scholarly journals Alternative Lengthening of Telomeres in the Budding Yeast Naumovozyma castellii

2019 ◽  
Vol 9 (10) ◽  
pp. 3345-3358 ◽  
Author(s):  
Marita Cohn ◽  
Ahu Karademir Andersson ◽  
Raquel Quintilla Mateo ◽  
Mirja Carlsson Möller
Keyword(s):  
Budding Yeast ◽  
Telomerase Activity ◽  
Telomeric Sequence ◽  
Wild Type ◽  
Alternative Lengthening Of Telomeres ◽  
Initiation Point ◽  
Interstitial Telomeric Sequence ◽  
Alternative Lengthening ◽  
Telomere Function ◽  
Linear Chromosomes

The enzyme telomerase ensures the integrity of linear chromosomes by maintaining telomere length. As a hallmark of cancer, cell immortalization and unlimited proliferation is gained by reactivation of telomerase. However, a significant fraction of cancer cells instead uses alternative telomere lengthening mechanisms to ensure telomere function, collectively known as Alternative Lengthening of Telomeres (ALT). Although the budding yeast Naumovozyma castellii (Saccharomyces castellii) has a proficient telomerase activity, we demonstrate here that telomeres in N. castellii are efficiently maintained by a novel ALT mechanism after telomerase knockout. Remarkably, telomerase-negative cells proliferate indefinitely without any major growth crisis and display wild-type colony morphology. Moreover, ALT cells maintain linear chromosomes and preserve a wild-type DNA organization at the chromosome termini, including a short stretch of terminal telomeric sequence. Notably, ALT telomeres are elongated by the addition of ∼275 bp repeats containing a short telomeric sequence and the subtelomeric DNA located just internally (TelKO element). Although telomeres may be elongated by several TelKO repeats, no dramatic genome-wide amplification occurs, thus indicating that the repeat addition may be regulated. Intriguingly, a short interstitial telomeric sequence (ITS) functions as the initiation point for the addition of the TelKO element. This implies that N. castellii telomeres are structurally predisposed to efficiently switch to the ALT mechanism as a response to telomerase dysfunction.

scholarly journals Molecular Basis for Telomere Repeat Divergence in Budding Yeast

2001 ◽  
Vol 21 (21) ◽  
pp. 7277-7286 ◽  
Author(s):  
Klaus Förstemann ◽  
Joachim Lingner
Keyword(s):  
Reverse Transcription ◽  
Budding Yeast ◽  
Repetitive Sequences ◽  
Telomerase Rna ◽  
Wild Type ◽  
Telomere Repeat ◽  
Telomere Binding Protein ◽  
Linear Chromosomes ◽  
Substrate Annealing

ABSTRACT Telomerase is a ribonucleoprotein enzyme that adds repetitive sequences to the ends of linear chromosomes, thereby counteracting nucleotide loss due to incomplete replication. A short region of the telomerase RNA subunit serves as template for nucleotide addition onto the telomere 3′ end. Although Saccharomyces cerevisiaecontains only one telomerase RNA gene, telomere repeat sequences are degenerate in this organism. Based on a detailed analysis of the telomere sequences specified by wild-type and mutant RNA templates in vivo, we show that the divergence of telomere repeats is due to abortive reverse transcription in the 3′ and 5′ regions of the template and due to the alignment of telomeres in multiple registers within the RNA template. Through the interpretation of wild-type telomere sequences, we identify nucleotides in the template that are not accessible for base pairing during substrate annealing. Rather, these positions become available as templates for reverse transcription only after alignment with adjacent nucleotides has occurred, indicating that a conformational change takes place upon substrate binding. We also infer that the central part of the template region is reverse transcribed processively. The inaccessibility of certain template positions for alignment and the processive polymerization of the central template portion may serve to reduce the possible repeat diversification and enhance the incorporation of binding sites for Rap1p, the telomere binding protein of budding yeast.

scholarly journals A translocation-defective telomerase with low levels of activity and processivity stabilizes short telomeres and confers immortalization

2013 ◽  
Vol 24 (9) ◽  
pp. 1469-1479 ◽  
Author(s):  
Yasmin D'Souza ◽  
Tsz Wai Chu ◽  
Chantal Autexier
Keyword(s):  
Dna Synthesis ◽  
Telomerase Activity ◽  
Human Cells ◽  
Telomerase Reverse Transcriptase ◽  
Wild Type ◽  
Telomeric Repeats ◽  
Telomeric Sequences ◽  
Telomere Function ◽  
Low Levels ◽  
Limited Lifespan

Short, repetitive, G-rich telomeric sequences are synthesized by telomerase, a ribonucleoprotein consisting of telomerase reverse transcriptase (TERT) and an integrally associated RNA. Human TERT (hTERT) can repetitively reverse transcribe its RNA template, acting processively to add multiple telomeric repeats onto the same substrate. We investigated whether certain threshold levels of telomerase activity and processivity are required to maintain telomere function and immortalize human cells with limited lifespan. We assessed hTERT variants with mutations in motifs implicated in processivity and interaction with DNA, namely the insertion in fingers domain (V791Y), and the E primer grip motif (W930F). hTERT-W930F and hTERT-V791Y reconstitute reduced levels of DNA synthesis and processivity compared with wild-type telomerase. Of interest, hTERT-W930F is more defective in translocation than hTERT-V791Y. Nonetheless, hTERT-W930F, but not hTERT-V791Y, immortalizes limited-lifespan human cells. Both hTERT-W930F– and hTERT-V791Y–expressing cells harbor short telomeres, measured as signal free ends (SFEs), yet SFEs persist only in hTERT-V791Y cells, which undergo apoptosis, likely as a consequence of a defect in recruitment of hTERT-V791Y to telomeres. Our study is the first to demonstrate that low levels of DNA synthesis—on the order of 20% of wild-type telomerase levels—and extension of as few as three telomeric repeats are sufficient to maintain functional telomeres and immortalize limited-lifespan human cells.

scholarly journals Deletion of Budding Yeast MAD2 Suppresses Clone-to-Clone Differences in Artificial Linear Chromosome Copy Numbers and Gives Rise to Higher Retention Rates

2020 ◽  
Vol 8 (10) ◽  
pp. 1495
Author(s):  
Scott C. Schuyler ◽  
Lin-Ing Wang ◽  
Yi-Shan Ding ◽  
Yi-Chieh Lee ◽  
Hsin-Yu Chen
Keyword(s):  
Budding Yeast ◽  
Retention Rate ◽  
Retention Rates ◽  
Yeast Cells ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Linear Chromosome ◽  
Copy Numbers ◽  
Clone Differences ◽  
Linear Chromosomes

Our goal was to investigate the changes in artificial short-linear chromosome average copy numbers per cell arising from partial or full loss of Mitotic Arrest-Deficient 2 (MAD2) spindle checkpoint function in budding yeast Saccharomyces cerevisiae. Average artificial linear chromosome copy numbers in a population of cells, as measured by quantitative polymerase chain reactions (qPCR), and retention rates, as measured by fluctuation analyses, were performed on a total of 62 individual wild type and mad2∆ mutant haploid and diploid clones. Wild type cells, both haploids and diploids, displayed phenotypically unique clone-to-clone differences: one group of 15 clones displayed low-copy numbers per cell and high retention rates, were 1 clone was found to have undergone a genomic integration event, and the second group of 15 clones displayed high copy numbers per cell and low retention rates, with the latter values being consistent with the previously published results where only a single clone had been measured. These chromosome states were observed to be unstable when propagated for 10 days under selection, where high copy-low retention rate clones evolved into low copy-high retention rate clones, but no evidence for integration events was observed. By contrast, mad2∆ haploid and mad2∆/mad2∆ diploids displayed a suppression of the clone-to-clone differences, where 20 out of 21 clones had mid-level artificial linear chromosome copy numbers per cell, but maintained elevated chromosome retention rates. The elevated levels in retention rates in mad2∆ and mad2∆/mad2∆ cells were also maintained even in the absence of selection during growth over 3 days. MAD2/mad2∆ heterozygous diploids displayed multiple clonal groups: 4 with low copy numbers, 5 with mid-level copy numbers, and 1 with a high copy number of artificial linear chromosomes, but all 10 clones uniformly displayed low retention rates. Our observations reveal that MAD2 function contributes to the ability of yeast cells to maintain a high number of artificial linear chromosomes per cell in some clones, but, counter-intuitively, mad2∆ suppresses clone-to-clone differences and leads to an improvement in artificial linear chromosome retention rates yielding a more uniform and stable clonal population with mid-level chromosome copy numbers per cell.

Evidence for Alternative Lengthening of Telomeres in Chronic Lymphocytic Leukemia Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1179-1179
Author(s):  
Rajendra N. Damle ◽  
Taraneh Banapour ◽  
Cristina Sison ◽  
Steven L. Allen ◽  
Kanti R. Rai ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Telomerase Activity ◽  
Lymphocytic Leukemia ◽  
Nuclear Bodies ◽  
Telomere Maintenance ◽  
Osteosarcoma Cell ◽  
Clonal Deletion ◽  
Alternative Lengthening Of Telomeres ◽  
Alternative Lengthening ◽  
V Genes

Abstract Telomere shortening is a consequence of repetitive clonal replication and leads to clonal deletion unless DNA extension and repair occur. All tumors must circumvent this problem by up-regulating mechanisms that lead to chromosomal lengthening. Two mechanisms have been identified that maintain chromosome ends- telomerase that does so by reverse transcription and alternative lengthening of telomeres (ALT) that occurs by homologous recombination. The latter function is characterized by the presence of promyelocytic leukemia protein-associated nuclear bodies (PML-NBs) and the presence of PML-NB is used to mark cells that use this process. B cell Chronic lymphocytic leukemia (B-CLL) cells with unmutated Ig V genes have shorter mean telomere lengths compared with those exhibiting mutated Ig V genes. In addition, cells with unmutated Ig V genes demonstrate more telomerase activity than their mutated counterparts. The mutated cases show long and heterogeneously elongated telomeres in spite of the absence, in most cases, of detectable telomerase activity. Therefore we determined whether the ALT pathway plays a role in telomere maintenance in B-CLL, using a monoclonal anti-PML antibody and a flow-cytometric assay for assessment of PML protein. Telomerase-expressing Jurkat T cells and murine fibroblasts-L cells served as negative controls for PML staining, whereas the ALT positive Osteosarcoma cell line U2-OS served as a positive control. In a cohort of 20 B-CLL cases, PML protein was detected in all cases regardless of Ig V mutation status. In addition, a similar percentage of cells within the clones contained PML (10 - 90% of the members of unmutated clones and 11–96% of mutated clones), whereas peripheral blood B cells from 6/6 elderly normal donors did not show any PML staining. PML expression was compared with telomere length and telomerase activity in the same cases. The percentage of cells showing PML expression inversely correlated with telomerase activity (r= −0.58; p=0.029). Although in most published reports telomere maintenance by ALT occurs in the absence of telomerase activity, we found ALT (as suggested by PML positive cells) in cells with telomerase activity (detected by the standard TRAP assay). Thus, B-CLL cases can express PML bodies and some B-CLL cells can contain both PML-NB and express telomerase activity. These findings suggest that B-CLL cells can use two distinct mechanisms to assure telomere maintenance and perpetuate clonal survival and expansion.

scholarly journals Telomerase and Alternative Lengthening of Telomeres coexist in the regenerating zebrafish caudal fins

2021 ◽  
Author(s):  
Maria L. Cayuela ◽  
Elena Martínez-Balsalobre ◽  
Monique Anchelin-Flageul ◽  
Francisca Alcaraz-Perez ◽  
Jesús García-Castillo ◽  
...  
Keyword(s):  
Telomere Length ◽  
Molecular Mechanisms ◽  
Regeneration Process ◽  
Wild Type ◽  
Alternative Lengthening Of Telomeres ◽  
Zebrafish Model ◽  
Alternative Lengthening ◽  
Vertebrate Model ◽  
New Treatments ◽  
Dependent Processes

Telomeres are essential for chromosome protection and genomic stability, and telomerase function is critical to organ homeostasis. Zebrafish has become a useful vertebrate model for understanding the cellular and molecular mechanisms of regeneration. The regeneration capacity of the caudal fin of wild-type zebrafish is not affected by repetitive amputation, but the behavior of telomeres during this process has not yet been studied. In this study, the regeneration process was characterized in a telomerase deficient zebrafish model. Moreover, the regenerative capacity after repetitive amputations and at different ages was studied. Regenerative efficiency decreases with aging in all genotypes and surprisingly, telomere length is maintained even in telomerase deficient genotypes. Our results suggest that telomere length can be maintained by the regenerating cells through the recombination-mediated Alternative Lengthening of Telomeres (ALT) pathway, which is likely to support high rates of cell proliferation during the tailfin regeneration process. As far as we know, this is the first animal model to study ALT mechanism in regeneration, which opens a wealth of possibilities to study new treatments of ALT dependent processes.

scholarly journals Spontaneous replication fork collapse regulates telomere length homeostasis in wild type cells

2020 ◽  
Author(s):  
Margherita Paschini ◽  
Cynthia M. Reyes ◽  
Abigail E. Gillespie ◽  
Karen A. Lewis ◽  
Leslie W. Glustrom ◽  
...  
Keyword(s):  
Dna Replication ◽  
Telomere Length ◽  
Replication Fork ◽  
Telomerase Activity ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomeric Repeats ◽  
Subsequent Response ◽  
Linear Chromosomes ◽  
Telomere Length Homeostasis

AbstractTelomeres present unique challenges for genomes with linear chromosomes, including the inability of the semi-conservative DNA replication machinery to fully duplicate the ends of linear molecules. This is solved in virtually all eukaryotes by the enzyme telomerase, through the addition of telomeric repeats onto chromosome ends. It is widely assumed that the primary site of action for telomerase is the single-stranded G-rich overhang at the ends of chromosomes, formed after DNA replication is complete. We show here that the preferred substrate for telomerase in wild type yeast is instead a collapsed fork generated during replication of duplex telomeric DNA. Furthermore, newly collapsed forks are extensively elongated by telomerase by as much as ∼200 nucleotides in a single cell division, indicating that a major source of newly synthesized telomeric repeats in wild type cells occurs at collapsed forks. Fork collapse and the subsequent response by telomerase are coordinated by the dual activities of a telomere-dedicated RPA-like complex, which facilitates replication of duplex telomeric DNA and also recruits telomerase to the fork, thereby ensuring a high probability of re-elongation if DNA replication fails. We further show that the ability of telomerase to elongate newly collapsed forks is dependent on the Rad51 protein, indicating that telomerase activity in response to fork collapse proceeds through a regulatory pathway distinct from how telomerase engages fully replicated chromosome termini. We propose a new model in which spontaneous replication fork collapse and the subsequent response by telomerase is a major determinant of telomere length homeostasis.

scholarly journals EXO1 Contributes to Telomere Maintenance in Both Telomerase-Proficient and Telomerase-Deficient Saccharomyces cerevisiae

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1651-1659 ◽  
Author(s):  
Alison A Bertuch ◽  
Victoria Lundblad
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Telomere Length ◽  
Budding Yeast ◽  
Telomere Maintenance ◽  
Wild Type ◽  
Rapid Loss ◽  
Telomere Shortening ◽  
Telomere Function

Abstract Previous work in budding yeast has indicated that telomeres are protected, at least in part, from the action of Exo1, which degrades the C-rich strand of partially uncapped telomeres. To explore this further, we examined the consequences of Exo1-mediated activity in strains that lacked Ku, telomerase, or both. Loss of Exo1 partially rescued the telomere length defect in a yku80Δ strain, demonstrating that exonuclease action can directly contribute to telomere shortening. The rapid loss of inviability displayed by a yku80Δ est2Δ strain was also partially alleviated by an exo1Δ mutation, further supporting the proposal that Exo1 is one target of the activities that normally protect wild-type telomeres. Conversely, however, Exo1 activity was also capable of enhancing telomere function and consequently cell proliferation, by contributing to a telomerase-independent pathway for telomere maintenance. The recovery of recombination-dependent survivors that arose in a yku80Δ est2Δ strain was partially dependent on Exo1 activity. Furthermore, the types of recombination events that facilitate telomerase-independent survival were influenced by Exo1 activity, in both est2Δ and yku80Δ est2Δ strains. These data demonstrate that Exo1 can make either positive or negative contributions to telomere function and cell viability, depending on whether telomerase or recombination is utilized to maintain telomere function.

scholarly journals Loss of Wild-Type ATRX Expression in Somatic Cell Hybrids Segregates with Activation of Alternative Lengthening of Telomeres

PLoS ONE ◽  
2012 ◽  
Vol 7 (11) ◽  
pp. e50062 ◽  
Author(s):  
Kylie Bower ◽  
Christine E. Napier ◽  
Sara L. Cole ◽  
Rebecca A. Dagg ◽  
Loretta M. S. Lau ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Hybrids ◽  
Wild Type ◽  
Alternative Lengthening Of Telomeres ◽  
Cell Hybrids ◽  
Alternative Lengthening

scholarly journals Coexistence of Alternative Lengthening of Telomeres and Telomerase in hTERT-Transfected GM847 Cells

2001 ◽  
Vol 21 (12) ◽  
pp. 3862-3875 ◽  
Author(s):  
Kilian Perrem ◽  
Lorel M. Colgin ◽  
Axel A. Neumann ◽  
Thomas R. Yeager ◽  
Roger R. Reddel
Keyword(s):  
Cell Line ◽  
Telomerase Activity ◽  
Promyelocytic Leukemia ◽  
Tumor Cell Lines ◽  
Hybrid Cells ◽  
Alternative Lengthening Of Telomeres ◽  
Positive Tumor Cell ◽  
Alternative Lengthening ◽  
Pml Bodies ◽  
Population Doublings

ABSTRACT It has been shown previously that some immortalized human cells maintain their telomeres in the absence of significant levels of telomerase activity by a mechanism referred to as alternative lengthening of telomeres (ALT). Cells utilizing ALT have telomeres of very heterogeneous length, ranging from very short to very long. Here we report the effect of telomerase expression in the ALT cell line GM847. Expression of exogenous hTERT in GM847 (GM847/hTERT) cells resulted in lengthening of the shortest telomeres; this is the first evidence that expression of hTERT in ALT cells can induce telomerase that is active at the telomere. However, rapid fluctuation in telomere length still occurred in the GM847/hTERT cells after more than 100 population doublings. Very long telomeres and ALT-associated promyelocytic leukemia (PML) bodies continued to be generated, indicating that telomerase activity induced by exogenous hTERT did not abolish the ALT mechanism. In contrast, when the GM847 cell line was fused with two different telomerase-positive tumor cell lines, the ALT phenotype was repressed in each case. These hybrid cells were telomerase positive, and the telomeres decreased in length, very rapidly at first and then at the rate seen in telomerase-negative normal cells. Additionally, ALT-associated PML bodies disappeared. After the telomeres had shortened sufficiently, they were maintained at a stable length by telomerase. Together these data indicate that the telomerase-positive cells contain a factor that represses the ALT mechanism but that this factor is unlikely to be telomerase. Further, the transfection data indicate that ALT and telomerase can coexist in the same cells.

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