequences or structures between nucleotides 208 and 330 of hTR (which include the conserved CR4-CR5 domain) were found to be important for hTERT-hTR interactions and for telomerase activity reconstitution. Human TERT carboxy-terminal amino acid deletions extending to motif E or the deletion of the first 280 amino acids abolished human telomerase activity without affecting the ability of hTERT to associate with hTR, suggesting that the RT and RNA binding functions of hTERT are separable. These results indicate that the reconstitution of human telomerase activity in vitro requires regions of hTERT that (i) are distinct from the conserved RT motifs and (ii) bind nucleotides distal to the hTR template sequence.

Expression of hTERT and hTR in cis reconstitutes and active human telomerase ribonucleoprotein.

Telomeres in eukaryotic cells are generally synthesized and maintained by the ribonucleoprotein (RNP) telomerase. This enzyme is composed of at least two subunits, the telomerase reverse transcriptase (TERT) and the telomerase RNA. Human telomerase activity can be reconstituted in vitro by the expression of the telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. The hTERT and hTR subunits are independently expressed in vivo, and little is known about the mechanism of their assembly. To facilitate recombinant telomerase RNP formation and reconstitution, we engineered a construct, termed hTERT-hTR cis, in which the 3' end of the hTERT coding sequence was extended by the addition of the sequence encoding hTR. Expression of the hTERT-hTR cis construct in vitro (in RRL) and in vivo (in the yeast Saccharomyces cerevisiae) produced hTERT-hTR transcripts of the predicted size. Active human telomerase was reconstituted by hTERT-hTR cis expression in both RRL and S. cerevisiae. Assembly of functional human telomerase by the bicistronic expression of the protein and RNA components may facilitate the overexpression and reconstitution of this enzyme in heterologous systems.

Functional reconstitution of human telomerase expressed in Saccharomyces cerevisiae.

Telomerase is a ribonucleoprotein enzyme complex that adds DNA repeats at the ends of chromosomes. In an effort to establish an in vivo heterologous expression system for active human telomerase, we expressed human telomerase reverse transcriptase (hTERT) in Saccharomyces cerevisiae and affinity-purified the protein as a fusion with glutathione S-transferase (GST). Addition of the GST moiety to the N terminus of hTERT did not interfere with telomerase activity when GST-hTERT was expressed in rabbit reticulocyte lysate (RRL) in the presence of the human telomerase RNA (hTR). Active human telomerase was immunoprecipitated from yeast lysates that co-expressed GST-hTERT and hTR. In addition, telomerase activity could be reconstituted in vitro by the addition of hTR to GST-hTERT that was immunoprecipitated from either RRL or S. cerevisiae lysates. The expression and reconstitution of human telomerase activity in yeast will provide powerful biochemical and genetic tools to study the various components required for the assembly and function of this enzyme.

Telomerase as a possible target for anticancer therapy.

Telomeres, the termini of chromosomes, provide essential stability to linear eukaryotic chromosomes. The enzyme telomerase is one mechanism that maintains telomeres, and is activated in 85% of human cancer cells. New studies on peptide nucleic acids (PNAs) that inhibit telomerase have demonstrated that unexpected regions of the enzyme can serve as targets for inhibitors. The novel delivery method used expands the utility of PNAs.

Tetrahymena telomerase ribonucleoprotein RNA-protein interactions.

Telomerase is an enzyme that is essential for the replication and maintenance of chromosomal termini. It is a ribonucleoprotein consisting of a catalytic subunit, one or more associated proteins, and an integral RNA subunit that serves as a template for the synthesisof telomeric repeats. We identified a Tetrahymena telomerase RNA-protein complex by an electrophoretic mobility shift assay, using telomerase partially purified from whole cell extracts and radiolabeled, in vitro transcribed wild-type Tetrahymena telomerase RNA. Complex formation was specific as unlabeled Tetra-hymena telomerase RNA, but not Escherichia coli ribo-somal RNAs, competitively inhibited complex formation. Binding required concentrations of MgCl2of at least 10 mM and occurred over a wide range of potassium glutamate concentrations (20-220 mM). The RNA-protein complex was optimally reconstituted with a 30 degrees C preincubation for

Mutational analysis of the Tetrahymena telomerase RNA: identification of residues affecting telomerase activity in vitro.

Telomere-specific repeat sequences are essential for chromosome end stability. Telomerase maintains telomere length by adding sequences de novo onto chromosome ends. The template domain of the telomerase RNA component dictates synthesis of species-specific telomeric repeats and other regions of the RNA have been suggested to be important for enzyme structure and/or catalysis. Using enzyme reconstituted in vitro with RNAs containing deletions or substitutions we identified nucleotides in the RNA component that are important for telomerase activity. Although many changes to conserved features in the RNA secondary structure did not abolish enzyme activity, levels of activity were often greatly reduced, suggesting that regions other than the template play a role in telomerase function. The template boundary was only altered by changes in stem II that affected the conserved region upstream of the template, not by changes in other regions, such as stems I, III and IV, consistent with a role of the conserved region in defining the 5' boundary of the template. Surprisingly, telomerase RNAs with substitutions or deletion of residues potentially abolishing the conserved pseudoknot structure had wild-type levels of telomerase activity. This suggests that this base pairing interaction may not be required for telomerase activity per se but may be conserved as a regulatory site for the enzyme in vivo.

Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA.

Telomerase is a ribonucleoprotein that catalyzes telomere elongation through the addition of TTAGGG repeats in humans. Activation of telomerase is often associated with immortalization of human cells and cancer. To dissect the human telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential 445 nucleotide human telomerase RNA (hTR) by micrococcal nuclease digestion of partially purified human telomerase, the addition of in vitro transcribed hTR reconstituted telomerase activity. The activity was dependent upon and specific to hTR. Using this assay, truncations at the 5' and 3' ends of hTR identified a functional region of hTR, similar in size to the full-length telomerase RNAs from ciliates. This region is located between positions 1-203. Furthermore, we found that residues 1-44, 5' to the template region (residues 46-56) are not essential for activity, indicating a minimal functional region is located between residues 44-203. Mutagenesis of full-length hTR between residues 170-179, 180-189 or 190-199 almost completely abolished the ability of the hTR to function in the reconstitution of telomerase activity, suggesting that sequences or structures within this 30 nucleotide region are required for activity, perhaps by binding telomerase protein components.

Telomerase and cancer: revisiting the telomere hypothesis.

Telomerase is a ribonucleoprotein DNA polymerase that elongates telomeres in eukaryotes. The telomere hypothesis implicates short telomere length and telomerase activation as critical players in cellular immortalization and cancer. In this review, we refine the original telomere hypothesis to address the results of recent studies on telomerase and telomere length regulation.

Boundary elements of the Tetrahymena telomerase RNA template and alignment domains.

Telomerase is a DNA polymerase fundamental to the replication and maintenance of telomere sequences at chromosome ends. The RNA component of telomerase is essential for the synthesis of telomere repeats. In vitro, the template domain (5'-CAACCCCAA-3') of the Tetrahymena telomerase RNA dictates the addition of Tetrahymena-specific telomere repeats d(TTGGGG)n, onto the 3' end of G-rich or telomeric substrates that are base-paired with the template and alignment regions of the RNA. Using a reconstituted in vitro system, we determined that altering the sequence of the alignment and template domains affects processivity of telomerase without abolishing telomerase activity. These results suggest that alternative template/alignment regions may be functional. In the ciliate telomerase RNAs, there is a conserved sequence 5'-(CU)GUCA-3', located two residues upstream of the template domain. The location and sequence of this conserved domain defined the 5' boundary of the template region. These data provide insights into the regulation of telomere synthesis by telomerase.

Secondary structure and interaction of phage D108 Ner repressor with a 61-base-pair operator: evidence for altered protein and DNA structures in the complex.

Ner repressors of the transposable phages Mu and D108 play a central role in regulating the expression of the early (transposase) operon and in ensuring that phage growth proceeds along a lytic pathway. The latter function is analogous to that performed by the Cro protein of phage lambda. Unlike lambda Cro, however, the structural basis of operator recognition is not known for the Ner repressors. In order to elucidate the structural features underlying operator recognition by Ner repressors, we have employed Raman spectroscopy as a probe of the solution secondary structures of both D108 Ner and Mu Ner. Additionally, we have obtained Raman spectra of the D108 Ner repressor when bound to a 61-base-pair oligodeoxynucleotide containing the 55-base-pair D108 ner binding site. Conformation-sensitive Raman bands show that both D108 and Mu Ner contain similar, highly alpha-helical (approximately 45%) secondary structures. The Raman markers also show that the substantial nonhelical secondary structure of both D108 Ner and Mu Ner is largely beta-stranded. The protein-free 61-bp D108 ner operator exhibits Raman marker bands diagnostic of an uninterrupted B DNA duplex. In the D108 Ner:DNA complex, we find the following: (i) B DNA stereochemistry is fully conserved, although with significant perturbations to the B form backbone geometry, particularly in AT-rich regions of the bound operator. (ii) The specific interactions that occur between Ner repressor and operator involve B DNA major groove sites. (iii) A small (8 +/- 3%) increase in alpha-helix content of the Ner repressor is detected upon operator binding. (iv) Finally, the local environments of many aromatic amino acids are substantially altered in the D108 Ner:DNA complex. We propose a molecular model for binding of D108 Ner to its operator that is consistent with both the present spectroscopic findings and the results of recent biochemical studies. Essential features of this model are bending of the DNA double helix and contact of operator sites with repressor domains bearing sequence homologies with the helix-turn-helix (HTH) motifs of other DNA-binding proteins. The Raman fingerprint of the Ner:DNA complex is shown to be clearly distinguishable from that of the lambda cI:DNA complex, even though both gene regulatory complexes are presumed to employ HTH recognition motifs. The unique Raman signatures observed for these repressor complexes suggest that the Raman methodology may be useful in discriminating different modes of operator recognition by the HTH motifs of regulatory proteins.

Functional reconstitution of wild-type and mutant Tetrahymena telomerase.

Telomerase is a ribonucleoprotein that catalyzes telomere elongation in vitro and in vivo. The 159-nucleotide RNA component of Tetrahymena telomerase contains the sequence 5'-CAACCCCAA-3' ("template region"), which serves as a template for the addition of the sequence d(TTGGGG)n to Tetrahymena telomeres. To dissect the Tetrahymena telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential telomerase RNA by micrococcal nuclease digestion of partially purified telomerase, the addition of in vitro-transcribed telomerase RNA reconstituted telomerase activity. The reconstituted activity was processive and showed the same primer specificities as native telomerase. Mutants in the RNA template region were tested in reconstitution assays to determine the role of the residues in this region in primer recognition and elongation. Two template mutants, encoding the sequences 5'-UAACCCCAA-3' and 5'-UAACCCUAA-3', specified the incorporation of dATP into the sequence d(TTAGGG). Telomerase reconstituted with a template mutant encoding the sequence 5'-CAACCCUAA-3' did not specify dATP incorporation and elongation by this mutant was not terminated by the addition of ddATP. In addition, a template mutant encoding the sequence 5'-CGGCCCCAA-3' specified the incorporation of ddCTP but not ddTTP while a mutant encoding the sequence 5'-CAACCCCGG-3' specified the incorporation of ddTTP but not ddCTP. These data suggest that only the most 5' six residues of the template region dictate the addition of telomeric repeats.

The Escherichia coli Mu/D108 phage ner homologue gene (nlp) is transcribed and evolutionarily conserved among the Enterobacteriaceae.

The Escherichia coli nlp gene is highly homologous to the regulatory ner genes of transposable coliphages, Mu and D108. It was discovered, via its action when overexpressed, as a positive activator of mal gene expression in a cya- crp*1 strain. Chromosomal disruption of the nlp gene by insertion of a promoterless luxAB reporter gene revealed that nlp is nonessential for E. coli viability. Light measurements from the resulting nlp::luxAB transcriptional fusion, plus RNA dot blot analysis, suggest that nlp is transcribed. Southern-blot analyses of DNAs from several bacterial species were performed and indicate that nlp is evolutionarily conserved, but only among closely related Enterobacteriaceae.

Modification of the suppressor phenotype of thymine requiring strains of Escherichia coli.

Thymine requiring strains of Escherichia coli suppress nonsense and frameshift mutations during translation. Strains with different genetic backgrounds exhibited different nonsense suppression spectra and showed differences in the apparent suppression efficiency. Part of this strain difference is due to a presumably novel gene (tsmA) mapping near 39 min. This gene affects the spectrum and apparent efficiency of suppression, and appears to affect the utilization of thymidine.

Characterization of the Pseudomonas aeruginosa transposable phage D3112 [corrected] left-end regulatory region.

The nucleotide sequence (2682 bp) of the left end of the Mu-like transposable bacteriophage D3112 cts15 from Pseudomonas aeruginosa was determined. A 720 bp open reading frame (ORF) is located on the bottom strand (positions 892-173), potentially encoding a polypeptide of 240 residues (Mr = 26,329). Specific binding of Escherichia coli Integration Host Factor (IHF) to a site located 907-922 bp from the D3112 left end suggests the existence of a P. aeruginosa IHF and its role, as in Mu, in the regulation of phage development.