Recognition and elongation of telomeres by telomerase

Telomeres stabilize chromosomal ends and allow their complete replication in vivo. In diverse eukaryotes, the essential telomeric DNA sequence consists of variable numbers of tandem repeats of simple, G + C rich sequences, with a strong strand bias of G residues on the strand oriented 5' to 3' toward the chromosomal terminus. This strand forms a protruding 3' over-hang at the chromosomal terminus in three different eukaryotes analyzed. Analysis of yeast and protozoan telomeres showed that telomeres are dynamic structures in vivo, being acted on by shortening and lengthening activities. We previously identified and partially purified an enzymatic activity, telomere terminal transferase, or telomerase, from the ciliate Tetrahymena. Telomerase is a ribonucleoprotein enzyme with essential RNA and protein components. This activity adds repeats of the Tetrahymena telomeric sequence, TTGGGG, onto the 3' end of a single-stranded DNA primer consisting of a few repeats of the G-rich strand of known telomeric, and telomere-like, sequences. The shortest oligonucleotide active as a primer was the decamer G4T2G4. Structural analysis of synthetic DNA oligonucleotides that are active as primers showed that they all formed discrete intramolecular foldback structures at temperatures below 40 degrees C. Addition of TTGGGG repeats occurs one nucleotide at a time by de novo synthesis, which is not templated by the DNA primer. Up to 8000 nucleotides of G4T2 repeats were added to the primer in vitro. We discuss the implications of this finding for regulation of telomerase in vivo and a model for telomere elongation by telomerase.     

Tetrahymena micronuclear sequences that function as telomeres in yeast.

We explored the ability of S. cerevisiae to utilize heterologous DNA sequences as telomeres by cloning germline (micronuclear) DNA from Tetrahymena thermophila on a linear yeast plasmid that selects for telomere function. The only Tetrahymena sequences that functioned in this assay were (C4A2)n repeats. Moreover, these repeats did not have to be derived from Tetrahymena telomeres, although we show that micronuclear telomeres (like macronuclear telomeres) of Tetrahymena terminate in (C4A2)n repeats. Chromosome-internal restriction fragments carrying (C4A2)n repeats also stabilized linear plasmids and were elongated by yeast telomeric repeats. In one case, the C4A2 repeat tract was approximately 1.5 kb from the end of the genomic Tetrahymena DNA fragment that was cloned, but this 1.5 kb of DNA was missing from the linear plasmid. Thus, yeast can utilize internally located tracts of telomere-like sequences, after the distal DNA is removed. The data provide an example of broken chromo-some healing, and underscore the importance of the telomeric repeat structure for recognition of functional telomeric DNA in vivo.     

Identification of the telomeric sequence of the acellular slime molds Didymium iridis and Physarum polycephalum.

We have determined the telomeric DNA sequence of the acellular slime molds Didymium iridis and Physarum polycephalum. In both organisms the telomeres consist of tandem repeats of the hexamer 5'(TTAGGG)3'. This sequence was determined by cloning and sequencing the telomeric fragment of the linear extrachromosomal ribosomal DNA from Didymium, as well as direct end labeling and sequencing the rDNA from both organisms. Interestingly, this sequence is identical to the telomeric DNA sequence of the flagellated protozoan Trypanosoma brucei, and suggests that despite the diversity of telomeric sequences previously determined in lower eukaryotes, the necessity to create functional telomeres has led to constraints on these sequences.     

Sequence-specific DNA primer effects on telomerase polymerization activity.

The ribonucleoprotein enzyme telomerase synthesizes one strand of telomeric DNA by copying a template sequence within the RNA moiety of the enzyme. Kinetic studies of this polymerization reaction were used to analyze the mechanism and properties of the telomerase from Tetrahymena thermophila. This enzyme synthesizes TTGGGG repeats, the telomeric DNA sequence of this species, by elongating a DNA primer whose 3' end base pairs with the template-forming domain of the RNA. The enzyme was found to act nonprocessively with short (10- to 12-nucleotide) primers but to become processive as TTGGGG repeats were added. Variation of the 5' sequences of short primers with a common 3' end identified sequence-specific effects which are distinct from those involving base pairing of the 3' end of the primer with the RNA template and which can markedly induce enzyme activity by increasing the catalytic rate of the telomerase polymerization reaction. These results identify an additional mechanistic basis for telomere and DNA end recognition by telomerase in vivo.     

Cloning of telomere-associated DNA using single-specific-primer polymerase chain reaction provides evidence for a conserved sequence directly adjacent to Theileria parva telomeric repeats

Telomere-associated (TA) DNA sequences of the intracellular protozoan parasite Theileria parva were isolated by a novel strategy using a modified version of single-specific-primer polymerase chain reaction (SSP-PCR). Nucleotide sequences of non-coding TA DNA from three telomeres (6017bp, 2435bp and 4859bp) contained no extensive tracts of repetitive DNA. Long open reading frames (ORFs) were present at the centromeric ends of two of the TA sequences, the 3' ends of the closest ORFs being only 2670bp and 2719bp from the telomeric repeats. There were regions of significant similarity between the nucleotide sequences of the non-coding regions of different telomeres. The longest region of similarity was a virtually identical 1650bp domain, located directly adjacent to the telomeric repeats of two separate telomeres. Comparison of the telomere proximal sequences defined in this study and two additional T. parva telomeres, whose sequences were determined previously, resulted in identification of a single copy 141bp conserved sequence directly adjacent to the telomeric repeats. The conserved sequence is present at all five T. parva telomeres that have been characterised. The only organism currently known to have a single copy conserved sequence located adjacent to the telomeric repeats is another intracellular protozoan, Leishmania braziliensis.     

Genetic dissection of the Kluyveromyces lactis telomere and evidence for telomere capping defects in TER1 mutants with long telomeres

In the yeast Kluyveromyces lactis, the telomeres are composed of perfect 25-bp repeats copied from a 30-nucleotide RNA template defined by 5-nucleotide terminal repeats. A genetic dissection of the K. lactis telomere was performed by using mutant telomerase RNA (TER1) alleles to incorporate mutated telomeric repeats. This analysis has shown that each telomeric repeat contains several functional regions, some of which may physically overlap. Mutations in the terminal repeats of the template RNA typically lead to telomere shortening, as do mutations in the right side of the Rap1p binding site. Mutations in the left half of the Rap1p binding site, however, lead to the immediate formation of long telomeres. When mutated, the region immediately 3' of the Rap1p binding site on the TG-rich strand of the telomere leads to telomeres that are initially short but eventually undergo extreme telomere elongation. Mutations between this region and the 3' terminal repeat cause elevated recombination despite the presence of telomeres of nearly wild-type length. Mutants with highly elongated telomeres were further characterized and exhibit signs of telomere capping defects, including elevated levels of subtelomeric recombination and the formation of extrachromosomal and single-stranded telomeric DNA. Lengthening caused by some Rap1 binding site mutations can be suppressed by high-copy-number RAP1. Mutated telomeric repeats from a delayed elongation mutant are shown to be defective at regulating telomere length in cells with wild-type telomerase, indicating that the telomeric repeats are defective at telomere length regulation.     

Dynamics of telomere length variation in Tetrahymena thermophila

We have analyzed the mechanism and dynamics of telomere length variation in the macronucleus of Tetrahymena thermophila. In a newly differentiated macronucleus, the average length of the telomeric repeated sequence, (C4A2 X T2G4)n, is closely regulated. In contrast, in vegetatively dividing cells in log phase, all macronuclear telomeric sequences lengthen coordinately by 3-10 bp per generation until up to 1000 bp are added. In both elongated and short telomeres, characteristic single-stranded breaks on both strands are distally located. Reduction of elongated telomeres to their original length involves either the appearance of a novel type of variant cell, incapable of net telomere elongation, or, under stationary phase conditions, a reversible removal of telomeric sequences. The demonstration that telomeres are dynamic structures provides evidence for a model of telomere length regulation by activities that add and remove telomeric repeats.     

Sequence specificity of inter- and intramolecular G-quadruplex formation by human telomeric DNA

Human telomeric DNA consists of tandem repeats of the sequence 5'-d(TTAGGG)-3'. Guanine-rich DNA, such as that seen at telomeres, forms G-quadruplex secondary structures. Alternative forms of G-quadruplex structures can have differential effects on activities involved in telomere maintenance. With this in mind, we analyzed the effect of sequence and length of human telomeric DNA on G-quadruplex structures by native polyacrylamide gel electrophoresis and circular dichroism. Telomeric oligonucleotides shorter than four, 5'-d(TTAGGG)-3' repeats formed intermolecular G-quadruplexes. However, longer telomeric repeats formed intramolecular structures. Altering the 5'-d(TTAGGG)-3' to 5'-d(TTAGAG)-3' in any one of the repeats of 5'-d(TTAGGG)(4)-3' converted an intramolecular structure to intermolecular G-quadruplexes with varying degrees of parallel or anti-parallel-stranded character, depending on the length of incubation time and DNA sequence. These structures were most abundant in K(+)-containing buffers. Higher-order structures that exhibited ladders on polyacrylamide gels were observed only for oligonucleotides with the first telomeric repeat altered. Altering the sequence of 5'-d(TTAGGG)(8)-3' did not result in the substantial formation of intermolecular structures even when the oligonucleotide lacked four consecutive telomeric repeats. However, many of these intramolecular structures shared common features with intermolecular structures formed by the shorter oligonucleotides. The wide variability in structure formed by human telomeric sequence suggests that telomeric DNA structure can be easily modulated by proteins, oxidative damage, or point mutations resulting in conversion from one form of G-quadruplex to another.     

Isolation of telomeric DNA from the filamentous fungus Podospora anserina and construction of a self-replicating linear plasmid showing high transformation frequency.

It has been previously shown that linear plasmids bearing Tetrahymena telomeric sequences are able to replicate autonomously in the filamentous fungus Podospora anserina (1). However, autonomous replication occurs in only 50-70% of the transformants, suggesting a defect in the recognition of the Tetrahymena telomeric template by the putative P. anserina telomerase so that only a fraction of entering DNA is stabilized into linear extrachromosomal molecules. We have cloned DNA sequences added to the Tetrahymena (T2G4)n ends of the linear plasmid. Nucleotide sequencing showed that these sequences are exclusively composed of T2AG3 repeat units. Hybridization experiments of Bal31 treated DNA showed that T2AG3 repeats are confined within 200 bp in chromosomal P. anserina telomeres. A new plasmid has been constructed so that after linearization, the terminal sequences contain T2AG3 repeats. This linear molecule transforms P. anserina with a high frequency (up to 1.75 x 10(4) transformants/micrograms), autonomous replication occurs in 100% of the transformants and the plasmid copy number is about 2-3 per nucleus. These results underscore the importance of the telomeric repeat nucleotide sequence for efficient recognition as functional telomeric DNA in vivo and provide the first step toward the development of an artificial chromosome cloning system for filamentous fungi.     

Recombinational telomere elongation promoted by DNA circles

Yeast mutants lacking telomerase are capable of maintaining telomeres by an alternate mechanism that depends on homologous recombination. We show here, by using Kluyveromyces lactis cells containing two types of telomeric repeats, that recombinational telomere elongation generates a repeating pattern common in most or all telomeres in survivors that retain both repeat types. We propose that these patterns arise from small circles of telomeric DNA being used as templates for rolling-circle gene conversion and that the sequence from the lengthened telomere is spread to other telomeres by additional, more typical gene conversion events. Consistent with this, artificially constructed circles of DNA containing telomeric repeats form long tandem arrays at telomeres when transformed into K. lactis cells. Mixing experiments done with two species of telomeric circles indicated that all of the integrated copies of the transforming sequence arise from a single original circular molecule.     

De novo telomere addition by Tetrahymena telomerase in vitro.

Previous molecular genetic studies have shown that during programmed chromosomal healing, telomerase adds telomeric repeats directly to non-telomeric sequences in Tetrahymena, forming de novo telomeres. However, the biochemical mechanism underlying this process is not well understood. Here, we show for the first time that telomerase activity is capable in vitro of efficiently elongating completely non-telomeric DNA oligonucleotide primers, consisting of natural telomere-adjacent or random sequences, at low primer concentrations. Telomerase activity isolated from mated or vegetative cells had indistinguishable specificities for nontelomeric and telomeric primers. Consistent with in vivo results, the sequence GGGGT... was the predominant initial DNA sequence added by telomerase in vitro onto the 3' end of the non-telomeric primers. The 3' and 5' sequences of the primer both influenced the efficiency and pattern of de novo telomeric DNA addition. Priming of telomerase by double-stranded primers with overhangs of various lengths showed a requirement for a minimal 3' overhang of 20 nucleotides. With fully single-stranded non-telomeric primers, primer length up to approximately 30 nucleotides strongly affected the efficiency of telomeric DNA addition. We propose a model for the primer binding site of telomerase for non-telomeric primers to account for these length and structural requirements. We also propose that programmed de novo telomere addition in vivo is achieved through a hitherto undetected intrinsic ability of telomerase to elongate completely non-telomeric sequences.     

Characterisation of the telomeres at opposite ends of a 3 Mb Theileria parva chromosome.

Bacteriophage lambda clones containing Theileria parva genomic DNA derived from two different telomeres were isolated and the nucleotide sequences of the telomeric repeats and adjacent telomere-associated (TAS) DNA were determined. The T.parva telomeric repeat sequences, a tandem array of TTTTAGGG or TTTAGGG interspersed with a few variant copies, showed a high degree of sequence identity to those of the photosynthetic algae Chlamydomonas reinhardtii (97% identity) and Chlorella vulgaris (87.7% identity) and the angiosperm Arabidopsis thaliana (84.4% identity). Unlike most organisms which have been studied, no significant repetitive sequences were found in the nucleotide sequences of TAS DNA located centromere-proximal to the telomeric repeats. Restriction mapping and hybridisation analysis of lambda EMBL3 clones containing 16 kilobases of TAS DNA derived from one telomere suggested that they did not contain long regions of repetitive DNA. The cloned TAS DNAs were mapped to T.parva Muguga genomic SfiI fragments 8 and 20, which are located at opposite ends of the largest T.parva chromosome. A 126 bp sequence located directly centromere-proximal to the telomeric repeats was 94% identical between the two cloned telomeres. The conserved 126 bp sequence was present on all T.parva Muguga telomeric SfiI fragments.     

Progressive alteration of telomeric sequences at one end of a yeast linear plasmid and its possible association with reduced plasmid stability

After selection for migration into the nucleus, a cytoplasmic yeast linear plasmid bearing an inverted terminal repeat (ITRs) at each end replicates in Saccharomyces cerevisiae in a linear form, called pTLU, which carries host telomeric repeats (TG(1-3))(n) of about 300-350 bp added to the ITR ends. We previously showed that the nucleotide composition of the added telomeric sequences varied among individual pTLU isolates, while those on the two ends of any given pTLU were always identical. The telomeric sequences of pTLU remained unchanged over numbers of cell generations when cells were selected for expression of the plasmid-borne nuclear marker. We report here that progressive alterations in telomeric sequences can be detected in cells which are grown under non-selective conditions. Surprisingly, in any given molecule, the telomeric alterations occur exclusively on one side, either the left or the right end, while the sequence at the opposite end remained identical to the original, suggesting a difference in the mode of DNA replication between the plasmid ends. These alterations occur over a broad area extending from the termini of telomeres to nucleotides near the junction between the telomeric sequences and the pTLU-ITR, implying that the plasmid ends undergo successive rounds of extension and contraction. Clonal analysis under non-selective conditions indicated that the alterations in telomeric sequences are generally associated with extreme instability of the pTLU plasmid.     

Macrostructure of the tomato telomeres.

The macrostructure of the tomato telomeres has been investigated by in situ hybridization, genomic sequencing, and pulsed-field gel electrophoresis. In situ hybridizations with a cloned telomeric sequence from Arabidopsis thaliana indicated that the telomeric repeat of tomato cross-hybridizes with that of Arabidopsis and is located at all telomeres. Bal31 digestion kinetics confirmed that the tomato telomeric repeat represents the outermost DNA sequence of each tomato chromosome. Genomic sequencing of enriched tomato telomeric sequences, using primers derived from the Arabidopsis sequence, revealed that the consensus sequence of the tomato telomeric repeat is TT(T/A)AGGG compared with the Arabidopsis consensus sequence of TTTAGGG. Furthermore, as shown by pulsed-field gel electrophoresis, the telomeric repeat of tomato is separated by not more than a few hundred kilobases from a previously described 162-base pair satellite DNA repeat of tomato (TGR I) at 20 of the 24 telomeres. Together, these sequences are found in the heterochromatic terminal knob observed in pachytene chromosomes. Therefore, these two repeats determine the structure of 20 of the 24 tomato chromosome ends over approximately 2% of the total chromosome length.     

Rare internal C4A4 repeats in the micronuclear genome of Oxytricha fallax.

Approximately 20,000 different short, linear, macronuclear DNA molecules are derived from micronuclear sequences of Oxytricha fallax after conjugation. These macronuclear DNAs are terminated at both ends by 20 base pairs of the sequence 5'-dC4A4-3'. Sequences homologous to this repeat (C4A4+) are also abundant in the micronuclear chromosomes, but most reside at their telomeres. Here we show that nontelomeric C4A4 clusters of 20 base pairs or longer exist in only a few hundred copies per micronuclear genome. This demonstrates that nearly none of the 20,000 sequence blocks of micronuclear DNA destined to be macronuclear DNA molecules can be flanked by full-length (20-base pair) C4A4 clusters, and therefore C4A4 repeats must be added to most, if not all, macronuclear telomeres during macronuclear development. Six internal micronuclear C4A4+ loci were cloned, and their structural relationships with macronuclear and micronuclear sequences were examined. The possible origins and functions of these rare, micronuclear internal C4A4 loci are discussed.     

Telomere sequences attached to nuclearly migrated yeast linear plasmid

The yeast linear plasmid pCLU1, derived from pGKL1, has terminal proteins (TPs) covalently attached at the 5' ends of inverted terminal repeats (ITRs) and replicates in the cytoplasm, presumably using the TP as a primer for DNA synthesis. In Saccharomyces cerevisiae, under certain conditions, pCLU1 migrated into the nucleus and replicated in either linear or circular form. The linear-form plasmid lacked TPs; instead it carried host-telomere repeats at the ITR ends. The present study showed that (1) the added telomere was primarily composed of the repeated tracts of TGTGTGGGTGTGG, which was complementary to the RNA template of yeast telomerase, (2) the telomeric addition occurred at the very end of the ITRs, and (3) the sequence composition of the added telomeres was diverse among individual plasmids, but symmetrically identical at both ends of each plasmid. A similar mode of telomere addition was also observed in cells defective in the RAD52 gene.     

In vitro interactions of Caenorhabditis elegans dystrophin with dystrobrevin and syntrophin

HeT-A elements are non-long terminal repeat retrotransposons added onto the Drosophila chromosome ends. We have investigated the formation in vitro of higher order structures by oligonucleotides derived from the 3' non-coding region of HeT-A elements and found that they are capable of forming G-quadruplex DNA. These results suggest that the 3' repeat region of HeT-A may structurally behave as the telomeric repeats common to a majority of eukaryotes. The presence of structural motifs shared by telomeres and centromeres and the implications of these findings for chromosome evolution are discussed.     

Identification and characterization of a putative telomere end-binding protein from Tetrahymena thermophila.

Telomeric DNA of Tetrahymena thermophila consists of a long stretch of (TTGGGG)n double-stranded repeats with a single-stranded (TTGGGG)2 3' overhang at the end of the chromosome. We have identified and characterized a protein that specifically binds to a synthetic telomeric substrate consisting of duplex DNA and the 3' telomeric repeat overhang. This protein is called TEP (telomere end-binding protein). A change from G to A in the third position of the TTGGGG overhang repeat converts the substrate to a human telomere analog and reduces the binding affinity approximately threefold. Changing two G's to C's in the TTGGGG repeats totally abolishes binding. However, permutation of the Tetrahymena repeat sequence has only a minor effect on binding. A duplex structure adjacent to the 3' overhang is required for binding, although the duplex need not contain telomeric repeats. TEP does not bind to G-quartet DNA, which is formed by many G-rich sequences. TEP has a greatly reduced affinity for RNA substrates. The copy number of TEP is at least 2 x 10(4) per cell, and it is present under different conditions of cell growth and development, although its level varies. UV cross-linking experiments show that TEP has an apparent molecular mass of approximately 65 kDa. Unlike other telomere end-binding proteins, TEP is sensitive to high salt concentrations.     

Characterization of Arabidopsis thaliana telomeres isolated in yeast.

In an effort to learn more about the genomic organization of chromosomal termini in plants we employed a functional complementation strategy to isolate Arabidopsis thaliana telomeres in the yeast, Saccharomyces cerevisiae. Eight yeast episomes carrying A. thaliana telomeric sequences were obtained. The plant sequences carried on two episomes, YpAtT1 and YpAtT7, were characterized in detail. The telomeric origins of YpAtT1 and YpAtT7 insert DNAs were confirmed by demonstrating that corresponding genomic sequences are preferentially degraded during exonucleolytic digestion. The isolated telomeric restriction fragments contain G-rich repeat arrays characteristic of A. thaliana telomeres, as well as subterminal telomere-associated sequences (TASs). DNA sequence analysis revealed the presence of variant telomeric repeats at the centromere-proximal border of the terminal block of telomere repeats. The TAS flanking the telomeric G-rich repeat in YpAtT7 corresponds to a repetitive element present at other A. thaliana telomeres, while more proximal sequences are unique to one telomere. The YpAtT1 TAS is unique in the Landsberg strain of A. thaliana from which the clone originated; however, the Landsberg TAS cross-hybridizes weakly to a second telomere in the strain Columbia. Restriction analysis with cytosine methylation-sensitive endonucleases indicated that both TASs are highly methylated in the genome.     

Developmentally controlled telomere addition in wild-type and mutant paramecia.

We analyzed sites of macronuclear telomere addition at a single genetic locus in Paramecium tetraurelia. We showed that in homozygous wild-type cells, differential genomic processing during macronuclear development resulted in the A surface antigen gene being located 8, 13, or 26 kilobases upstream from a macronuclear telomere. We describe variable rearrangements that occurred at the telomere 8 kilobases from the A gene. A mutant (d48) that forms a telomere near the 5' end of the A gene was also analyzed. This mutant was shown to create simple terminal deletions; telomeric repeats were added directly to the truncated wild-type A gene sequence. In both the mutant and wild-type cells, the telomeric sequences (a mixture of C4A2 and C3A3 repeats) were added to various sequences within a specific 200- to 500-base-pair region rather than to a single site. No similarities were found in the primary sequences surrounding the telomere addition sites. The mutation in d48 changed the region of telomere addition at the A gene locus; this is the first example in ciliates of a mutation that affects the site of telomere addition.