Long Telomeres Produced by Telomerase-Resistant Recombination Are Established from a Single Source and Are Subject to Extreme Sequence Scrambling

Considerable evidence now supports the idea that the moderate telomere lengthening produced by recombinational telomere elongation (RTE) in a Kluyveromyces lactis telomerase deletion mutant occurs through a roll-and-spread mechanism. However, it is unclear whether this mechanism can account for other forms of RTE that produce much longer telomeres such as are seen in human alternative lengthening of telomere (ALT) cells or in the telomerase-resistant type IIR “runaway” RTE such as occurs in the K. lactis stn1-M1 mutant. In this study we have used mutationally tagged telomeres to examine the mechanism of RTE in an stn1-M1 mutant both with and without telomerase. Our results suggest that the establishment stage of the mutant state in newly generated stn1-M1 ter1-Δ mutants surprisingly involves a first stage of sudden telomere shortening. Our data also show that, as predicted by the roll-and-spread mechanism, all lengthened telomeres in a newly established mutant cell commonly emerge from a single telomere source. However, in sharp contrast to the RTE of telomerase deletion survivors, we show that the RTE of stn1-M1 ter1-Δ cells produces telomeres whose sequences undergo continuous intense scrambling via recombination. While telomerase was not necessary for the long telomeres in stn1-M1 cells, its presence during their establishment was seen to interfere with the amplification of repeats via recombination, a result consistent with telomerase retaining its ability to add repeats during active RTE. Finally, we observed that the presence of active mismatch repair or telomerase had important influences on telomeric amplification and/or instability.     

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.     

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.     

Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination

Some human cancers maintain their telomeres using the alternative lengthening of telomeres (ALT) mechanism; a process thought to involve recombination. Different types of recombinational telomere elongation pathways have been identified in yeasts. In senescing yeast telomerase deletion (ter1-Δ) mutants with very short telomeres, it has been hypothesized that copying a tiny telomeric circle (t-circle) by a rolling circle mechanism is the key event in telomere elongation. In other cases more closely resembling ALT cells, such as the stn1-M1 mutant of Kluyveromyces lactis, the telomeres appear to be continuously unstable and routinely reach very large sizes. By employing two-dimensional gel electrophoresis and electron microscopy, we show that stn1-M1 cells contain abundant double stranded t-circles ranging from ∼100 to 30 000 bp in size. We also observed small single-stranded t-circles, specifically composed of the G-rich telomeric strand and tailed circles resembling rolling circle replication intermediates. The t-circles most likely arose from recombination events that also resulted in telomere truncations. The findings strengthen the possibility that t-circles contribute to telomere maintenance in stn1-M1 and ALT cells.     

Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops

A prerequisite for cellular immortalization in human cells is the elongation of telomeres through the upregulation of telomerase or by the alternative lengthening of telomeres (ALT) pathway. In this study, telomere structure in multiple ALT cell lines was examined by electron microscopy. Nuclei were isolated from GM847, GM847-Tert, and WI-38 VA13 ALT cells, psoralen photo-cross-linked in situ, and the telomere restriction fragments were purified by gel filtration chromatography. Examination of telomere-enriched fractions revealed frequent extrachromosomal circles, ranging from 0.7 to 56.8 kb. t-loops were also observed, with the loop portion ranging from 0.5 to 70.2 kb. The total length of the loop plus tail of the t-loops corresponded to the telomere restriction fragment length from the ALT cell lines as determined by pulsed-field gel electrophoresis. The presence of extrachromosomal circles containing telomeric DNA was confirmed by two-dimensional pulsed-field gel electrophoresis. These results show that extrachromosomal telomeric DNA circles are present in ALT nuclei and suggest a roll-and-spread mechanism of telomere elongation similar to that seen in previous observations of multiple yeast species. Results presented here also indicate that expression of telomerase in GM847 cells does not affect t-loop or extrachromosomal circle formation.     

Recombination can either help maintain very short telomeres or generate longer telomeres in yeast cells with weak telomerase activity

Yeast mutants lacking telomerase are able to elongate their telomeres through processes involving homologous recombination. In this study, we investigated telomeric recombination in several mutants that normally maintain very short telomeres due to the presence of a partially functional telomerase. The abnormal colony morphology present in some mutants was correlated with especially short average telomere length and with a requirement for RAD52 for indefinite growth. Better-growing derivatives of some of the mutants were occasionally observed and were found to have substantially elongated telomeres. These telomeres were composed of alternating patterns of mutationally tagged telomeric repeats and wild-type repeats, an outcome consistent with amplification occurring via recombination rather than telomerase. Our results suggest that recombination at telomeres can produce two distinct outcomes in the mutants we studied. In occasional cells, recombination generates substantially longer telomeres, apparently through the roll-and-spread mechanism. However, in most cells, recombination appears limited to helping to maintain very short telomeres. The latter outcome likely represents a simplified form of recombinational telomere maintenance that is independent of the generation and copying of telomeric circles.     

Mutant Telomeric Repeats in Yeast Can Disrupt the Negative Regulation of Recombination-Mediated Telomere Maintenance and Create an Alternative Lengthening of Telomeres-Like Phenotype

Some human cancers maintain telomeres using alternative lengthening of telomeres (ALT), a process thought to be due to recombination. In Kluyveromyces lactis mutants lacking telomerase, recombinational telomere elongation (RTE) is induced at short telomeres but is suppressed once telomeres are moderately elongated by RTE. Recent work has shown that certain telomere capping defects can trigger a different type of RTE that results in much more extensive telomere elongation that is reminiscent of human ALT cells. In this study, we generated telomeres composed of either of two types of mutant telomeric repeats, Acc and SnaB, that each alter the binding site for the telomeric protein Rap1. We show here that arrays of both types of mutant repeats present basally on a telomere were defective in negatively regulating telomere length in the presence of telomerase. Similarly, when each type of mutant repeat was spread to all chromosome ends in cells lacking telomerase, they led to the formation of telomeres produced by RTE that were much longer than those seen in cells with only wild-type telomeric repeats. The Acc repeats produced the more severe defect in both types of telomere maintenance, consistent with their more severe Rap1 binding defect. Curiously, although telomerase deletion mutants with telomeres composed of Acc repeats invariably showed extreme telomere elongation, they often also initially showed persistent very short telomeres with few or no Acc repeats. We suggest that these result from futile cycles of recombinational elongation and truncation of the Acc repeats from the telomeres. The presence of extensive 3′ overhangs at mutant telomeres suggests that Rap1 may normally be involved in controlling 5′ end degradation.     

Amplification of telomeric arrays via rolling-circle mechanism

Alternative (telomerase-independent) lengthening of telomeres mediated through homologous recombination is often accompanied by a generation of extrachromosomal telomeric circles (t-circles), whose role in direct promotion of recombinational telomere elongation has been recently demonstrated. Here we present evidence that t-circles in a natural telomerase-deficient system of mitochondria of the yeast Candida parapsilosis replicate independently of the linear chromosome via a rolling-circle mechanism. This is supported by an observation of (i) single-stranded DNA consisting of concatameric arrays of telomeric sequence, (ii) lasso-shaped molecules representing rolling-circle intermediates, and (iii) preferential incorporation of deoxyribonucleotides into telomeric fragments and t-circles. Analysis of naturally occurring variant t-circles revealed conserved motifs with potential function in driving the rolling-circle replication. These data indicate that extrachromosomal t-circles observed in a wide variety of organisms, including yeasts, plants, Xenopus laevis, and certain human cell lines, may represent independent replicons generating telomeric sequences and, thus, actively participating in telomere dynamics. Moreover, because of the promiscuous occurrence of t-circles across phyla, the results from yeast mitochondria have implications related to the primordial system of telomere maintenance, providing a paradigm for evolution of telomeres in nuclei of early eukaryotes.     

Maintenance of very long telomeres by recombination in the Kluyveromyces lactis stn1-M1 mutant involves extreme telomeric turnover, telomeric circles, and concerted telomeric amplification

Some cancers utilize the recombination-dependent process of alternative lengthening of telomeres (ALT) to maintain long heterogeneous telomeres. Here, we studied the recombinational telomere elongation (RTE) of the Kluyveromyces lactis stn1-M1 mutant. We found that the total amount of the abundant telomeric DNA in stn1-M1 cells is subject to rapid variation and that it is likely to be primarily extrachromosomal. Rad50 and Rad51, known to be required for different RTE pathways in Saccharomyces cerevisiae, were not essential for the production of either long telomeres or telomeric circles in stn1-M1 cells. Circles of DNA containing telomeric repeats (t-circles) either present at the point of establishment of long telomeres or introduced later into stn1-M1 cells each led to the formation of long tandem arrays of the t-circle's sequence, which were incorporated at multiple telomeres. These tandem arrays were extraordinarily unstable and showed evidence of repeated rounds of concerted amplification. Our results suggest that the maintenance of telomeres in the stn1-M1 mutant involves extreme turnover of telomeric sequences from processes including both large deletions and the copying of t-circles.     

Human POT1 facilitates telomere elongation by telomerase

Mammalian telomeric DNA is mostly composed of double-stranded 5'-TTAGGG-3' repeats and ends with a single-stranded 3' overhang. Telomeric proteins stabilize the telomere by protecting the overhang from degradation or by remodeling the telomere into a T loop structure. Telomerase is a ribonucleoprotein that synthesizes new telomeric DNA. In budding yeast, other proteins, such as Cdc13p, that may help maintain the telomere end by regulating the recruitment or local activity of telomerase have been identified. Pot1 is a single-stranded telomeric DNA binding protein first identified in fission yeast, where it was shown to protect telomeres from degradation [10]. Human POT1 (hPOT1) protein is known to bind specifically to the G-rich telomere strand. We now show that hPOT1 can act as a telomerase-dependent, positive regulator of telomere length. Three splice variants of hPOT1 were overexpressed in a telomerase-positive human cell line. All three variants lengthened telomeres, and splice variant 1 was the most effective. hPOT1 was unable to lengthen the telomeres of telomerase-negative cells unless telomerase activity was induced. These data suggest that a normal function of hPOT1 is to facilitate telomere elongation by telomerase.     

Tobacco GTBP1, a Homolog of Human Heterogeneous Nuclear Ribonucleoprotein, Protects Telomeres from Aberrant Homologous Recombination

Telomeres are nucleoprotein complexes essential for the integrity of eukaryotic chromosomes. Cellular roles of single-stranded telomeric DNA binding proteins have been extensively described in yeast and animals, but our knowledge about plant single-strand telomeric factors is rudimentary. Here, we investigated Nicotiana tabacum G-strand-specific single-stranded telomere binding proteins (GTBPs), homologs of a human heterogeneous nuclear ribonucleoprotein. GTBPs bound specifically to the plant single-stranded (TTTAGGG)₄ telomeric repeat element in vitro and were associated with telomeric sequences in tobacco BY-2 suspension cells. Transgenic plants (35S:RNAi-GTBP1), in which GTBP1 was suppressed, exhibited severe developmental anomalies. In addition, the chromosomes of 35S:RNAi-GTBP1 cells displayed elongated telomeres, frequent formation of extrachromosomal telomeric circles, and numerous abnormal anaphase bridges, indicating that GTBP1 knockdown tobacco plants experienced genome instability. GTBP1 inhibited strand invasion, an initial step in interchromosomal homologous recombination. We propose that GTBP1 plays a critical role in telomere structure and function by preventing aberrant interchromosomal telomeric homologous recombination in tobacco.     

Factors influencing the recombinational expansion and spread of telomeric tandem arrays in Kluyveromyces lactis

We have previously shown that DNA circles containing telomeric repeats and a marker gene can promote the recombinational elongation of telomeres in Kluyveromyces lactis by a mechanism proposed to involve rolling-circle DNA synthesis. Wild-type cells acquire a long tandem array at a single telomere, while telomerase deletion (ter1-Δ) cells, acquire an array and also spread it to multiple telomeres. In this study, we further examine the factors that affect the formation and spread of telomeric tandem arrays. We show that a telomerase⁺ strain with short telomeres and high levels of subtelomeric gene conversion can efficiently form and spread arrays, while a telomere fusion mutant is not efficient at either process. This indicates that an elevated level of gene conversion near telomeres is required for spreading but that growth senescence and a tendency to elongate telomeres in the absence of exogenously added circles are not. Surprisingly, telomeric repeats are frequently deleted from a transforming URA3-telomere circle at or prior to the time of array formation by a mechanism dependent upon the presence of subtelomeric DNA in the circle. We further show that in a ter1-Δ strain, long tandem arrays can arise from telomeres initially containing a single-copy insert of the URA3-telomere sequence. However, the reduced rate of array formation in such strains suggests that single-copy inserts are not typical intermediates in arrays formed from URA3-telomere circles. Using heteroduplex circles, we have demonstrated that either strand of a URA3-telomere circle can be utilized to form telomeric tandem arrays. Consistent with this, we demonstrate that 100-nucleotide single-stranded telomeric circles of either strand can promote recombinational telomere elongation.     

Telomere loops and homologous recombination-dependent telomeric circles in a Kluyveromyces lactis telomere mutant strain

The Kluyveromyces lactis ter1-16T strain contains mutant telomeres that are poorly bound by Rap1, resulting in a telomere-uncapping phenotype and significant elongation of the telomeric DNA. The elongated telomeres of ter1-16T allowed the isolation and examination of native yeast telomeric DNA by electron microscopy. In the telomeric DNA isolated from ter1-16T, looped molecules were observed with the physical characteristics of telomere loops (t-loops) previously described in mammalian and plant cells. ter1-16T cells were also found to contain free circular telomeric DNA molecules (t-circles) ranging up to the size of an entire telomere. When the ter1-16T uncapping phenotype was repressed by overexpression of RAP1 or recombination was inhibited by deletion of rad52, the isolated telomeric DNA contained significantly fewer t-loops and t-circles. These results suggest that disruption of Rap1 results in elevated recombination at telomeres, leading to increased strand invasion of the 3′ overhang within t-loop junctions and resolution of the t-loop junctions into free t-circles.     

DNA recognition and binding by the Euplotes telomere protein.

The 51-kDa telomere protein from Euplotes crassus binds to the extreme terminus of macronuclear telomeres, generating a very salt-stable telomeric DNA-protein complex. The protein recognizes both the sequence and the structure of the telomeric DNA. To explore how the telomere protein recognizes and binds telomeric DNA, we have examined the DNA-binding specificity of the purified protein using oligonucleotides that mimic natural and mutant versions of Euplotes telomeres. The protein binds very specifically to the 3' terminus of single-stranded oligonucleotides with the sequence (T4G4) > or = 3 T4G2; even slight modifications to this sequence reduce binding dramatically. The protein does not bind oligonucleotides corresponding to the complementary C4A4 strand of the telomere or to double-stranded C4A4.T4G4-containing sequences. Digestion of the telomere protein with trypsin generates an N-terminal protease-resistant fragment of approximately 35 kDa. This 35-kDa peptide appears to comprise the DNA-binding domain of the telomere protein as it retains most of the DNA-binding characteristics of the native 51-kDa protein. For example, the 35-kDa peptide remains bound to telomeric DNA in 2 M KCl. Additionally, the peptide binds well to single-stranded oligonucleotides that have the same sequence as the T4G4 strand of native telomeres but binds very poorly to mutant telomeric DNA sequences and double-stranded telomeric DNA. Removal of the C-terminal 15 kDa from the telomere protein does diminish the ability of the protein to bind only to the terminus of a telomeric DNA molecule.     

Recombination can cause telomere elongations as well as truncations deep within telomeres in wild-type Kluyveromyces lactis cells

In this study, we examined the role of recombination at the telomeres of the yeast Kluyveromyces lactis. We demonstrated that an abnormally long and mutationally tagged telomere was subject to high rates of telomere rapid deletion (TRD) that preferentially truncated the telomere to near-wild-type size. Unlike the case in Saccharomyces cerevisiae, however, there was not a great increase in TRD in meiosis. About half of mitotic TRD events were associated with deep turnover of telomeric repeats, suggesting that telomeres were often cleaved to well below normal length prior to being reextended by telomerase. Despite its high rate of TRD, the long telomere showed no increase in the rate of subtelomeric gene conversion, a highly sensitive test of telomere dysfunction. We also showed that the long telomere was subject to appreciable rates of becoming elongated substantially further through a recombinational mechanism that added additional tagged repeats. Finally, we showed that the deep turnover that occurs within normal-length telomeres was diminished in the absence of RAD52. Taken together, our results suggest that homologous recombination is a significant process acting on both abnormally long and normally sized telomeres in K. lactis.     

The role of nonhomologous end-joining components in telomere metabolism in Kluyveromyces lactis

The relationship between telomeres and nonhomologous end-joining (NHEJ) is paradoxical, as NHEJ proteins are part of the telomere cap, which serves to differentiate telomeres from DNA double-strand breaks. We explored these contradictory functions for NHEJ proteins by investigating their role in Kluyveromyces lactis telomere metabolism. The ter1-4LBsr allele of the TER1 gene resulted in the introduction of sequence altered telomeric repeats and subsequent telomere-telomere fusions (T-TFs). In this background, Lig4 and Ku80 were necessary for T-TFs to form. Nej1, essential for NHEJ at internal positions, was not. Hence, T-TF formation was mediated by an unusual NHEJ mechanism. Rad50 and mre11 strains exhibited stable short telomeres, suggesting that Rad50 and Mre11 were required for telomerase recruitment. Introduction of the ter1-4LBsr allele into these strains failed to result in telomere elongation as normally observed with the ter1-4LBsr allele. Thus, the role of Rad50 and Mre11 in the formation of T-TFs was unclear. Furthermore, rad50 and mre11 mutants had highly increased subtelomeric recombination rates, while ku80 and lig4 mutants displayed moderate increases. Ku80 mutant strains also contained extended single-stranded 3' telomeric overhangs. We concluded that NHEJ proteins have multiple roles at telomeres, mediating fusions of mutant telomeres and ensuring end protection of normal telomeres.     

Characterization of two telomeric DNA processing reactions in Saccharomyces cerevisiae.

We have investigated two reactions that occur on telomeric sequences introduced into Saccharomyces cerevisiae cells by transformation. The elongation reaction added repeats of the yeast telomeric sequence C1-3A to telomeric sequences at the end of linear DNA molecules. The reaction worked on the Tetrahymena telomeric sequence C4A2 and also on the simple repeat CA. The reaction was orientation specific: it occurred only when the GT-rich strand ran 5' to 3' towards the end of the molecule. Telomere elongation occurred by non-template-directed DNA synthesis rather than any type of recombination with chromosomal telomeres, because C1-3A repeats could be added to unrelated DNA sequences between the CA-rich repeats and the terminus of the transforming DNA. The elongation reaction was very efficient, and we believe that it was responsible for maintaining an average telomere length despite incomplete replication by template-directed DNA polymerase. The resolution reaction processed a head-to-head inverted repeat of telomeric sequences into two new telomeres at a frequency of 10(-2) per cell division.     

Processing of telomeric DNA ends requires the passage of a replication fork.

During telomere replication in yeast, chromosome ends acquire a long single-stranded extension of the strand making the 3' end. Previous work showed that these 3' tails are generated late in S-phase, when conventional replication is virtually complete. In addition, the extensions were also observed in cells that lacked telomerase. Therefore, a model was proposed that predicted an activity that recessed the 5' ends at yeast telomeres after conventional replication was complete. Here, we demonstrate that this processing activity is dependent on the passage of a replication fork through yeast telomeres. A non-replicating linear plasmid with telomeres at each end does not acquire single-stranded extensions, while an identical construct containing an origin of replication does. Thus, the processing activity could be associated with the enzymes at the replication fork itself, or the passage of the fork through the telomeric sequences allows a transient access for the activity to the telomeres. We therefore propose that there is a mechanistic link between the conventional replication machinery and telomere maintenance.     

Sequencing of Saccharomyces telomeres cloned using T4 DNA polymerase reveals two domains.

By using T4 DNA polymerase rather than S1 or Bal31 nuclease to clone yeast telomeres, very little telomeric DNA is lost. These clones were used to determine the DNA sequence of virtually the entire telomeric tract. Our results demonstrated that a slightly modified version, C2-3A(CA)1-6, of the consensus derived from sequence analysis of more-internal regions (J. Shampay, J. W. Szostak, and E. H. Blackburn, Nature [London] 310:154-157, 1984) extends to the very end of the chromosome. The sequence analysis also suggests that yeast telomeres consist of two domains: the proximal 120 to 150 base pairs, which appear to be protected from processes such as recombination, degradation, and elongation, and the distal portion of the telomere, which is more susceptible to these events.