scholarly journals Genetic Dissection of the Kluyveromyces lactis Telomere and Evidence for Telomere Capping Defects in TER1 Mutants with Long Telomeres

2004 ◽  
Vol 3 (2) ◽  
pp. 369-384 ◽  
Author(s):  
Dana H. Underwood ◽  
Coleen Carroll ◽  
Michael J. McEachern
Keyword(s):  
Binding Site ◽  
Telomere Length ◽  
Kluyveromyces Lactis ◽  
Genetic Dissection ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomeric Repeats ◽  
Functional Regions ◽  
Telomere Capping ◽  
Terminal Repeats

ABSTRACT 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.

scholarly journals The Rap1p-Telomere Complex Does Not Determine the Replicative Capacity of Telomerase-Deficient Yeast

2003 ◽  
Vol 23 (23) ◽  
pp. 8729-8739 ◽  
Author(s):  
Sarit Smolikov ◽  
Anat Krauskopf
Keyword(s):  
Telomere Length ◽  
Replicative Senescence ◽  
Budding Yeast ◽  
Kluyveromyces Lactis ◽  
Dna Binding Protein ◽  
Replicative Capacity ◽  
Telomeric Dna ◽  
Telomeric Repeats ◽  
Telomere Lengthening

ABSTRACT Telomeres are nucleoprotein structures that cap the ends of chromosomes and thereby protect their stability and integrity. In the presence of telomerase, the enzyme that synthesizes telomeric repeats, telomere length is controlled primarily by Rap1p, the budding yeast telomeric DNA binding protein which, through its C-terminal domain, nucleates a protein complex that limits telomere lengthening. In the absence of telomerase, telomeres shorten with every cell division, and eventually, cells enter replicative senescence. We have set out to identify the telomeric property that determines the replicative capacity of telomerase-deficient budding yeast. We show that in cells deficient for both telomerase and homologous recombination, replicative capacity is dependent on telomere length but not on the binding of Rap1p to the telomeric repeats. Strikingly, inhibition of Rap1p binding or truncation of the C-terminal tail of Rap1p in Kluyveromyces lactis and deletion of the Rap1p-recruited complex in Saccharomyces cerevisiae lead to a dramatic increase in replicative capacity. The study of the role of telomere binding proteins and telomere length on replicative capacity in yeast may have significant implications for our understanding of cellular senescence in higher organisms.

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 Template Requirements for Telomerase Translocation in Kluyveromyces lactis

2004 ◽  
Vol 24 (2) ◽  
pp. 912-923 ◽  
Author(s):  
Dana H. Underwood ◽  
Robert P. Zinzen ◽  
Michael J. McEachern
Keyword(s):  
Reverse Transcriptase ◽  
Telomere Length ◽  
Kluyveromyces Lactis ◽  
Telomerase Rna ◽  
Wild Type ◽  
Telomeric Repeats

ABSTRACT Telomeres are synthesized by telomerase, a specialized reverse transcriptase, which contains a template in its intrinsic RNA component. In Kluyveromyces lactis, the repeats synthesized by the wild-type telomerase are 25 nucleotides (nt) in length and uniform in sequence. To determine the role of the 5-nt repeats defining the ends of the K. lactis telomerase RNA template in telomerase translocation, we have made mutations in and around them and observed their effects on telomere length and the sequence of newly made telomeric repeats. These template mutations typically result in telomeres that are shorter than those of wild-type cells. The mismatches between the telomerase template and the telomeric tip that occur after telomerase-mediated incorporation of the mutations are normally not removed. Instead, the mutations lead to the synthesis of aberrant repeats that range in size from 31 to 13 bp. Therefore, the specificity with which the telomeric tip aligns with the telomere is critical for the production of the uniform repeats seen in K. lactis. In addition, the region immediately 3′ of the template may play an important role in translocation of the enzyme.

Dynamics of Telomeric DNA Turnover in Yeast

Genetics ◽  
2002 ◽  
Vol 160 (1) ◽  
pp. 63-73
Author(s):  
Michael J McEachern ◽  
Dana Hager Underwood ◽  
Elizabeth H Blackburn
Keyword(s):  
Kluyveromyces Lactis ◽  
Mutant Gene ◽  
Dna Repeats ◽  
Wild Type ◽  
Telomeric Dna ◽  
Cell Divisions ◽  
Dna Turnover ◽  
Length Regulation ◽  
Turn Over

Abstract Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template. We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template. Each initially produced normally regulated telomeres. One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats. This mutant differs from previously studied delayed elongation mutants in a number of properties. The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity. TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions. Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres. These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover.

scholarly journals Recombination Can either Help Maintain Very Short Telomeres or Generate Longer Telomeres in Yeast Cells with Weak Telomerase Activity

2011 ◽  
Vol 10 (8) ◽  
pp. 1131-1142 ◽  
Author(s):  
Evelina Basenko ◽  
Zeki Topcu ◽  
Michael J. McEachern
Keyword(s):  
Homologous Recombination ◽  
Telomere Length ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Yeast Cells ◽  
Colony Morphology ◽  
Wild Type ◽  
Telomeric Repeats ◽  
Yeast Mutants ◽  
Derivatives Of

ABSTRACT 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.

scholarly journals Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

2008 ◽  
Vol 28 (7) ◽  
pp. 2380-2390 ◽  
Author(s):  
Hong Ji ◽  
Christopher J. Adkins ◽  
Bethany R. Cartwright ◽  
Katherine L. Friedman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Specific Binding ◽  
Telomerase Activity ◽  
Negative Regulator ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomere Length Homeostasis

ABSTRACT In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.

scholarly journals Murine Pif1 Interacts with Telomerase and Is Dispensable for Telomere Function In Vivo

2006 ◽  
Vol 27 (3) ◽  
pp. 1017-1026 ◽  
Author(s):  
Bryan E. Snow ◽  
Maria Mateyak ◽  
Jana Paderova ◽  
Andrew Wakeham ◽  
Caterina Iorio ◽  
...  
Keyword(s):  
Telomere Length ◽  
Chromosomal Rearrangements ◽  
Dna Helicase ◽  
Negative Regulator ◽  
Wild Type ◽  
Genetic Redundancy ◽  
Telomeric Dna ◽  
Dna Helicases

ABSTRACT Pif1 is a 5′-to-3′ DNA helicase critical to DNA replication and telomere length maintenance in the budding yeast Saccharomyces cerevisiae. ScPif1 is a negative regulator of telomeric repeat synthesis by telomerase, and recombinant ScPif1 promotes the dissociation of the telomerase RNA template from telomeric DNA in vitro. In order to dissect the role of mPif1 in mammals, we cloned and disrupted the mPif1 gene. In wild-type animals, mPif1 expression was detected only in embryonic and hematopoietic lineages. mPif1 − / − mice were viable at expected frequencies, displayed no visible abnormalities, and showed no reproducible alteration in telomere length in two different null backgrounds, even after several generations. Spectral karyotyping of mPif1 − / − fibroblasts and splenocytes revealed no significant change in chromosomal rearrangements. Furthermore, induction of apoptosis or DNA damage revealed no differences in cell viability compared to what was found for wild-type fibroblasts and splenocytes. Despite a novel association of mPif1 with telomerase, mPif1 did not affect the elongation activity of telomerase in vitro. Thus, in contrast to what occurs with ScPif1, murine telomere homeostasis or genetic stability does not depend on mPif1, perhaps due to fundamental differences in the regulation of telomerase and/or telomere length between mice and yeast or due to genetic redundancy with other DNA helicases.

scholarly journals Role of Mammalian Rad54 in Telomere Length Maintenance

2003 ◽  
Vol 23 (16) ◽  
pp. 5572-5580 ◽  
Author(s):  
Isabel Jaco ◽  
Purificación Muñoz ◽  
Fermín Goytisolo ◽  
Joanna Wesoly ◽  
Susan Bailey ◽  
...  
Keyword(s):  
Dna Repair ◽  
Telomere Length ◽  
Essential Role ◽  
Repair Pathway ◽  
Wild Type ◽  
Putative Role ◽  
Telomere Capping ◽  
Chromosome Fusions ◽  
Deficient Mice

ABSTRACT The homologous recombination (HR) DNA repair pathway participates in telomere length maintenance in yeast but its putative role at mammalian telomeres is unknown. Mammalian Rad54 is part of the HR machinery, and Rad54-deficient mice show a reduced HR capability. Here, we show that Rad54-deficient mice also show significantly shorter telomeres than wild-type controls, indicating that Rad54 activity plays an essential role in telomere length maintenance in mammals. Rad54 deficiency also resulted in an increased frequency of end-to-end chromosome fusions involving telomeres compared to the controls, suggesting a putative role of Rad54 in telomere capping. Finally, the study of mice doubly deficient for Rad54 and DNA-PKcs showed that telomere fusions due to DNA-PKcs deficiency were not rescued in the absence of Rad54, suggesting that they are not mediated by Rad54 activity.

scholarly journals Expression of Mutated Paramecium Telomerase RNAs In Vivo Leads to Templating Errors That Resemble Those Made by Retroviral Reverse Transcriptase

1999 ◽  
Vol 19 (4) ◽  
pp. 2887-2894 ◽  
Author(s):  
Amanda J. Ye ◽  
W. John Haynes ◽  
Daniel P. Romero
Keyword(s):  
Reverse Transcriptase ◽  
De Novo ◽  
Telomerase Rna ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomeric Repeats ◽  
Immunodeficiency Virus ◽  
In The Wild

ABSTRACT Telomeric DNA consists of short, tandemly repeated sequences at the ends of chromosomes. Telomeric DNA in the ciliate Paramecium tetraurelia is synthesized by an error-prone telomerase with an RNA template specific for GGGGTT repeats. We have previously shown that misincorporation of TTP residues at the telomerase RNA templating nucleotide C52 accounts for the 30% GGGTTT repeats randomly distributed in wild-type telomeres. To more completely characterize variable repeat synthesis in P. tetraurelia, telomerase RNA genes mutated at C52 (A, U, and G) were expressed in vivo. De novo telomeric repeats from transformants indicate that the predominant TTP misincorporation error seen in the wild-type telomerase is dependent on the presence of a C residue at template position 52. Paradoxically, the effects of various other telomerase RNA template and alignment region mutations on de novo telomeres include significant changes in fidelity, as well as the synthesis of aberrant, 5-nucleotide telomeric repeats. The occurrence of deletion errors and the altered fidelity of mutatedP. tetraurelia telomerase, in conjunction with misincorporation by the wild-type enzyme, suggest that the telomerase RNA template domain may be analogous to homopolymeric mutational hot spots that lead to similar errors by the human immunodeficiency virus proofreading-deficient reverse transcriptase.

scholarly journals Uncapping and Deregulation of Telomeres Lead to Detrimental Cellular Consequences in Yeast

1999 ◽  
Vol 145 (2) ◽  
pp. 203-214 ◽  
Author(s):  
Christopher D. Smith ◽  
Elizabeth H. Blackburn
Keyword(s):  
Dna Sequences ◽  
Tandem Repeats ◽  
Function Analysis ◽  
Kluyveromyces Lactis ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomere Function ◽  
Morphological Defects ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Structures

Telomeres are the protein–nucleic acid structures at the ends of eukaryote chromosomes. Tandem repeats of telomeric DNA are templated by the RNA component (TER1) of the ribonucleoprotein telomerase. These repeats are bound by telomere binding proteins, which are thought to interact with other factors to create a higher-order cap complex that stabilizes the chromosome end. In the budding yeast Kluyveromyces lactis, the incorporation of certain mutant DNA sequences into telomeres leads to uncapping of telomeres, manifested by dramatic telomere elongation and increased length heterogeneity (telomere deregulation). Here we show that telomere deregulation leads to enlarged, misshapen “monster” cells with increased DNA content and apparent defects in cell division. However, such deregulated telomeres became stabilized at their elongated lengths upon addition of only a few functionally wild-type telomeric repeats to their ends, after which the frequency of monster cells decreased to wild-type levels. These results provide evidence for the importance of the most terminal repeats at the telomere in maintaining the cap complex essential for normal telomere function. Analysis of uncapped and capped telomeres also show that it is the deregulation resulting from telomere uncapping, rather than excessive telomere length per se, that is associated with DNA aberrations and morphological defects.

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