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 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 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.

scholarly journals RAD50 and RAD51 Define Two Pathways That Collaborate to Maintain Telomeres in the Absence of Telomerase

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 143-152 ◽  
Author(s):  
Siyuan Le ◽  
J Kent Moore ◽  
James E Haber ◽  
Carol W Greider
Keyword(s):  
Cell Death ◽  
Telomere Length ◽  
Gene Conversion ◽  
De Novo ◽  
Dna Recombination ◽  
Telomere Maintenance ◽  
Triple Mutant ◽  
Yeast Telomerase ◽  
Telomere Lengthening

Abstract Telomere length is maintained by the de novo addition of telomere repeats by telomerase, yet recombination can elongate telomeres in the absence of telomerase. When the yeast telomerase RNA component, TLC1, is deleted, telomeres shorten and most cells die. However, gene conversion mediated by the RAD52 pathway allows telomere lengthening in rare survivor cells. To further investigate the role of recombination in telomere maintenance, we assayed telomere length and the ability to generate survivors in several isogenic DNA recombination mutants, including rad50, rad51, rad52, rad54, rad57, xrs2, and mre11. The rad51, rad52, rad54, and rad57 mutations increased the rate of cell death in the absence of TLC1. In contrast, although the rad50, xrs2, and mre11 strains initially had short telomeres, double mutants with tlc1 did not affect the rate of cell death, and survivors were generated at later times than tlc1 alone. While none of the double mutants of recombination genes and tlc1 (except rad52 tlc1) blocked the ability to generate survivors, a rad50 rad51 tlc1 triple mutant did not allow the generation of survivors. Thus RAD50 and RAD51 define two separate pathways that collaborate to allow cells to survive in the absence of telomerase.

scholarly journals Spermatozoa telomeres determine telomere length in early embryos and offspring

Reproduction ◽  
2016 ◽  
Vol 151 (1) ◽  
pp. 1-7 ◽  
Author(s):  
C de Frutos ◽  
A P López-Cardona ◽  
N Fonseca Balvís ◽  
R Laguna-Barraza ◽  
D Rizos ◽  
...  
Keyword(s):  
Telomere Length ◽  
Mus Musculus ◽  
Mus Spretus ◽  
Cell Stage ◽  
Young Males ◽  
Early Embryos ◽  
Parent Of Origin ◽  
Telomere Lengthening ◽  
Zygote Stage

Offspring telomere length (TL) has been correlated with paternal TL, but the mechanism for this parent of origin-specific inheritance remains unclear. The objective of this study has been to determine the role of spermatozoa TL in embryonic telomere lengthening by using two mouse models showing dimorphism in their spermatozoa TL: Mus musculus vs Mus spretus and old vs young Mus musculus. Mus spretus spermatozoa displayed a shorter TL than Mus musculus. Hybrid offspring exhibited lower TL compared with Mus musculus starting at the two-cell stage, before the onset of telomerase expression. To analyze the role of spermatozoa telomeres in early telomere lengthening, we compared the TL in oocytes, zygotes, two-cell embryos and blastocysts produced by parthenogenesis or by fertilization with Mus musculus or Mus spretus spermatozoa. TL was significantly higher in spermatozoa compared with oocytes, and it increased significantly from the oocyte to the zygote stage in those embryos fertilized with Mus musculus spermatozoa, but not in those fertilized with Mus spretus spermatozoa or produced by parthenogenesis. A further increase was noted from the zygote to the two-cell stage in fertilized Mus musculus embryos, whereas hybrid embryos maintained the oocyte TL. Spermatozoa TL shortened with age in Mus musculus and the offspring from young males showed a significantly higher TL compared with that fathered by old males. These significant differences were already noticeable at the two-cell stage. These results suggest that spermatozoa telomeres act as a guide for telomerase-independent telomere lengthening resulting in differences in TL that persist after birth.Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/151/1/1/suppl/DC1.

scholarly journals The Function of DNA Polymerase α at Telomeric G Tails Is Important for Telomere Homeostasis

2000 ◽  
Vol 20 (3) ◽  
pp. 786-796 ◽  
Author(s):  
Aegina Adams Martin ◽  
Isabelle Dionne ◽  
Raymund J. Wellinger ◽  
Connie Holm
Keyword(s):  
Telomere Length ◽  
Dna Polymerase ◽  
Telomere Maintenance ◽  
Replication Machinery ◽  
Telomeric Dna ◽  
Dna Polymerase Α ◽  
Telomeric Silencing ◽  
Pass Through ◽  
Telomere Lengthening ◽  
Lagging Strand

ABSTRACT Telomere length control is influenced by several factors, including telomerase, the components of telomeric chromatin structure, and the conventional replication machinery. Although known components of the replication machinery can influence telomere length equilibrium, little is known about why mutations in certain replication proteins cause dramatic telomere lengthening. To investigate the cause of telomere elongation in cdc17/pol1 (DNA polymerase α) mutants, we examined telomeric chromatin, as measured by its ability to repress transcription on telomere-proximal genes, and telomeric DNA end structures in pol1-17 mutants. pol1-17 mutants with elongated telomeres show a dramatic loss of the repression of telomere-proximal genes, or telomeric silencing. In addition,cdc17/pol1 mutants grown under telomere-elongating conditions exhibit significant increases in single-stranded character in telomeric DNA but not at internal sequences. The single strandedness is manifested as a terminal extension of the G-rich strand (G tails) that can occur independently of telomerase, suggesting thatcdc17/pol1 mutants exhibit defects in telomeric lagging-strand synthesis. Interestingly, the loss of telomeric silencing and the increase in the sizes of the G tails at the telomeres temporally coincide and occur before any detectable telomere lengthening is observed. Moreover, the G tails observed incdc17/pol1 mutants incubated at the semipermissive temperature appear only when the cells pass through S phase and are processed by the time cells reach G1. These results suggest that lagging-strand synthesis is coordinated with telomerase-mediated telomere maintenance to ensure proper telomere length control.

scholarly journals The Role of Rif1 in telomere length regulation is separable from its role in origin firing

2020 ◽  
Author(s):  
Calla B. Shubin ◽  
Carol W. Greider
Keyword(s):  
Telomere Length ◽  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Origin Firing ◽  
Replication Complex ◽  
Telomere Elongation ◽  
Length Regulation ◽  
Telomere Length Regulation ◽  
Telomere Lengthening

AbstractTo examine the established link between DNA replication and telomere length, we tested whether firing of telomeric origins would cause telomere lengthening. We found that RIF1 mutants that block Protein Phosphatase 1 (PP1) binding activated telomeric origins but did not elongate telomeres. In a second approach, we found overexpression of ΔN-Dbf4 and Cdc7 increased DDK activity and activated telomeric origins, yet telomere length was unchanged. We tested a third mechanism to activate origins using the sld3-A mcm5-bob1 mutant that deregulates the pre-replication complex, and again saw no change in telomere length. Finally, we tested whether mutations in RIF1 that cause telomere elongation would affect origin firing. We found that neither rif1-Δ1322 nor rif1HOOK affected firing of telomeric origins. We conclude that telomeric origin firing does not cause telomere elongation, and the role of Rif1 in regulating origin firing is separable from its role in regulating telomere length.

scholarly journals The role of Rif1 in telomere length regulation is separable from its role in origin firing

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Calla B Shubin ◽  
Carol W Greider
Keyword(s):  
Telomere Length ◽  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Origin Firing ◽  
Replication Complex ◽  
Telomere Elongation ◽  
Length Regulation ◽  
Telomere Length Regulation ◽  
Telomere Lengthening

To examine the established link between DNA replication and telomere length, we tested whether firing of telomeric origins would cause telomere lengthening. We found that RIF1 mutants that block Protein Phosphatase 1 (PP1) binding activated telomeric origins but did not elongate telomeres. In a second approach, we found overexpression of ∆N-Dbf4 and Cdc7 increased DDK activity and activated telomeric origins, yet telomere length was unchanged. We tested a third mechanism to activate origins using the sld3-A mcm5-bob1 mutant that de-regulates the pre-replication complex, and again saw no change in telomere length. Finally, we tested whether mutations in RIF1 that cause telomere elongation would affect origin firing. We found that neither rif1-∆1322 nor rif1HOOK affected firing of telomeric origins. We conclude that telomeric origin firing does not cause telomere elongation, and the role of Rif1 in regulating origin firing is separable from its role in regulating telomere length.

scholarly journals Telomere Length, Telomerase Activity, and Replicative Potential in HIV Infection: Analysis of CD4+ and CD8+T Cells from HIV-discordant Monozygotic Twins

1997 ◽  
Vol 185 (7) ◽  
pp. 1381-1386 ◽  
Author(s):  
Larry D. Palmer ◽  
Nan-ping Weng ◽  
Bruce L. Levine ◽  
Carl H. June ◽  
H. Clifford Lane ◽  
...  
Keyword(s):  
T Cells ◽  
Hiv Infection ◽  
Telomere Length ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Replicative Senescence ◽  
Monozygotic Twins ◽  
Telomerase Activity ◽  
Replicative Capacity

To address the possible role of replicative senescence in human immunodeficiency virus (HIV) infection, telomere length, telomerase activity, and in vitro replicative capacity were assessed in peripheral blood T cells from HIV+ and HIV− donors. Genetic and age-specific effects on these parameters were controlled by studying HIV-discordant pairs of monozygotic twins. Telomere terminal restriction fragment (TRF) lengths from CD4+ T cells of HIV+ donors were significantly greater than those from HIV− twins. In contrast, telomere lengths in CD8+ T cells from HIV+ donors were shorter than in HIV− donors. The in vitro replicative capacity of CD4+ cells from HIV+ donors was equivalent to that of HIV− donors in response to stimulation through T cell receptor CD3 and CD28. Little or no telomerase activity was detected in freshly isolated CD4+ or CD8+ lymphocytes from HIV+ or HIV− donors, but was induced by in vitro stimulation of both HIV+ and HIV− donor cells. These results suggest that HIV infection is associated with alterations in the population dynamics of both CD4+ and CD8+ T cells, but fail to provide evidence for clonal exhaustion or replicative senescence as a mechanism underlying the decline in CD4+ T cells of HIV-infected donors.

scholarly journals Selective Elimination of Osteosarcoma Cell Lines with Short Telomeres by ATR Inhibitors

2020 ◽  
Author(s):  
Tomas Goncalves ◽  
Georgia Zoumpoulidou ◽  
Carlos Alvarez-Mendoza ◽  
Caterina Mancusi ◽  
Laura C. Collopy ◽  
...  
Keyword(s):  
Telomere Length ◽  
Cell Lines ◽  
Replicative Senescence ◽  
Telomere Maintenance ◽  
Osteosarcoma Cell ◽  
Cancer Evolution ◽  
Induced Apoptosis ◽  
Chromosome Bridges ◽  
Atr Kinase ◽  
Telomere Lengthening

AbstractTo avoid replicative senescence or telomere-induced apoptosis, cancers employ telomere maintenance mechanisms (TMMs) involving either the upregulation of telomerase or the acquisition of recombination-based alternative telomere lengthening (ALT). The choice of TMM may differentially influence cancer evolution and be exploitable in targeted therapies. Here, we examine TMMs in a panel of seventeen osteosarcoma-derived cell lines defining three separate groups according to TMM. Eight were ALT-positive, including the previously uncharacterised lines, KPD and LM7. ALT-negative cell lines were further classified into two groups according to their telomere length. HOS-MNNG, OHSN, SJSA-1, HAL, 143b and HOS displayed sub-normally short telomere length, while MG-63, MHM and HuO-3N1 displayed long telomeres. Importantly, sub-normally short telomeres were significantly associated with hypersensitivity to three different therapeutics targeting the ataxia telangiectasia and Rad3-related (ATR) kinase - AZD-6738/Ceralasertib, VE-822/Berzoserib and BAY-1895344 - compared to long telomeres, maintained via ALT or telomerase. Within 24 hours of ATR inhibition, cells with short but not long telomeres displayed chromosome bridges and underwent cell death, indicating a selective dependency on ATR for chromosome stability. Collectively, our work provides a resource to identify links between TMMs and drug sensitivity in osteosarcoma and indicates that telomere length predicts ATR-inhibitor sensitivity in cancer.

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