scholarly journals Mechanism of processive telomerase catalysis revealed by high-resolution optical tweezers

2019 ◽  
Author(s):  
Eric M. Patrick ◽  
Joseph Slivka ◽  
Bramyn Payne ◽  
Matthew J. Comstock ◽  
Jens C. Schmidt
Keyword(s):  
High Resolution ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Telomeric Dna ◽  
Telomeric Repeats ◽  
G Quadruplex ◽  
Stable Substrate ◽  
Template Region

Telomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled. How telomerase remains associated with substrate DNA during this critical translocation step remains unknown. Using a newly developed single-molecule telomerase activity assay utilizing high-resolution optical tweezers, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. After release of multiple telomeric repeats from telomerase, we observed folding of product DNA into G-quadruplex structures. Our results provide detailed mechanistic insights into telomerase catalysis, a process of critical importance in aging and cancer.

scholarly journals Extreme mechanical diversity of human telomeric DNA revealed by fluorescence-force spectroscopy

2019 ◽  
Vol 116 (17) ◽  
pp. 8350-8359 ◽  
Author(s):  
Jaba Mitra ◽  
Monika A. Makurath ◽  
Thuy T. M. Ngo ◽  
Alice Troitskaia ◽  
Yann R. Chemla ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Force Spectroscopy ◽  
Telomeric Dna ◽  
Telomeric Sequences ◽  
Substitution Mutant

G-quadruplexes (GQs) can adopt diverse structures and are functionally implicated in transcription, replication, translation, and maintenance of telomere. Their conformational diversity under physiological levels of mechanical stress, however, is poorly understood. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to measure human telomeric sequences under tension. Abrupt GQ unfolding with K+in solution occurred at as many as four discrete levels of force. Added to an ultrastable state and a gradually unfolding state, there were six mechanically distinct structures. Extreme mechanical diversity was also observed with Na+, although GQs were mechanically weaker. Our ability to detect small conformational changes at low forces enabled the determination of refolding forces of about 2 pN. Refolding was rapid and stochastically redistributed molecules to mechanically distinct states. A single guanine-to-thymine substitution mutant required much higher ion concentrations to display GQ-like unfolding and refolded via intermediates, contrary to the wild type. Contradicting an earlier proposal, truncation to three hexanucleotide repeats resulted in a single-stranded DNA-like mechanical behavior under all conditions, indicating that at least four repeats are required to form mechanically stable structures.

scholarly journals The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans

2019 ◽  
Vol 116 (49) ◽  
pp. 24542-24550 ◽  
Author(s):  
Jiarui Song ◽  
Dhenugen Logeswaran ◽  
Claudia Castillo-González ◽  
Yang Li ◽  
Sreyashree Bose ◽  
...  
Keyword(s):  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Telomerase Rna ◽  
Telomeric Dna ◽  
Structural Element ◽  
Stem Loop ◽  
Evolutionary Pathway ◽  
Pol Iii

Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3′ long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.

scholarly journals G-quadruplex Stabilization Fuels the ALT Pathway in ALT-positive Osteosarcoma Cells

Genes ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 304 ◽  
Author(s):  
Roberta Amato ◽  
Martina Valenzuela ◽  
Francesco Berardinelli ◽  
Erica Salvati ◽  
Carmen Maresca ◽  
...  
Keyword(s):  
Dna Damage ◽  
Sister Chromatid Exchanges ◽  
Telomere Maintenance ◽  
Osteosarcoma Cell ◽  
Telomeric Dna ◽  
Replicative Stress ◽  
Alternative Lengthening ◽  
G Quadruplex ◽  
Reduce Cell Proliferation ◽  
The Impact

Most human tumors maintain telomere lengths by telomerase, whereas a portion of them (10–15%) uses a mechanism named alternative lengthening of telomeres (ALT). The telomeric G-quadruplex (G4) ligand RHPS4 is known for its potent antiproliferative effect, as shown in telomerase-positive cancer models. Moreover, RHPS4 is also able to reduce cell proliferation in ALT cells, although the influence of G4 stabilization on the ALT mechanism has so far been poorly investigated. Here we show that sensitivity to RHPS4 is comparable in ALT-positive (U2OS; SAOS-2) and telomerase-positive (HOS) osteosarcoma cell lines, unlinking the telomere maintenance mechanism and RHPS4 responsiveness. To investigate the impact of G4 stabilization on ALT, the cardinal ALT hallmarks were analyzed. A significant induction of telomeric doublets, telomeric clusterized DNA damage, ALT-associated Promyelocytic Leukaemia-bodies (APBs), telomere sister chromatid exchanges (T-SCE) and c-circles was found exclusively in RHPS4-treated ALT cells. We surmise that RHPS4 affects ALT mechanisms through the induction of replicative stress that in turn is converted in DNA damage at telomeres, fueling recombination. In conclusion, our work indicates that RHPS4-induced telomeric DNA damage promotes overactivation of telomeric recombination in ALT cells, opening new questions on the therapeutic employment of G4 ligands in the treatment of ALT positive tumors.

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 Step-by-step evolution of telomeres: lessons from yeasts

2020 ◽  
Author(s):  
Filip Červenák ◽  
Regina Sepšiová ◽  
Jozef Nosek ◽  
Ľubomír Tomáška
Keyword(s):  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Rapid Evolution ◽  
Evolutionary Process ◽  
Telomere Maintenance ◽  
Evolutionary Origin ◽  
Special Focus ◽  
Telomeric Dna ◽  
Telomeric Repeats ◽  
Ascomycetous Yeasts

Abstract In virtually every eukaryotic species, the ends of nuclear chromosomes are protected by telomeres, nucleoprotein structures counteracting the end-replication problem and suppressing recombination and undue DNA repair. Although in most cases, the primary structure of telomeric DNA is conserved, there are several exceptions to this rule. One is represented by the telomeric repeats of ascomycetous yeasts, which encompass a great variety of sequences, whose evolutionary origin has been puzzling for several decades. At present, the key questions concerning the driving force behind their rapid evolution and the means of co-evolution of telomeric repeats and telomere-binding proteins remain largely unanswered. Previously published studies addressed mostly the general concepts of the evolutionary origin of telomeres, key properties of telomeric proteins as well as the molecular mechanisms of telomere maintenance, however, the evolutionary process itself has not been analyzed thoroughly. Here, we aimed to inspect the evolution of telomeres in ascomycetous yeasts from the subphyla Saccharomycotina and Taphrinomycotina, with special focus on the evolutionary origin of species-specific telomeric repeats. We analyzed the sequences of telomeric repeats from 204 yeast species classified into 20 families and as a result, we propose a step-by-step model, which integrates the diversity of telomeric repeats, telomerase RNAs, telomere-binding protein complexes and explains a propensity of certain species to generate the repeat heterogeneity within a single telomeric array.

scholarly journals Telomere Maintenance in Telomerase-Deficient Mouse Embryonic Stem Cells: Characterization of an Amplified Telomeric DNA

2000 ◽  
Vol 20 (11) ◽  
pp. 4115-4127 ◽  
Author(s):  
Hiroyuki Niida ◽  
Yoichi Shinkai ◽  
M. Prakash Hande ◽  
Takehisa Matsumoto ◽  
Shoko Takehara ◽  
...  
Keyword(s):  
Growth Rate ◽  
Mammalian Cells ◽  
Chromosomal Instability ◽  
Es Cells ◽  
Embryonic Stem ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Telomeric Dna ◽  
Rapid Growth Rate

ABSTRACT Telomere dynamics, chromosomal instability, and cellular viability were studied in serial passages of mouse embryonic stem (ES) cells in which the telomerase RNA (mTER) gene was deleted. These cells lack detectable telomerase activity, and their growth rate was reduced after more than 300 divisions and almost zero after 450 cell divisions. After this growth crisis, survivor cells with a rapid growth rate did emerge. Such survivors were found to maintain functional telomeres in a telomerase-independent fashion. Although telomerase-independent telomere maintenance has been reported for some immortalized mammalian cells, its molecular mechanism has not been elucidated. Characterization of the telomeric structures in one of the survivor mTER −/− cell lines showed amplification of the same tandem arrays of telomeric and nontelomeric sequences at most of the chromosome ends. This evidence implicatescis/trans amplification as one mechanism for the telomerase-independent maintenance of telomeres in mammalian cells.

scholarly journals TERRA G-quadruplex RNA interaction with TRF2 GAR domain is required for telomere integrity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yang Mei ◽  
Zhong Deng ◽  
Olga Vladimirova ◽  
Nitish Gulve ◽  
F. Brad Johnson ◽  
...  
Keyword(s):  
Telomeric Repeat ◽  
Telomere Maintenance ◽  
Structural Basis ◽  
Telomeric Dna ◽  
Recognition Element ◽  
Age Related ◽  
Binding Factor ◽  
G Quadruplex ◽  
Genetic Deletion ◽  
Rna Interaction

AbstractTelomere dysfunction causes chromosomal instability which is associated with many cancers and age-related diseases. The non-coding telomeric repeat-containing RNA (TERRA) forms a structural and regulatory component of the telomere that is implicated in telomere maintenance and chromosomal end protection. The basic N-terminal Gly/Arg-rich (GAR) domain of telomeric repeat-binding factor 2 (TRF2) can bind TERRA but the structural basis and significance of this interaction remains poorly understood. Here, we show that TRF2 GAR recognizes G-quadruplex features of TERRA. We show that small molecules that disrupt the TERRA-TRF2 GAR complex, such as N-methyl mesoporphyrin IX (NMM) or genetic deletion of TRF2 GAR domain, result in the loss of TERRA, and the induction of γH2AX-associated telomeric DNA damage associated with decreased telomere length, and increased telomere aberrations, including telomere fragility. Taken together, our data indicates that the G-quadruplex structure of TERRA is an important recognition element for TRF2 GAR domain and this interaction between TRF2 GAR and TERRA is essential to maintain telomere stability.

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