scholarly journals Investigating the Effect of Mono- and Dimeric 360A G-Quadruplex Ligands on Telomere Stability by Single Telomere Length Analysis (STELA)

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 577 ◽  
Author(s):  
In Hwang ◽  
Patrick Mailliet ◽  
Virginie Hossard ◽  
Jean-Francois Riou ◽  
Anthony Bugaut ◽  
...  
Keyword(s):  
Telomere Length ◽  
Dna Binding Protein ◽  
Replication Machinery ◽  
Telomeric Dna ◽  
Full Spectrum ◽  
G Quadruplex ◽  
The Mean ◽  
Length Analysis ◽  
First Time

Telomeres are nucleoprotein structures that cap and protect the natural ends of chromosomes. Telomeric DNA G-rich strands can form G-quadruplex (or G4) structures. Ligands that bind to and stabilize G4 structures can lead to telomere dysfunctions by displacing shelterin proteins and/or by interfering with the replication of telomeres. We previously reported that two pyridine dicarboxamide G4 ligands, 360A and its dimeric analogue (360A)2A, were able to displace in vitro hRPA (a single-stranded DNA-binding protein of the replication machinery) from telomeric DNA by stabilizing the G4 structures. In this paper, we perform for the first time single telomere length analysis (STELA) to investigate the effect of G4 ligands on telomere length and stability. We used the unique ability of STELA to reveal the full spectrum of telomere lengths at a chromosome terminus in cancer cells treated with 360A and (360A)2A. Upon treatment with these ligands, we readily detected an increase of ultrashort telomeres, whose lengths are significantly shorter than the mean telomere length, and that could not have been detected by other methods.

scholarly journals RTEL1 influences the abundance and localization of TERRA RNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fiorella Ghisays ◽  
Aitor Garzia ◽  
Hexiao Wang ◽  
Claudia Canasto-Chibuque ◽  
Marcel Hohl ◽  
...  
Keyword(s):  
Cell Viability ◽  
Telomere Length ◽  
Human Cells ◽  
Helicase Domain ◽  
Telomere Repeat ◽  
Disease Phenotypes ◽  
G Quadruplex ◽  
Non Coding Rnas ◽  
Subtelomeric Regions

AbstractTelomere repeat containing RNAs (TERRAs) are a family of long non-coding RNAs transcribed from the subtelomeric regions of eukaryotic chromosomes. TERRA transcripts can form R-loops at chromosome ends; however the importance of these structures or the regulation of TERRA expression and retention in telomeric R-loops remain unclear. Here, we show that the RTEL1 (Regulator of Telomere Length 1) helicase influences the abundance and localization of TERRA in human cells. Depletion of RTEL1 leads to increased levels of TERRA RNA while reducing TERRA-containing R loops at telomeres. In vitro, RTEL1 shows a strong preference for binding G-quadruplex structures which form in TERRA. This binding is mediated by the C-terminal region of RTEL1, and is independent of the RTEL1 helicase domain. RTEL1 binding to TERRA appears to be essential for cell viability, underscoring the importance of this function. Degradation of TERRA-containing R-loops by overexpression of RNAse H1 partially recapitulates the increased TERRA levels and telomeric instability associated with RTEL1 deficiency. Collectively, these data suggest that regulation of TERRA is a key function of the RTEL1 helicase, and that loss of that function may contribute to the disease phenotypes of patients with RTEL1 mutations.

scholarly journals Telomere length analysis in crustacean species: Metapenaeus macleayi, Sagmariasus verreauxi, and Jasus edwardsii

2011 ◽  
Vol 68 (10) ◽  
pp. 2053-2058 ◽  
Author(s):  
Rosamond M. Godwin ◽  
Stewart Frusher ◽  
Steven S. Montgomery ◽  
Jennifer Ovenden
Keyword(s):  
Telomere Length ◽  
Life Histories ◽  
Age Determination ◽  
Age And Growth ◽  
Telomeric Dna ◽  
Test Species ◽  
Crustacean Species ◽  
Jasus Edwardsii ◽  
Length Analysis ◽  
Sagmariasus Verreauxi

Abstract Godwin, R. M., Frusher, S., Montgomery, S. S., and Ovenden, J. 2011. Telomere length analysis in crustacean species: Metapenaeus macleayi, Sagmariasus verreauxi, and Jasus edwardsii. – ICES Journal of Marine Science, 68: 2053–2058. Estimates of age and growth in crustaceans have been historically problematic and presented significant challenges to researchers. Current techniques of age determination provide valuable data, but also suffer from disadvantages. Telomeric DNA has been proposed as an age biomarker because it shortens with age in some species. In this study, the feasibility of using telomere length (TL) to estimate age was examined in the school prawn Metapenaeus macleayi and the spiny lobsters Sagmariasus verreauxi and Jasus edwardsii. Carapace length (CL) was used as a surrogate for age, and terminal restriction fragment assays were used to test the relationship between TL and size. Degradation of telomeric DNA with time during storage significantly influenced TL estimates, particularly for M. macleayi. TLs obtained from species in this study were 10–20 kb. No relationship between CL and TL was detected for any of the test species, and TL did not differ between male and female M. macleayi. TLs of J. edwardsii pueruli were unexpectedly shorter than those of J. edwardsii adults. The suitability of TL as an age biomarker in crustaceans may be limited, but further research is needed to elucidate telomere dynamics in these species with their different life histories and lifespans.

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 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 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 Functional Diversification of Replication Protein A Paralogs and Telomere Length Maintenance in Arabidopsis

Genetics ◽  
2020 ◽  
Vol 215 (4) ◽  
pp. 989-1002
Author(s):  
Behailu B. Aklilu ◽  
François Peurois ◽  
Carole Saintomé ◽  
Kevin M. Culligan ◽  
Daniela Kobbe ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Telomere Length ◽  
Protein A ◽  
Replication Protein A ◽  
Telomerase Activity ◽  
Replication Protein ◽  
Telomeric Dna ◽  
Telomere Shortening ◽  
Replication Group

Replication protein A (RPA) is essential for many facets of DNA metabolism. The RPA gene family expanded in Arabidopsis thaliana with five phylogenetically distinct RPA1 subunits (RPA1A-E), two RPA2 (RPA2A and B), and two RPA3 (RPA3A and B). RPA1 paralogs exhibit partial redundancy and functional specialization in DNA replication (RPA1B and RPA1D), repair (RPA1C and RPA1E), and meiotic recombination (RPA1A and RPA1C). Here, we show that RPA subunits also differentially impact telomere length set point. Loss of RPA1 resets bulk telomeres at a shorter length, with a functional hierarchy for replication group over repair and meiosis group RPA1 subunits. Plants lacking RPA2A, but not RPA2B, harbor short telomeres similar to the replication group. Telomere shortening does not correlate with decreased telomerase activity or deprotection of chromosome ends in rpa mutants. However, in vitro assays show that RPA1B2A3B unfolds telomeric G-quadruplexes known to inhibit replications fork progression. We also found that ATR deficiency can partially rescue short telomeres in rpa2a mutants, although plants exhibit defects in growth and development. Unexpectedly, the telomere shortening phenotype of rpa2a mutants is completely abolished in plants lacking the RTEL1 helicase. RTEL1 has been implicated in a variety of nucleic acid transactions, including suppression of homologous recombination. Thus, the lack of telomere shortening in rpa2a mutants upon RTEL1 deletion suggests that telomere replication defects incurred by loss of RPA may be bypassed by homologous recombination. Taken together, these findings provide new insight into how RPA cooperates with replication and recombination machinery to sustain telomeric DNA.

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 Control of Human Telomere Length by TRF1 and TRF2

2000 ◽  
Vol 20 (5) ◽  
pp. 1659-1668 ◽  
Author(s):  
Agata Smogorzewska ◽  
Bas van Steensel ◽  
Alessandro Bianchi ◽  
Stefan Oelmann ◽  
Matthias R. Schaefer ◽  
...  
Keyword(s):  
Telomere Length ◽  
Negative Regulator ◽  
Loop Model ◽  
Telomeric Dna ◽  
Telomere Elongation ◽  
Original Length ◽  
Population Doublings ◽  
Ttaggg Repeat

ABSTRACT Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3′ telomere terminus in TRF1- and TRF2-induced telomeric loops.

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