scholarly journals Tetrahymena Mutants With Short Telomeres

Genetics ◽  
1998 ◽  
Vol 150 (2) ◽  
pp. 643-650
Author(s):  
Shawn Ahmed ◽  
Hong Sheng ◽  
Luming Niu ◽  
Eric Henderson
Keyword(s):  
Growth Rate ◽  
Telomere Length ◽  
Genetic Defect ◽  
Telomerase Rna ◽  
Wild Type ◽  
Cell Type ◽  
Variant Cell ◽  
Length Regulation ◽  
Telomere Length Regulation

Abstract Telomere length is dynamic in many organisms. Genetic screens that identify mutants with altered telomere lengths are essential if we are to understand how telomere length is regulated in vivo. In Tetrahymena thermophila, telomeres become long at 30°, and growth rate slows. A slow-growing culture with long telomeres is often overgrown by a variant cell type with short telomeres and a rapid-doubling rate. Here we show that this variant cell type with short telomeres is in fact a mutant with a genetic defect in telomere length regulation. One of these telomere growth inhibited forever (tgi) mutants was heterozygous for a telomerase RNA mutation, and this mutant telomerase RNA caused telomere shortening when overexpressed in wild-type cells. Several other tgi mutants were also likely to be heterozygous at their mutant loci, since they reverted to wild type when selective pressure for short telomeres was removed. These results illustrate that telomere length can regulate growth rate in Tetrahymena and that this phenomenon can be exploited to identify genes involved in telomere length regulation.

scholarly journals Cdc13 N-Terminal Dimerization, DNA Binding, and Telomere Length Regulation

2010 ◽  
Vol 30 (22) ◽  
pp. 5325-5334 ◽  
Author(s):  
Meghan T. Mitchell ◽  
Jasmine S. Smith ◽  
Mark Mason ◽  
Sandy Harper ◽  
David W. Speicher ◽  
...  
Keyword(s):  
Dna Binding ◽  
Telomere Length ◽  
Functional Characterization ◽  
Point Mutations ◽  
Telomeric Dna ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Length Regulation ◽  
Telomere Length Regulation

ABSTRACT The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that is directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.

scholarly journals Telomerase-Associated Protein TEP1 Is Not Essential for Telomerase Activity or Telomere Length Maintenance In Vivo

2000 ◽  
Vol 20 (21) ◽  
pp. 8178-8184 ◽  
Author(s):  
Yie Liu ◽  
Bryan E. Snow ◽  
M. Prakash Hande ◽  
Gabriela Baerlocher ◽  
Valerie A. Kickhoefer ◽  
...  
Keyword(s):  
Telomere Length ◽  
Rna Binding ◽  
Es Cells ◽  
Embryonic Stem ◽  
Telomerase Activity ◽  
Telomerase Rna ◽  
Wild Type ◽  
Rnp Complex ◽  
Successive Generations

ABSTRACT TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. ThemTep1-deficient (mTep1 −/−) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues frommTep1 −/− mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1 −/− mice. Telomere length, even in later generations ofmTep1 −/− mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.

scholarly journals Expression of Telomerase RNA Template, but Not Telomerase Reverse Transcriptase, Is Limiting for Telomere Length Maintenance In Vivo

2004 ◽  
Vol 24 (16) ◽  
pp. 7024-7031 ◽  
Author(s):  
Y. Jeffrey Chiang ◽  
Michael T. Hemann ◽  
Karen S. Hathcock ◽  
Lino Tessarollo ◽  
Lionel Feigenbaum ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Telomere Length ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Gene Copy ◽  
Telomerase Reverse Transcriptase ◽  
Telomerase Rna ◽  
Wild Type ◽  
Telomere Elongation

ABSTRACT Telomerase consists of two essential components, the telomerase RNA template (TR) and telomerase reverse transcriptase (TERT). The haplo-insufficiency of TR was recently shown to cause one form of human dyskeratosis congenita, an inherited disease marked by abnormal telomere shortening. Consistent with this finding, we recently reported that mice heterozygous for inactivation of mouse TR exhibit a similar haplo-insufficiency and are deficient in the ability to elongate telomeres in vivo. To further assess the genetic regulation of telomerase activity, we have compared the abilities of TR-deficient and TERT-deficient mice to maintain or elongate telomeres in interspecies crosses. Homozygous TERT knockout mice had no telomerase activity and failed to maintain telomere length. In contrast, TERT+/− heterozygotes had no detectable defect in telomere elongation compared to wild-type controls, whereas TR+/− heterozygotes were deficient in telomere elongation. Levels of TERT mRNA in heterozygous mice were one-third to one-half the levels expressed in wild-type mice, similar to the reductions in telomerase RNA observed in TR heterozygotes. These findings indicate that both TR and TERT are essential for telomere maintenance and elongation but that gene copy number and transcriptional regulation of TR, but not TERT, are limiting for telomerase activity under the in vivo conditions analyzed.

scholarly journals Swc4 positively regulates telomere length independently of its roles in NuA4 and SWR1 complexes

2020 ◽  
Vol 48 (22) ◽  
pp. 12792-12803
Author(s):  
Jia-Cheng Liu ◽  
Qian-Jin Li ◽  
Ming-Hong He ◽  
Can Hu ◽  
Pengfei Dai ◽  
...  
Keyword(s):  
Telomere Length ◽  
Essential Gene ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Direct Role ◽  
Telomere Shortening ◽  
Length Regulation ◽  
Telomere Length Regulation

Abstract Telomeres at the ends of eukaryotic chromosomes are essential for genome integrality and stability. In order to identify genes that sustain telomere maintenance independently of telomerase recruitment, we have exploited the phenotype of over-long telomeres in the cells that express Cdc13-Est2 fusion protein, and examined 195 strains, in which individual non-essential gene deletion causes telomere shortening. We have identified 24 genes whose deletion results in dramatic failure of Cdc13-Est2 function, including those encoding components of telomerase, Yku, KEOPS and NMD complexes, as well as quite a few whose functions are not obvious in telomerase activity regulation. We have characterized Swc4, a shared subunit of histone acetyltransferase NuA4 and chromatin remodeling SWR1 (SWR1-C) complexes, in telomere length regulation. Deletion of SWC4, but not other non-essential subunits of either NuA4 or SWR1-C, causes significant telomere shortening. Consistently, simultaneous disassembly of NuA4 and SWR1-C does not affect telomere length. Interestingly, inactivation of Swc4 in telomerase null cells accelerates both telomere shortening and senescence rates. Swc4 associates with telomeric DNA in vivo, suggesting a direct role of Swc4 at telomeres. Taken together, our work reveals a distinct role of Swc4 in telomere length regulation, separable from its canonical roles in both NuA4 and SWR1-C.

scholarly journals Rif1 regulates telomere length through conserved HEAT repeats

2021 ◽  
Vol 49 (7) ◽  
pp. 3967-3980
Author(s):  
Calla B Shubin ◽  
Rini Mayangsari ◽  
Ariel D Swett ◽  
Carol W Greider
Keyword(s):  
Dna Binding ◽  
Telomere Length ◽  
Functional Role ◽  
Conserved Residues ◽  
Binding Interface ◽  
Binding Function ◽  
Length Regulation ◽  
Telomere Length Regulation ◽  
Telomere Regulation ◽  
Conserved Amino Acids

AbstractIn budding yeast, Rif1 negatively regulates telomere length, but the mechanism of this regulation has remained elusive. Previous work identified several functional domains of Rif1, but none of these has been shown to mediate telomere length. To define Rif1 domains responsible for telomere regulation, we localized truncations of Rif1 to a single specific telomere and measured telomere length of that telomere compared to bulk telomeres. We found that a domain in the N-terminus containing HEAT repeats, Rif1177–996, was sufficient for length regulation when tethered to the telomere. Charged residues in this region were previously proposed to mediate DNA binding. We found that mutation of these residues disrupted telomere length regulation even when Rif1 was tethered to the telomere. Mutation of other conserved residues in this region, which were not predicted to interact with DNA, also disrupted telomere length maintenance, while mutation of conserved residues distal to this region did not. Our data suggest that conserved amino acids in the region from 436 to 577 play a functional role in telomere length regulation, which is separate from their proposed DNA binding function. We propose that the Rif1 HEAT repeats region represents a protein-protein binding interface that mediates telomere length regulation.

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 Regulating telomere length from the inside out: The replication fork model

2016 ◽  
Author(s):  
Carol W Greider
Keyword(s):  
Telomere Length ◽  
Alternative Model ◽  
Replication Fork ◽  
Molecular Level ◽  
Origin Firing ◽  
Length Regulation ◽  
Telomere Length Regulation ◽  
Inside Out ◽  
Counting Model

Telomere length is regulated around an equilibrium set point. Telomeres shorten during replication and are lengthened by telomerase. Disruption of the length equilibrium leads to disease, thus it is important to understand the mechanisms that regulate length at the molecular level. The prevailing protein counting model for regulating telomerase access to elongate the telomere does not explain accumulating evidence of a role of DNA replication in telomere length regulation. Here I present an alternative model: the replication fork model that can explain how passage of a replication fork and regulation of origin firing affect telomere length.

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.

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