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Sample records for telomeric g-quadruplex structures

  1. Oligonucleotide Models of Telomeric DNA and RNA Form a Hybrid G-quadruplex Structure as a Potential Component of Telomeres*

    Science.gov (United States)

    Xu, Yan; Ishizuka, Takumi; Yang, Jie; Ito, Kenichiro; Katada, Hitoshi; Komiyama, Makoto; Hayashi, Tetsuya

    2012-01-01

    Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends. PMID:23012368

  2. Structure of human telomeric RNA (TERRA): stacking of two G-quadruplex blocks in K(+) solution.

    Science.gov (United States)

    Martadinata, Herry; Phan, Anh Tuân

    2013-04-02

    Telomeric repeat-containing RNAs (TERRA) are transcription products of the telomeres. Human TERRA sequences containing UUAGGG repeats can form parallel-stranded G-quadruplexes. The stacking interaction of such structures was shown to be important for ligand targeting and higher-order arrangement of G-quadruplexes in long TERRA sequences. Here we report on the first high-resolution structure of a stacked G-quadruplex formed by the 10-nucleotide human TERRA sequence r(GGGUUAGGGU) in potassium solution. This structure comprises two dimeric three-layer parallel-stranded G-quadruplex blocks, which stack on each other at their 5'-ends. The adenine in each UUA loop is nearly coplanar with the 5'-end G-tetrad forming an A·(G·G·G·G)·A hexad, thereby increasing the stacking contacts between the two blocks. Interestingly, this stacking and loop conformation is different from all structures previously reported for the free human TERRA but resembles the structure previously determined for a complex between a human TERRA sequence and an acridine ligand. This stacking conformation is a potential target for drugs that recognize or induce the stacking interface.

  3. The effect of the TRF2 N-terminal and TRFH regions on telomeric G-quadruplex structures

    OpenAIRE

    Pedroso, Ilene M.; Hayward, William; Fletcher, Terace M.

    2009-01-01

    The sequence of human telomeric DNA consists of tandem repeats of 5?-d(TTAGGG)-3?. This guanine-rich DNA can form G-quadruplex secondary structures which may affect telomere maintenance. A current model for telomere protection by the telomere-binding protein, TRF2, involves the formation of a t-loop which is stabilized by a strand invasion-like reaction. This type of reaction may be affected by G-quadruplex structures. We analyzed the influence of the arginine-rich, TRF2 N-terminus (TRF2B), a...

  4. Interaction of hnRNP A1 with telomere DNA G-quadruplex structures studied at the single molecule level

    DEFF Research Database (Denmark)

    Krüger, Asger Christian; Raarup, Merete Krog; Nielsen, Morten Muhlig

    2010-01-01

    the interaction of hnRNP A1 with G-quadruplex DNA structures containing the human telomere repeat (TTAGGG) by gel retardation assays, ensemble fluorescence energy transfer (FRET) spectroscopy, and single molecule FRET microscopy. Our biochemical experiments show that hnRNP A1 binds well to the G......-quadruplex telomeric DNA. Ensemble and single molecule FRET measurements provide further insight into molecular conformation: the telomeric DNA overhang is found to be in a folded state in the absence of hnRNP A1 and to remain predominantly in a compact state when complexed with hnRNP A1. This finding is in contrast...

  5. The effect of the TRF2 N-terminal and TRFH regions on telomeric G-quadruplex structures.

    Science.gov (United States)

    Pedroso, Ilene M; Hayward, William; Fletcher, Terace M

    2009-04-01

    The sequence of human telomeric DNA consists of tandem repeats of 5'-d(TTAGGG)-3'. This guanine-rich DNA can form G-quadruplex secondary structures which may affect telomere maintenance. A current model for telomere protection by the telomere-binding protein, TRF2, involves the formation of a t-loop which is stabilized by a strand invasion-like reaction. This type of reaction may be affected by G-quadruplex structures. We analyzed the influence of the arginine-rich, TRF2 N-terminus (TRF2(B)), as well as this region plus the TRFH domain of TRF2 (TRF2(BH)), on the structure of G-quadruplexes. Circular dichroism results suggest that oligonucleotides with 4, 7 and 8 5'-d(TTAGGG)-3' repeats form hybrid structures, a mix of parallel/antiparallel strand orientation, in K(+). TRF2(B) stimulated the formation of parallel-stranded structures and, in some cases, intermolecular structures. TRF2(BH) also stimulated intermolecular but not parallel-stranded structures. Only full-length TRF2 and TRF2(BH) stimulated uptake of a telomeric single-stranded oligonucleotide into a plasmid containing telomeric DNA in the presence of K(+). The results in this study suggest that G-quadruplex formation inhibits oligonucleotide uptake into the plasmid, but the inhibition can be overcome by TRF2. This study is the first analysis of the effects of TRF2 domains on G-quadruplex structures and has implications for the role of G-quadruplexes and TRF2 in the formation of t-loops.

  6. Expression of Telomere-Associated Proteins is Interdependent to Stabilize Native Telomere Structure and Telomere Dysfunction by G-Quadruplex Ligand Causes TERRA Upregulation.

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    Sadhukhan, Ratan; Chowdhury, Priyanka; Ghosh, Sourav; Ghosh, Utpal

    2017-11-13

    Telomere DNA can form specialized nucleoprotein structure with telomere-associated proteins to hide free DNA ends or G-quadruplex structures under certain conditions especially in presence of G-quadruplex ligand. Telomere DNA is transcribed to form non-coding telomere repeat-containing RNA (TERRA) whose biogenesis and function is poorly understood. Our aim was to find the role of telomere-associated proteins and telomere structures in TERRA transcription. We silenced four [two shelterin (TRF1, TRF2) and two non-shelterin (PARP-1, SLX4)] telomere-associated genes using siRNA and verified depletion in protein level. Knocking down of one gene modulated expression of other telomere-associated genes and increased TERRA from 10q, 15q, XpYp and XqYq chromosomes in A549 cells. Telomere was destabilized or damaged by G-quadruplex ligand pyridostatin (PDS) and bleomycin. Telomere dysfunction-induced foci (TIFs) were observed for each case of depletion of proteins, treatment with PDS or bleomycin. TERRA level was elevated by PDS and bleomycin treatment alone or in combination with depletion of telomere-associated proteins.

  7. Kinetic partitioning modulates human telomere DNA G-quadruplex structural polymorphism.

    Directory of Open Access Journals (Sweden)

    Xi Long

    Full Text Available Telomeres are specialized chromatin structures found at the end of chromosomes and are crucial to the maintenance of eukaryotic genome stability. Human telomere DNA is comprised of the repeating sequence (T2AG3n, which is predominantly double-stranded but terminates with a 3' single-stranded tail. The guanine-rich tail can fold into secondary structures known as a G-quadruplexes (GQs that may exist as a polymorphic mixture of anti-parallel, parallel, and several hybrid topological isomers. Using single-molecule Förster resonance energy transfer (smFRET, we have reconstructed distributions of telomere DNA GQ conformations generated by an in situ refolding protocol commonly employed in single-molecule studies of GQ structure, or using a slow cooling DNA annealing protocol typically used in the preparation of GQ samples for ensemble biophysical analyses. We find the choice of GQ folding protocol has a marked impact on the observed distributions of DNA conformations under otherwise identical buffer conditions. A detailed analysis of the kinetics of GQ folding over timescales ranging from minutes to hours revealed the distribution of GQ structures generated by in situ refolding gradually equilibrates to resemble the distribution generated by the slow cooling DNA annealing protocol. Interestingly, conditions of low ionic strength, which promote transient GQ unfolding, permit the fraction of folded DNA molecules to partition into a distribution that more closely approximates the thermodynamic folding equilibrium. Our results are consistent with a model in which kinetic partitioning occurs during in situ folding at room temperature in the presence of K(+ ions, producing a long-lived non-equilibrium distribution of GQ structures in which the parallel conformation predominates on the timescale of minutes. These results suggest that telomere DNA GQ folding kinetics, and not just thermodynamic stability, likely contributes to the physiological ensemble GQ

  8. An Investigation of G-Quadruplex Structural Polymorphism in the Human Telomere Using a Combined Approach of Hydrodynamic Bead Modeling and Molecular Dynamics Simulation

    Science.gov (United States)

    2015-01-01

    Guanine-rich oligonucleotides can adopt noncanonical tertiary structures known as G-quadruplexes, which can exist in different forms depending on experimental conditions. High-resolution structural methods, such as X-ray crystallography and NMR spectroscopy, have been of limited usefulness in resolving the inherent structural polymorphism associated with G-quadruplex formation. The lack of, or the ambiguous nature of, currently available high-resolution structural data, in turn, has severely hindered investigations into the nature of these structures and their interactions with small-molecule inhibitors. We have used molecular dynamics in conjunction with hydrodynamic bead modeling to study the structures of the human telomeric G-quadruplex-forming sequences at the atomic level. We demonstrated that molecular dynamics can reproduce experimental hydrodynamic measurements and thus can be a powerful tool in the structural study of existing G-quadruplex sequences or in the prediction of new G-quadruplex structures. PMID:24779348

  9. Polyethylene glycol binding alters human telomere G-quadruplex structure by conformational selection.

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    Buscaglia, Robert; Miller, M Clarke; Dean, William L; Gray, Robert D; Lane, Andrew N; Trent, John O; Chaires, Jonathan B

    2013-09-01

    Polyethylene glycols (PEGs) are widely used to perturb the conformations of nucleic acids, including G-quadruplexes. The mechanism by which PEG alters G-quadruplex conformation is poorly understood. We describe here studies designed to determine how PEG and other co-solutes affect the conformation of the human telomeric quadruplex. Osmotic stress studies using acetonitrile and ethylene glycol show that conversion of the 'hybrid' conformation to an all-parallel 'propeller' conformation is accompanied by the release of about 17 water molecules per quadruplex and is energetically unfavorable in pure aqueous solutions. Sedimentation velocity experiments show that the propeller form is hydrodynamically larger than hybrid forms, ruling out a crowding mechanism for the conversion by PEG. PEGs do not alter water activity sufficiently to perturb quadruplex hydration by osmotic stress. PEG titration experiments are most consistent with a conformational selection mechanism in which PEG binds more strongly to the propeller conformation, and binding is coupled to the conformational transition between forms. Molecular dynamics simulations show that PEG binding to the propeller form is sterically feasible and energetically favorable. We conclude that PEG does not act by crowding and is a poor mimic of the intranuclear environment, keeping open the question of the physiologically relevant quadruplex conformation.

  10. Structure of long human telomeric RNA (TERRA): G-quadruplexes formed by four and eight UUAGGG repeats are stable building blocks.

    Science.gov (United States)

    Martadinata, Herry; Heddi, Brahim; Lim, Kah Wai; Phan, Anh Tuân

    2011-07-26

    The discovery of long RNA transcripts of telomeric repeats (TERRA) and their potential to form G-quadruplexes stimulated studies on the possible arrangements of G-quadruplexes along TERRA. Here we performed ribonuclease protection assay to investigate the structures formed by long human TERRA. We found that G-quadruplexes comprising four and eight UUAGGG repeats were most resistant to RNase T1 digestion, presumably with the former adopting an all-parallel-stranded propeller-type conformation and the latter forming a structure with two tandemly stacked G-quadruplex subunits each containing three G-tetrad layers. Molecular dynamics simulations of eight-repeat human TERRA sequences consisting of different stacking interfaces between the two G-quadruplex subunits, i.e., 5'-5', 3'-3', 3'-5', and 5'-3', demonstrated stacking feasibility for all but the 5'-3' arrangement. A continuous stacking of the loop bases from one G-quadruplex subunit to the next was observed for the 5'-5' stacking conformation. We also put forward other possible stacking arrangements that involve more than one linker connecting the two G-quadruplex subunits. On the basis of these results, we propose a "beads-on-a-string"-like arrangement along human TERRA, whereby each bead is made up of either four or eight UUAGGG repeats in a one- or two-block G-quadruplex arrangement, respectively. © 2011 American Chemical Society

  11. Photoreactivity of the linker region of two consecutive G-quadruplexes formed by human telomeric DNA.

    Science.gov (United States)

    Li, Yue; Sugiyama, Hiroshi

    2015-05-25

    We report the application of a photoreaction method for probing two consecutive G-quadruplexes formed by human telomeric DNA. This method can discriminate the loop structure located between two consecutive G-quadruplexes formed by eight TTAGGG repeats in K(+) and Na(+) solutions.

  12. An Individual Nanocube-Based Plasmonic Biosensor for Real-Time Monitoring the Structural Switch of the Telomeric G-Quadruplex.

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    Tian, Yuanyuan; Zhang, Lei; Shen, Jingjing; Wu, Lingzhi; He, Hongzhang; Ma, Dik-Lung; Leung, Chung-Hang; Wu, Weibing; Fan, Quli; Huang, Wei; Wang, Lianhui

    2016-06-01

    Promoted by the localized surface plasmon resonance nanotechnology, a simple and sensitive plasmonic aptamer nanosensor (nanoaptasensor) on an individual Au@Ag core-shell nanocube (Au@Ag NC) has been proposed for real-time monitoring of the formation process of G-quadruplex structures and label-free analysis of potassium ions (K(+) ). In particular, the analysis of the thermodynamic parameters indicates that there are two types of binding states accompanied with a remarkable change of free energy (ΔG) in the sequential folding process of telomere DNA sequence. This nanoaptasensor has raised promising applications in monitoring the dynamic process of the structural switch of the G-quadruplex. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Selective recognition and stabilization of new ligands targeting the potassium form of the human telomeric G-quadruplex DNA

    Science.gov (United States)

    Lin, Yi-Hwa; Chuang, Show-Mei; Wu, Pei-Ching; Chen, Chun-Liang; Jeyachandran, Sivakamavalli; Lo, Shou-Chen; Huang, Hsu-Shan; Hou, Ming-Hon

    2016-08-01

    The development of a ligand that is capable of distinguishing among the wide variety of G-quadruplex structures and targeting telomeres to treat cancer is particularly challenging. In this study, the ability of two anthraquinone telomerase inhibitors (NSC749235 and NSC764638) to target telomeric G-quadruplex DNA was probed. We found that these ligands specifically target the potassium form of telomeric G-quadruplex DNA over the DNA counterpart. The characteristic interaction with the telomeric G-quadruplex DNA and the anticancer activities of these ligands were also explored. The results of this present work emphasize our understanding of the binding selectivity of anthraquinone derivatives to G-quadruplex DNA and assists in future drug development for G-quadruplex-specific ligands.

  14. Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration.

    Science.gov (United States)

    Gilbert-Girard, Shella; Gravel, Annie; Artusi, Sara; Richter, Sara N; Wallaschek, Nina; Kaufer, Benedikt B; Flamand, Louis

    2017-07-15

    Human herpesviruses 6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes using a mechanism that remains poorly understood. To achieve a better understanding of the HHV-6A/B integration mechanism, we made use of BRACO-19, a compound that stabilizes G-quadruplex secondary structures and prevents telomere elongation by the telomerase complex. First, we analyzed the folding of telomeric sequences into G-quadruplex structures and their binding to BRACO-19 using G-quadruplex-specific antibodies and surface plasmon resonance. Circular dichroism studies indicate that BRACO-19 modifies the conformation and greatly stabilizes the G-quadruplexes formed in G-rich telomeric DNA. Subsequently we assessed the effects of BRACO-19 on the HHV-6A initial phase of infection. Our results indicate that BRACO-19 does not affect entry of HHV-6A DNA into cells. We next investigated if stabilization of G-quadruplexes by BRACO-19 affected HHV-6A's ability to integrate its genome into host chromosomes. Incubation of telomerase-expressing cells with BRACO-19, such as HeLa and MCF-7, caused a significant reduction in the HHV-6A integration frequency ( P integration frequency in U2OS cells that lack telomerase activity and elongate their telomeres through alternative lengthening mechanisms. Our data suggest that the fluidity of telomeres is important for efficient chromosomal integration of HHV-6A and that interference with telomerase activity negatively affects the generation of cellular clones containing integrated HHV-6A. IMPORTANCE HHV-6A/B can integrate their genomes into the telomeres of infected cells. Telomeres consist of repeated hexanucleotides (TTAGGG) of various lengths (up to several kilobases) and end with a single-stranded 3' extension. To avoid recognition and induce a DNA damage response, the single-stranded overhang folds back on itself and forms a telomeric loop (T-loop) or adopts a tertiary structure, referred to as a G-quadruplex. In the

  15. Chiral metallohelices enantioselectively target hybrid human telomeric G-quadruplex DNA

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    Zhao, Andong; Howson, Suzanne E.; Ren, Jinsong; Scott, Peter; Wang, Chunyu

    2017-01-01

    Abstract The design and synthesis of metal complexes that can specifically target DNA secondary structure has attracted considerable attention. Chiral metallosupramolecular complexes (e.g. helicates) in particular display unique DNA-binding behavior, however until recently few examples which are both water-compatible and enantiomerically pure have been reported. Herein we report that one metallohelix enantiomer Δ1a, available from a diastereoselective synthesis with no need for resolution, can enantioselectively stabilize human telomeric hybrid G-quadruplex and strongly inhibit telomerase activity with IC50 of 600 nM. In contrast, no such a preference is observed for the mirror image complex Λ1a. More intriguingly, neither of the two enantiomers binds specifically to human telomeric antiparallel G-quadruplex. To the best of our knowledge, this is the first example of one pair of enantiomers with contrasting selectivity for human telomeric hybrid G-quadruplex. Further studies show that Δ1a can discriminate human telomeric G-quadruplex from other telomeric G-quadruplexes. PMID:28398500

  16. Spectral Analysis of Interaction between Human Telomeric G-Quadruplex and Liliflorin A, the First Lignan Derivative Interacted with G-Quadruplex DNA.

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    Liu, Ting-ting; Zhou, Shuang; Jia, Qian-lan; Wang, Wen-shu; Yan, Xiao-qian; Zhang, Wen-hao; Wang, Shuai-qi; Jiao, Yu-guo

    2016-03-01

    Human telomeric G-quadruplex is a four-stranded structure folded by guanines (G) via Hoogsteen hydrogen bonding. The ligands which stabilize the G-quadruplex are often telomerase inhibitors and may become antitumor agents. Here, the interaction between a lignan derivative liliflorin A and human telomeric sequence dGGG (TTAGGG)3G-quadruplex HTG21 were examined by CD, FRET, and NMR spectroscopic methods. In addition, Molecular Docking was used to study the binding of liliflorin A to dTAGGG (TTAGGG)3 G-quadruplex HTG23. The CD data showed that liliflorin A enhanced HTG21 T(m). The T(m) value of G-quadruplex was enhanced 3.2 degrees C by 4.0 μmol x L(-1) liliflorin A in FRET. The NMR spectra of HTG21 showed vivid alteration after reacting with liliflorin A in 3 hours. Molecular Docking suggested liliflorin A bound to the wide groove of HTG23 at G9, G10, G16 and G17. Liliflorin A was the first lignan derivative that could stabilize HTG21 selectively and provided a new candidate for antitumor drug design targeting on human telomeric G-quadruplex.

  17. Calculation of Hydrodynamic Properties for G-Quadruplex Nucleic Acid Structures from in silico Bead Models

    Science.gov (United States)

    Le, Huy T.; Buscaglia, Robert; Dean, William L.; Chaires, Jonathan B.; Trent, John O.

    2012-01-01

    Nucleic acids enriched in guanine bases can adopt unique quadruple helical tertiary structures known as G-quadruplexes. G-quadruplexes have emerged as attractive drug targets as many G-quadruplex-forming sequences have been discovered in functionally critical sites within the human genome, including the telomere, oncogene promoters, and mRNA processing sites. A single G-quadruplex-forming sequence can adopt one of multiple folding topologies often resulting in a lack of a single definitive atomic-level resolution structure for many of these sequences and a major challenge to the discovery of G-quadruplex-selective small molecule drugs. Low-resolution techniques employed to study G-quadruplex structures (e.g. CD spectroscopy) are often unable to discern between G-quadruplex structural ensembles while high-resolution techniques (e.g. NMR spectroscopy) can be overwhelmed by a highly polymorphic system. Hydrodynamic bead modeling is an approach to studying G-quadruplex structures that could bridge the gap between low-resolution techniques and high-resolution molecular models. Here, we present a discussion of hydrodynamic bead modeling in the context of studying G-quadruplex structures, highlighting recent successes and limitations to this approach, as well as an example featuring a G-quadruplex structure formed from the human telomere. This example can easily be adapted to the investigation of any other G-quadruplex-forming sequences. PMID:22886555

  18. X-ray characterization of mesophases of human telomeric G-quadruplexes and other DNA analogues

    Science.gov (United States)

    Yasar, Selcuk; Schimelman, Jacob B.; Aksoyoglu, M. Alphan; Steinmetz, Nicole F.; French, Roger H.; Parsegian, V. Adrian; Podgornik, Rudolf

    2016-06-01

    Observed in the folds of guanine-rich oligonucleotides, non-canonical G-quadruplex structures are based on G-quartets formed by hydrogen bonding and cation-coordination of guanosines. In dilute 5‧-guanosine monophosphate (GMP) solutions, G-quartets form by the self-assembly of four GMP nucleotides. We use x-ray diffraction to characterize the columnar liquid-crystalline mesophases in concentrated solutions of various model G-quadruplexes. We then probe the transitions between mesophases by varying the PEG solution osmotic pressure, thus mimicking in vivo molecular crowding conditions. Using the GMP-quadruplex, built by the stacking of G-quartets with no covalent linking between them, as the baseline, we report the liquid-crystalline phase behaviors of two other related G-quadruplexes: (i) the intramolecular parallel-stranded G-quadruplex formed by the 22-mer four-repeat human telomeric sequence AG3(TTAG3)3 and (ii) the intermolecular parallel-stranded G-quadruplex formed by the TG4T oligonucleotides. Finally, we compare the mesophases of the G-quadruplexes, under PEG-induced crowding conditions, with the corresponding mesophases of the canonical duplex and triplex DNA analogues.

  19. G-quadruplex structure and stability illuminated by 2-aminopurine phasor plots.

    Science.gov (United States)

    Buscaglia, Robert; Jameson, David M; Chaires, Jonathan B

    2012-05-01

    The use of time-resolved fluorescence measurements in studies of telomeric G-quadruplex folding and stability has been hampered by the complexity of fluorescence lifetime distributions in solution. The application of phasor diagrams to the analysis of time-resolved fluorescence measurements, collected from either frequency-domain or time-domain instrumentation, allows for rapid characterization of complex lifetime distributions. Phasor diagrams are model-free graphical representations of transformed time-resolved fluorescence results. Simplification of complex fluorescent decays by phasor diagrams is demonstrated here using a 2-aminopurine substituted telomeric G-quadruplex sequence. The application of phasor diagrams to complex systems is discussed with comparisons to traditional non-linear regression model fitting. Phasor diagrams allow for the folding and stability of the telomeric G-quadruplex to be monitored in the presence of either sodium or potassium. Fluorescence lifetime measurements revealed multiple transitions upon folding of the telomeric G-quadruplex through the addition of potassium. Enzymatic digestion of the telomeric G-quadruplex structure, fluorescence quenching and Förster resonance energy transfer were also monitored through phasor diagrams. This work demonstrates the sensitivity of time-resolved methods for monitoring changes to the telomeric G-quadruplex and outlines the phasor diagram approach for analysis of complex time-resolved results that can be extended to other G-quadruplex and nucleic acid systems.

  20. Platinum(II) phenanthroimidazole G-quadruplex ligand induces selective telomere shortening in A549 cancer cells.

    Science.gov (United States)

    Mancini, Johanna; Rousseau, Philippe; Castor, Katherine J; Sleiman, Hanadi F; Autexier, Chantal

    2016-02-01

    Telomere maintenance, achieved by the binding of protective shelterin capping proteins to telomeres and by either telomerase or a recombination-based alternative lengthening of telomere (ALT) mechanism, is critical for cell proliferation and survival. Extensive telomere shortening or loss of telomere integrity activates DNA damage checkpoints, leading to cell senescence or death. Although telomerase upregulation is an attractive target for anti-cancer therapy, the lag associated with telomere shortening and the potential activation of ALT pose a challenge. An alternative approach is to modify telomere interactions with binding proteins (telomere uncapping). G-quadruplex ligands stabilize structures generated from single-stranded G-rich 3'-telomere end (G-quadruplex) folding, which in principle, cannot be elongated by telomerase, thus leading to telomere shortening. Ligands can also mediate rapid anti-proliferative effects by telomere uncapping. We previously reported that the G-quadruplex ligand, phenylphenanthroimidazole ethylenediamine platinum(II) (PIP), inhibits telomerase activity in vitro[47]. In the current study, a long-term seeding assay showed that PIP significantly inhibited the seeding capacity of A549 lung cancer cells and to a lesser extent primary MRC5 fibroblast cells. Importantly, treatment with PIP caused a significant dose- and time-dependent decrease in average telomere length of A549 but not MRC5 cells. Moreover, cell cycle analysis revealed a significant increase in G1 arrest upon treatment of A549 cells, but not MRC5 cells. Both apoptosis and cellular senescence may contribute to the anti-proliferative effects of PIP. Our studies validate the development of novel and specific therapeutic ligands targeting telomeric G-quadruplex structures in cancer cells. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  1. Mechanochemical properties of individual human telomeric RNA (TERRA) G-quadruplexes.

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    Yangyuoru, Philip M; Zhang, Amy Y Q; Shi, Zhe; Koirala, Deepak; Balasubramanian, Shankar; Mao, Hanbin

    2013-10-11

    Potential functions: By following the unfolding and refolding of individual human RNA telomeric (TERRA) G-quadruplexes (GQs) in laser tweezers, the mechanical stability and transition kinetics of RNA GQs are obtained. Comparison between TERRA and DNA GQs suggests their different regulatory capacities for processes associated with human telomeres. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Studies of G-quadruplex DNA structures at the single molecule level

    DEFF Research Database (Denmark)

    Kragh, Sofie Louise

    2015-01-01

    Folding of G-quaduplex structures adopted by the human telomeric repeat is here studied by single molecule FRET microscopy. This method allows for the investigation of G-quadruplex structures and their conformational dynamic. Telomeres are located at the ends of our chromosomes and end in a single...... range. FRET spectroscopy can be performed on an ensemble of molecules, or on the single molecule level. In single molecule FRET experiments it is possible to follow the behaviour in time for each molecule independently, allowing insight into both dynamically and statistically heterogeneous molecular...... populations and thus providing more information than traditional ensemble experiments. Using single molecule FRET microscopy different aspects of G-quadruplex folding were investigated. We have obtained direct insight into G-quadruplex structural polymorphism both in K+ and Na+ solutions. Polymorphism have...

  3. Molecular crowding effects on conformation and stability of G-quadruplex DNA structure: insights from molecular dynamics simulation.

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    Verdian Doghaei, A; Housaindokht, M R; Bozorgmehr, M R

    2015-01-07

    Intracellular space is highly crowded with different biomolecules such as proteins, nucleic acids and ions. Therefore molecular crowding is a crucial factor in determining the structure, stability and function of G-quadruplexs. The effect of crowding on the DNA G-quadruplexes structure and stability has been studied by experimental methods, but it hasn't been known how crowding agents stabilize the G-quadruplex structure in molecular level yet. Here, we present a molecular dynamics investigation over the effect of molecular crowding, imitated here by ethanol, on the stability of G-quadruplex structure both in presence and absence of stabilizing K(+) cations. It was demonstrated that G-quadruplex structure in the water collapses in the absence of this cation, while ethanol stabilizes the structure of G-quadruplex by the excluded volume and decreases the water activity. The presence of ethanol can increase the stability of the Hoogsteen hydrogen bonds within the G-quartet. To understand the importance of cations, simulation has been performed on the cation containing G-quadruplex in the presence of ethanol with different concentrations. Molecular dynamics simulations of the structure and stability of human telomeric G-quadruplex in ethanol containing solution has enhanced our understanding on how the parallel-stranded G-quadruplex is affected in a condition where the water content is reduced. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. The G-quadruplex ligand telomestatin impairs binding of topoisomerase IIIalpha to G-quadruplex-forming oligonucleotides and uncaps telomeres in ALT cells.

    Directory of Open Access Journals (Sweden)

    Nassima Temime-Smaali

    Full Text Available In Alternative Lengthening of Telomeres (ALT cell lines, specific nuclear bodies called APBs (ALT-associated PML bodies concentrate telomeric DNA, shelterin components and recombination factors associated with telomere recombination. Topoisomerase IIIalpha (Topo III is an essential telomeric-associated factor in ALT cells. We show here that the binding of Topo III to telomeric G-overhang is modulated by G-quadruplex formation. Topo III binding to G-quadruplex-forming oligonucleotides was strongly inhibited by telomestatin, a potent and specific G-quadruplex ligand. In ALT cells, telomestatin treatment resulted in the depletion of the Topo III/BLM/TRF2 complex and the disruption of APBs and led to the segregation of PML, shelterin components and Topo III. Interestingly, a DNA damage response was observed at telomeres in telomestatin-treated cells. These data indicate the importance of G-quadruplex stabilization during telomere maintenance in ALT cells. The function of TRF2/Topo III/BLM in the resolution of replication intermediates at telomeres is discussed.

  5. Photocrosslinking of human telomeric G-quadruplex loops by anti cyclobutane thymine dimer formation.

    Science.gov (United States)

    Su, Dian G T; Fang, Huafeng; Gross, Michael L; Taylor, John-Stephen A

    2009-08-04

    The unusual structural forms of telomere DNA, which protect the ends of chromosomes during replication, may render it vulnerable to unprecedented photodamage, possibly involving nonadjacent bases that are made proximate by folding. The G-quadruplex for the human telomere sequence consisting of a repeating d(TTAGGG) is one unusual form. Tel22, d[AGGG(TTAGGG)(3)], forms a basket structure in the presence of Na(+) and may form multiple equilibrating structures in the presence of K(+) with hybrid-type structures predominating. UVB irradiation of d[AGGG(TTAGGG)(3)] in the presence of Na(+) results in a cis,syn thymine dimer between two adjacent Ts in a TTA loop and a mixture of nonadjacent anti thymine dimers between various loops. Irradiation in the presence of K(+), however, produces, in addition to these same products, a large amount of specific anti thymine dimers formed between either T in loop 1 and the central T in loop 3. These latter species were not observed in the presence of Na(+). Interloop-specific anti thymine dimers are incompatible with hybrid-type structures, but could arise from a chair or basket-type structure or from triplex intermediates involved in interconverting these structures. If these unique nonadjacent anti thymine dimer photoproducts also form in vivo, they would constitute a previously unrecognized type of DNA photodamage that may interfere with telomere replication and present a unique challenge to DNA repair. Furthermore, these unusual anti photoproducts may be used to establish the presence of G-quadruplex or quadruplex-like structures in vivo.

  6. Intramolecular telomeric G-quadruplexes dramatically inhibit DNA synthesis by replicative and translesion polymerases, revealing their potential to lead to genetic change.

    Directory of Open Access Journals (Sweden)

    Deanna N Edwards

    Full Text Available Recent research indicates that hundreds of thousands of G-rich sequences within the human genome have the potential to form secondary structures known as G-quadruplexes. Telomeric regions, consisting of long arrays of TTAGGG/AATCCC repeats, are among the most likely areas in which these structures might form. Since G-quadruplexes assemble from certain G-rich single-stranded sequences, they might arise when duplex DNA is unwound such as during replication. Coincidentally, these bulky structures when present in the DNA template might also hinder the action of DNA polymerases. In this study, single-stranded telomeric templates with the potential to form G-quadruplexes were examined for their effects on a variety of replicative and translesion DNA polymerases from humans and lower organisms. Our results demonstrate that single-stranded templates containing four telomeric GGG runs fold into intramolecular G-quadruplex structures. These intramolecular G quadruplexes are somewhat dynamic in nature and stabilized by increasing KCl concentrations and decreasing temperatures. Furthermore, the presence of these intramolecular G-quadruplexes in the template dramatically inhibits DNA synthesis by various DNA polymerases, including the human polymerase δ employed during lagging strand replication of G-rich telomeric strands and several human translesion DNA polymerases potentially recruited to sites of replication blockage. Notably, misincorporation of nucleotides is observed when certain translesion polymerases are employed on substrates containing intramolecular G-quadruplexes, as is extension of the resulting mismatched base pairs upon dynamic unfolding of this secondary structure. These findings reveal the potential for blockage of DNA replication and genetic changes related to sequences capable of forming intramolecular G-quadruplexes.

  7. Origin of Ion Specificity of Telomeric DNA G-Quadruplexes Investigated by Free-Energy Simulations.

    Science.gov (United States)

    Siebenmorgen, Till; Zacharias, Martin

    2017-06-06

    Telomeric DNA consists of tandem repeats of the sequence d(TTAGGG) that form G-quadruplex structures made of stacked guanines with monovalent cations bound at a central cavity. Although different ions can stabilize a G-quadruplex structure, the preferred bound ions are typically K(+) or Na(+). Several different strand-folding topologies have been reported for Q-quadruplexes formed from telomeric repeats depending on DNA length and ion solution condition. This suggests a possible dependence of the ion selectivity of the central pore on the folding topology of the quadruplex. Molecular dynamics free energy perturbation has been employed to systematically study the relative affinity of the central quadruplex pore for different cation types and the associated energetic and solvation contributions to ion selectivity. The calculations have been performed on two different common quadruplex folding topologies. For both topologies, the same ion selectivity was found with a preference for K(+) followed by Rb(+) and Na(+), which agrees with the experimentally determined preference for most investigated quadruplexes. The selectivity is determined by a balance between attractive Coulomb interactions and loss of hydration but also modulated by van der Waals contributions. Specificity is mediated by the central guanines and no significant contribution of the nucleic acid backbone. The simulations indicate that different topologies might be stabilized by ions bound at the surface or alternative sites of the quadruplex because the ion specificity of the central pore does not depend on the strand folding topology. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  8. Understanding the stability of DNA G-quadruplex units in long human telomeric strands.

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    Bugaut, Anthony; Alberti, Patrizia

    2015-06-01

    Human telomeric DNA is composed of GGGTTA repeats. The presence of consecutive guanines makes the telomeric G-strand prone to fold into contiguous (or tandem) G-quadruplexes (G4s). The aim of this study was to provide a clarified picture of the stability of telomeric tandem G4 structures as a function of the number of G4 units and of boundary sequences, and an understanding of the diversity of their melting behaviors in terms of the single G4 units composing them. To this purpose we undertook an UV-spectroscopic investigation of the structure and stability of telomeric repeats potentially able to fold into up to four contiguous G4s, flanked or not by TTA sequences at their 5' and 3' extremities. We explain why the stability of (GGGTTA)4m-1GGG structures (m = 2, 3, 4 …) decreases with increasing the number m of G4 units, whereas the stability of TTA-(GGGTTA)4m-1GGG-TTA structures does not. Our results support that the inner G4 units have similar stabilities, whereas the stabilities of the terminal G4 units are modulated by their flanking nucleotides: in a TTA-(GGGTTA)4m-1GGG-TTA tandem context, the terminal G4 units are roughly as stable as the inner G4 units; while in a (GGGTTA)4m-1GGG tandem context, the G4 at the 5' extremity is more stable than the G4 at the 3' extremity, which in turn is more stable than an inner G4. Our study provides new information about the global and local stability of telomeric tandem G4 structures under near physiological conditions. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  9. An assay for human telomeric G-quadruplex DNA binding drugs

    Science.gov (United States)

    Watkins, Derrick; Ranjan, Nihar; Kumar, Sunil; Gong, Changjun; Arya, Dev P.

    2014-01-01

    Compounds that stabilize the G-quadruplexes formed by human telomeres can inhibit the telomerase activity and are potential cancer therapies. We have developed an assay for the screening of compounds with high affinity for human telomeric G-quadruplexes (HTG). The assay uses a thiazole orange fluorescent reporter molecule conjugated to the aminoglycoside, neomycin, as a probe in a fluorescence displacement assay. The conjugation of the planar base stacking thiazole orange with the groove binding neomycin results in high affinity probe that can determine the relative binding affinity of high affinity HTG binding drugs in a high throughput format. The robust assay is applicable for the determination of the binding affinity of HTG in the presence of K+ or Na+. PMID:24246738

  10. Stable G-quadruplex structure in a hydrated ion pair: cholinium cation and dihydrogen phosphate anion.

    Science.gov (United States)

    Fujita, Kyoko; Ohno, Hiroyuki

    2012-06-11

    A combination of cholinium cations and dihydrogen phosphate anions is indispensable for the G-quadruplex structure regardless of water content. No G-quadruplex structure was observed in most salts even containing either cholinium cations or dihydrogen phosphate anions.

  11. Coarse-Grained Simulations Complemented by Atomistic Molecular Dynamics Provide New Insights into Folding and Unfolding of Human Telomeric G-Quadruplexes.

    Science.gov (United States)

    Stadlbauer, Petr; Mazzanti, Liuba; Cragnolini, Tristan; Wales, David J; Derreumaux, Philippe; Pasquali, Samuela; Šponer, Jiří

    2016-12-13

    G-quadruplexes are the most important noncanonical DNA architectures. Many quadruplex-forming sequences, including the human telomeric sequence d(GGGTTA)n, have been investigated due to their implications in cancer and other diseases, and because of their potential in DNA-based nanotechnology. Despite the availability of atomistic structural studies of folded G-quadruplexes, their folding pathways remain mysterious, and mutually contradictory models of folding coexist in the literature. Recent experiments convincingly demonstrated that G-quadruplex folding often takes days to reach thermodynamic equilibrium. Based on atomistic simulations of diverse classes of intermediates in G-quadruplex folding, we have suggested that the folding is an extremely multipathway process combining a kinetic partitioning mechanism with conformational diffusion. However, complete G-quadruplex folding is far beyond the time scale of atomistic simulations. Here we use high-resolution coarse-grained simulations to investigate potential unfolding intermediates, whose structural dynamics are then further explored with all-atom simulations. This multiscale approach indicates how various pathways are interconnected in a complex network. Spontaneous conversions between different folds are observed. We demonstrate the inability of simple order parameters, such as radius of gyration or the number of native H-bonds, to describe the folding landscape of the G-quadruplexes. Our study also provides information relevant to further development of the coarse-grained force field.

  12. Selection of G-quadruplex folding topology with LNA-modified human telomeric sequences in K+ solution

    DEFF Research Database (Denmark)

    Pradhan, Devranjan; Hansen, Lykke H; Vester, Birte

    2011-01-01

    this problem by examining the impact of LNA (locked nucleic acid) modifications on the folding topology of the dimeric model system of the human telomere sequence. In solution, this DNA G-quadruplex forms a mixture of G-quadruplexes with antiparallel and parallel topologies. Using CD and NMR spectroscopies, we......G-rich nucleic acid oligomers can form G-quadruplexes built by G-tetrads stacked upon each other. Depending on the nucleotide sequence, G-quadruplexes fold mainly with two topologies: parallel, in which all G-tracts are oriented parallel to each other, or antiparallel, in which one or more G......-tracts are oriented antiparallel to the other G-tracts. In the former topology, all glycosidic bond angles conform to anti conformations, while in the latter topology they adopt both syn and anti conformations. It is of interest to understand the molecular forces that govern G-quadruplex folding. Here, we approach...

  13. Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes

    Science.gov (United States)

    Bončina, Matjaž; Podlipnik, Črtomir; Piantanida, Ivo; Eilmes, Julita; Teulade-Fichou, Marie-Paule; Vesnaver, Gorazd; Lah, Jurij

    2015-01-01

    Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolinium ligands (Phen-DC3, 360A-Br) to the ht-DNA fragment (Tel22) AGGG(TTAGGG)3 using isothermal titration calorimetry, CD and fluorescence spectroscopy, gel electrophoresis and molecular modeling. By global thermodynamic analysis of experimental data we show that the driving forces characterized by contributions of specific interactions, changes in solvation and conformation differ significantly for binding of ligands with low quadruplex selectivity over duplexes (Net, DP77, DP78, TMPyP4; KTel22 ≈ KdsDNA). These contributions are in accordance with the observed structural features (changes) and suggest that upon binding Net, DP77, DP78 and TMPyP4 select hybrid-1 and/or hybrid-2 conformation while Phen-DC3 and 360A-Br induce the transition of hybrid-1 and hybrid-2 to the structure with characteristics of antiparallel or hybrid-3 type conformation. PMID:26546516

  14. Single-molecule analysis of thymine dimer-containing G-quadruplexes formed from the human telomere sequence.

    Science.gov (United States)

    Wolna, Anna H; Fleming, Aaron M; Burrows, Cynthia J

    2014-12-09

    The human telomere plays crucial roles in maintaining genome stability. In the presence of suitable cations, the repetitive 5'-TTAGGG-3' human telomere sequence can fold into G-quadruplexes that adopt the hybrid, basket, or propeller fold. The telomere sequence is hypersensitive to UV-induced thymine dimer (T=T) formation, yet it does not cause telomere shortening. In this work, the potential structural disruption and thermodynamic stability of the T=T-containing natural telomere sequences were studied to understand why this damage is tolerated in telomeres. First, established methods, such as thermal melting measurements, electrophoretic mobility shift assays, and circular dichroism spectroscopy, were utilized to determine the effects of the damage on these structures. Second, a single-molecule ion channel recording technique using α-hemolysin (α-HL) was employed to examine further the structural differences between the damaged sequences. It was observed that the damage caused slightly lower thermal stabilities and subtle changes in the circular dichroism spectra for hybrid and basket folds. The α-HL experiments determined that T=Ts disrupt double-chain reversal loop formation but are tolerated in edgewise and diagonal loops. The largest change was observed for the T=T-containing natural telomere sequence when the propeller fold (all double-chain reversal loops) was studied. On the basis of the α-HL experiments, it was determined that a triplexlike structure exists under conditions that favor a propeller structure. The biological significance of these observations is discussed.

  15. Effects of a halogenated G-quadruplex ligand from the pyridine dicarboxamide series on the terminal sequence of XpYp telomere in HT1080 cells.

    Science.gov (United States)

    Sidibe, Assitan; Hamon, Florian; Largy, Eric; Gomez, Dennis; Teulade-Fichou, Marie-Paule; Trentesaux, Chantal; Riou, Jean-François

    2012-12-01

    Non-canonical four-stranded structures called G-quadruplexes can form among telomere repeats during its replication. Small molecule ligands able to interact and to stabilize G-quadruplexes were shown to disrupt the binding of essential telomeric components, such as POT1 and to trigger a telomeric dysfunction associated with a delayed growth arrest in tumor cells. We describe here the chemical synthesis and the G-quadruplex binding properties of three halogenated analogs of the 360A ligand that belongs to the 2,6 pyridine dicarboxamide series. 360A is now commonly used as a benchmark both for biophysical and cellular assays as this compound was shown to display a potent affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA and to induce delayed growth inhibition in HT1080 tumor cell line. Two biophysical assays indicate that, in most cases, the presence of the halogen atom seems to slightly improve the interaction with the telomeric quadruplex. For stability reasons, the bromo derivative (360A-Br) was selected for the cellular assays. Since POT1 participates to the fine tuning of the C-strand end resection during telomere replication, we investigated the effect of 360A-Br to alter the terminal nucleotide composition of XpYp telomere in HT1080 cells using C-STELA. HT1080 cells treated for up to 24 days with 360A-Br presented some minor but significant variations of C-strand terminal nucleotide composition, also observed with a partial siRNA depletion of POT1. The relevance of these minor modifications of the telomeric C-strand resection induced by 360A-Br in HT1080 cells are discussed. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  16. Plastic roles of phenylalanine and tyrosine residues of TLS/FUS in complex formation with the G-quadruplexes of telomeric DNA and TERRA.

    Science.gov (United States)

    Kondo, Keiko; Mashima, Tsukasa; Oyoshi, Takanori; Yagi, Ryota; Kurokawa, Riki; Kobayashi, Naohiro; Nagata, Takashi; Katahira, Masato

    2018-02-12

    The length of a telomere is regulated via elongation and shortening processes. Telomeric DNA and telomeric repeat-containing RNA (TERRA), which both contain G-rich repeated sequences, form G-quadruplex structures. Previously, translocated in liposarcoma (TLS) protein, also known as fused in sarcoma (FUS) protein, was found to form a ternary complex with the G-quadruplex structures of telomeric DNA and TERRA. We then showed that the third RGG motif of TLS, the RGG3 domain, is responsible for the complex formation. However, the structural basis for their binding remains obscure. Here, NMR-based binding assaying revealed the interactions in the binary and ternary complexes of RGG3 with telomeric DNA or/and TERRA. In the ternary complex, tyrosine bound exclusively to TERRA, while phenylalanine bound exclusively to telomeric DNA. Thus, tyrosine and phenylalanine each play a central role in the recognition of TERRA and telomeric DNA, respectively. Surprisingly in the binary complexes, RGG3 used both tyrosine and phenylalanine residues to bind to either TERRA or telomeric DNA. We propose that the plastic roles of tyrosine and phenylalanine are important for RGG3 to efficiently form the ternary complex, and thereby regulate the telomere shortening.

  17. Atomistic picture for the folding pathway of a hybrid-1 type human telomeric DNA G-quadruplex.

    Directory of Open Access Journals (Sweden)

    Yunqiang Bian

    2014-04-01

    Full Text Available In this work we studied the folding process of the hybrid-1 type human telomeric DNA G-quadruplex with solvent and K(+ ions explicitly modeled. Enabled by the powerful bias-exchange metadynamics and large-scale conventional molecular dynamic simulations, the free energy landscape of this G-DNA was obtained for the first time and four folding intermediates were identified, including a triplex and a basically formed quadruplex. The simulations also provided atomistic pictures for the structures and cation binding patterns of the intermediates. The results showed that the structure formation and cation binding are cooperative and mutually supporting each other. The syn/anti reorientation dynamics of the intermediates was also investigated. It was found that the nucleotides usually take correct syn/anti configurations when they form native and stable hydrogen bonds with the others, while fluctuating between two configurations when they do not. Misfolded intermediates with wrong syn/anti configurations were observed in the early intermediates but not in the later ones. Based on the simulations, we also discussed the roles of the non-native interactions. Besides, the formation process of the parallel conformation in the first two G-repeats and the associated reversal loop were studied. Based on the above results, we proposed a folding pathway for the hybrid-1 type G-quadruplex with atomistic details, which is new and more complete compared with previous ones. The knowledge gained for this type of G-DNA may provide a general insight for the folding of the other G-quadruplexes.

  18. G-quadruplex secondary structure from circular dichroism spectroscopy.

    Science.gov (United States)

    Del Villar-Guerra, Rafael; Trent, John O; Chaires, Jonathan B

    2017-10-26

    A curated library of circular dichroism spectra of 23 G-quadruplexes of known structure was built and analyzed. The goal of this study was to use this reference library to develop an algorithm to derive quantitative estimates of the secondary structure content of quadruplexes from their experimental CD spectra. Principal component analysis and singular value decomposition were used to characterize the reference spectral library. CD spectra were successfully fit to obtain estimates of the amounts of base steps in anti-anti, syn-anti or anti-syn conformations, in diagonal or lateral loops or in other conformations. The results show that CD spectra of nucleic acids can be analyzed to obtain quantitative structural information about secondary structure content in an analogous way to methods used to analyze protein CD spectra. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. A multi-functional guanine derivative for studying the DNA G-quadruplex structure.

    Science.gov (United States)

    Ishizuka, Takumi; Zhao, Pei-Yan; Bao, Hong-Liang; Xu, Yan

    2017-10-23

    In the present study, we developed a multi-functional guanine derivative, 8FG, as a G-quadruplex stabilizer, a fluorescent probe for the detection of G-quadruplex formation, and a 19F sensor for the observation of the G-quadruplex. We demonstrate that the functional nucleoside bearing a 3,5-bis(trifluoromethyl)benzene group at the 8-position of guanine stabilizes the DNA G-quadruplex structure and fluoresces following the G-quadruplex formation. Furthermore, we show that the functional sensor can be used to directly observe DNA G-quadruplexes by 19F-NMR in living cells. To our knowledge, this is the first study showing that the nucleoside derivative simultaneously allows for three kinds of functions at a single G-quadruplex DNA. Our results suggest that the multi-functional nucleoside derivative can be broadly used for studying the G-quadruplex structure and serves as a powerful tool for examining the molecular basis of G-quadruplex formation in vitro and in living cells.

  20. Spectroscopic investigation on the interaction of copper porphyrazines and phthalocyanine with human telomeric G-quadruplex DNA.

    Science.gov (United States)

    Hassani, Leila; Hakimian, Fatemeh; Safaei, Elham

    2014-01-01

    The G-quadruplex DNA is a novel target for anticancer drug discovery and many scientific groups are investigating interaction of small molecules with G-quadruplex DNA to discover therapeutic agents for cancer. Here, interaction of a phthalocyanine (Cu(PcTs)) and two tetrapyridinoporphyrazines ([Cu(2,3-tmtppa)](4+) and [Cu(3,4-tmtppa)](4+)) with Na(+) and K(+) forms of human telomeric G-quadruplex DNA has been investigated by spectroscopic techniques. The results indicated that interaction of the cationic porphyrazines is remarkably stronger than the anionic phthalocyanine and they presumably bind to the G-quadruplex DNA through end-stacking. Fluorescent intercalator displacement assay implied the displacement ability of the complexes with thiazole orange. In addition, circular dichroism spectra of both quadruplex forms converge to the Na(+) isoform after binding to the porphyrazines. In conclusion, the porphyrazines as the complexes that bind to the G-quadruplex DNA, could be suitable candidates for further investigations about inhibition of telomerase enzyme. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Synthesis and structural characterization of stable branched DNA g-quadruplexes using the trebler phosphoramidite.

    Science.gov (United States)

    Ferreira, Rubén; Alvira, Margarita; Aviñó, Anna; Gómez-Pinto, Irene; González, Carlos; Gabelica, Valérie; Eritja, Ramon

    2012-04-01

    Guanine (G)-rich sequences can form a noncanonical four-stranded structure known as the G-quadruplex. G-quadruplex structures are interesting because of their potential biological properties and use in nanosciences. Here, we describe a method to prepare highly stable G-quadruplexes by linking four G-rich DNA strands to form a monomolecular G-quadruplex. In this method, one strand is synthesized first, and then a trebler molecule is added to simultaneously assemble the remaining three strands. This approach allows the introduction of specific modifications in only one of the strands. As a proof of concept, we prepared a quadruplex where one of the chains includes a change in polarity. A hybrid quadruplex is observed in ammonium acetate solutions, whereas in the presence of sodium or potassium, a parallel G-quadruplex structure is formed. In addition to the expected monomolecular quadruplexes, we observed the presence of dimeric G-quadruplex structures. We also applied the method to prepare G-quadruplexes containing a single 8-aminoguanine substitution and found that this single base stabilizes the G-quadruplex structure when located at an internal position.

  2. Confined space facilitates G-quadruplex formation

    Science.gov (United States)

    Shrestha, Prakash; Jonchhe, Sagun; Emura, Tomoko; Hidaka, Kumi; Endo, Masayuki; Sugiyama, Hiroshi; Mao, Hanbin

    2017-07-01

    Molecular simulations suggest that the stability of a folded macromolecule increases in a confined space due to entropic effects. However, due to the interactions between the confined molecular structure and the walls of the container, clear-cut experimental evidence for this prediction is lacking. Here, using DNA origami nanocages, we show the pure effect of confined space on the property of individual human telomeric DNA G-quadruplexes. We induce targeted mechanical unfolding of the G-quadruplex while leaving the nanocage unperturbed. We find that the mechanical and thermodynamic stabilities of the G-quadruplex inside the nanocage increase with decreasing cage size. Compared to the case of diluted or molecularly crowded buffer solutions, the G-quadruplex inside the nanocage is significantly more stable, showing a 100 times faster folding rate. Our findings suggest the possibility of co-replicational or co-transcriptional folding of G-quadruplex inside the polymerase machinery in cells.

  3. Discovery of selective ligands for telomeric RNA G-quadruplexes (TERRA) through 19F-NMR based fragment screening.

    Science.gov (United States)

    Garavís, Miguel; López-Méndez, Blanca; Somoza, Alvaro; Oyarzabal, Julen; Dalvit, Claudio; Villasante, Alfredo; Campos-Olivas, Ramón; González, Carlos

    2014-07-18

    Telomeric repeat-containing RNA (TERRA) is a novel and very attractive antitumoral target. Here, we report the first successful application of (19)F-NMR fragment-based screening to identify chemically diverse compounds that bind to an RNA molecule such as TERRA. We have built a library of 355 fluorinated fragments, and checked their interaction with a long telomeric RNA as a target molecule. The screening resulted in the identification of 20 hits (hit rate of 5.6%). For a number of binders, their interaction with TERRA was confirmed by (19)F- and (1)H NMR as well as by CD melting experiments. We have also explored the selectivity of the ligands for RNA G-quadruplexes and found that some of the hits do not interact with other nucleic acids such as tRNA and duplex DNA and, most importantly, favor the propeller-like parallel conformation in telomeric DNA G-quadruplexes. This suggests a selective recognition of this particular quadruplex topology and that different ligands may recognize specific sites in propeller-like parallel G-quadruplexes. Such features make some of the resulting binders promising lead compounds for fragment based drug discovery.

  4. Effect of pressure on thermal stability of g-quadruplex DNA and double-stranded DNA structures.

    Science.gov (United States)

    Takahashi, Shuntaro; Sugimoto, Naoki

    2013-10-29

    Pressure is a thermodynamic parameter that can induce structural changes in biomolecules due to a volumetric decrease. Although most proteins are denatured by pressure over 100 MPa because they have the large cavities inside their structures, the double-stranded structure of DNA is stabilized or destabilized only marginally depending on the sequence and salt conditions. The thermal stability of the G-quadruplex DNA structure, an important non-canonical structure that likely impacts gene expression in cells, remarkably decreases with increasing pressure. Volumetric analysis revealed that human telomeric DNA changed by more than 50 cm3 mol-1 during the transition from a random coil to a quadruplex form. This value is approximately ten times larger than that for duplex DNA under similar conditions. The volumetric analysis also suggested that the formation of G-quadruplex DNA involves significant hydration changes. The presence of a cosolute such as poly(ethylene glycol) largely repressed the pressure effect on the stability of G-quadruplex due to alteration in stabilities of the interactions with hydrating water. This review discusses the importance of local perturbations of pressure on DNA structures involved in regulation of gene expression and highlights the potential for application of high-pressure chemistry in nucleic acid-based nanotechnology.

  5. Effect of Pressure on Thermal Stability of G-Quadruplex DNA and Double-Stranded DNA Structures

    Directory of Open Access Journals (Sweden)

    Shuntaro Takahashi

    2013-10-01

    Full Text Available Pressure is a thermodynamic parameter that can induce structural changes in biomolecules due to a volumetric decrease. Although most proteins are denatured by pressure over 100 MPa because they have the large cavities inside their structures, the double-stranded structure of DNA is stabilized or destabilized only marginally depending on the sequence and salt conditions. The thermal stability of the G-quadruplex DNA structure, an important non-canonical structure that likely impacts gene expression in cells, remarkably decreases with increasing pressure. Volumetric analysis revealed that human telomeric DNA changed by more than 50 cm3 mol−1 during the transition from a random coil to a quadruplex form. This value is approximately ten times larger than that for duplex DNA under similar conditions. The volumetric analysis also suggested that the formation of G-quadruplex DNA involves significant hydration changes. The presence of a cosolute such as poly(ethylene glycol largely repressed the pressure effect on the stability of G-quadruplex due to alteration in stabilities of the interactions with hydrating water. This review discusses the importance of local perturbations of pressure on DNA structures involved in regulation of gene expression and highlights the potential for application of high-pressure chemistry in nucleic acid-based nanotechnology.

  6. Complicated behavior of G-quadruplexes and evaluating G-quadruplexes' ligands in various systems mimicking cellular circumstance

    Directory of Open Access Journals (Sweden)

    Shi-Ke Wang

    2016-03-01

    Full Text Available Environments surrounding G-rich sequences remarkably affect the conformations of these structures. A proper evaluation system mimicking the crowded environment in a cell with macromolecules should be developed to perform structural and functional studies on G-quadruplexes. In this study, the topology and stability of a G-quadruplex formed by human telomeric repeat sequences were investigated in a macromolecule-crowded environment created by polyethylene glycol 200 (PEG200, tumor cell extract, and Xenopus laevis egg extract. The interactions between small molecules and telomeric G-quadruplexes were also evaluated in the different systems. The results suggested that the actual behavior of G-quadruplex structures in cells extract is quite different from that in the PEG crowding system, and proteins or other factors in extracts might play a very important role in G-quadruplex structures.

  7. Interactions of DNA binding proteins with G-Quadruplex structures at the single molecule level

    Science.gov (United States)

    Ray, Sujay

    Guanine-rich nucleic acid (DNA/RNA) sequences can form non-canonical secondary structures, known as G-quadruplex (GQ). Numerous in vivo and in vitro studies have demonstrated formation of these structures in telomeric and non-telomeric regions of the genome. Telomeric GQs protect the chromosome ends whereas non-telomeric GQs either act as road blocks or recognition sites for DNA metabolic machinery. These observations suggest the significance of these structures in regulation of different metabolic processes, such as replication and repair. GQs are typically thermodynamically more stable than the corresponding Watson-Crick base pairing formed by G-rich and C-rich strands, making protein activity a crucial factor for their destabilization. Inside the cell, GQs interact with different proteins and their enzymatic activity is the determining factor for their stability. We studied interactions of several proteins with GQs to understand the underlying principles of protein-GQ interactions using single-molecule FRET and other biophysical techniques. Replication Protein-A (RPA), a single stranded DNA (ssDNA) binding protein, is known to posses GQ unfolding activity. First, we compared the thermal stability of three potentially GQ-forming DNA sequences (PQS) to their stability against RPA-mediated unfolding. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQS in the genome. The thermal stability of these structures do not necessarily correlate with their stability against protein-mediated unfolding. We conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. To determine the critical structural factors that influence protein-GQ interactions we studied two groups of GQ structures that have systematically varying loop lengths and number of G-tetrad layers. We

  8. Targeting G-quadruplex DNA Structures by EMICORON has a strong antitumor efficacy against advanced models of human colon cancer

    DEFF Research Database (Denmark)

    Porru, Manuela; Artuso, Simona; Salvati, Erica

    2015-01-01

    We previously identified EMICORON as a novel G-quadruplex (G4) ligand showing high selectivity for G4 structures over the duplex DNA, causing telomere damage and inhibition of cell proliferation in transformed and tumor cells. Here, we evaluated the antitumoral effect of EMICORON on advanced models...... of DNA damage and impairment of proliferation and angiogenesis are proved to be key determinants of EMICORON antitumoral activity. Altogether, our results, performed on advanced experimental models of human colon cancer that bridge the translational gap between preclinical and clinical studies...

  9. Identification of New Natural DNA G-Quadruplex Binders Selected by a Structure-Based Virtual Screening Approach

    Directory of Open Access Journals (Sweden)

    Stefano Alcaro

    2013-09-01

    Full Text Available The G-quadruplex DNA structures are mainly present at the terminal portion of telomeres and can be stabilized by ligands able to recognize them in a specific manner. The recognition process is usually related to the inhibition of the enzyme telomerase indirectly involved and over-expressed in a high percentage of human tumors. There are several ligands, characterized by different chemical structures, already reported in the literature for their ability to bind and stabilize the G-quadruplex structures. Using the structural and biological information available on these structures; we performed a high throughput in silico screening of commercially natural compounds databases by means of a structure-based approach followed by docking experiments against the human telomeric sequence d[AG3(T2AG33]. We identified 12 best hits characterized by different chemical scaffolds and conformational and physicochemical properties. All of them were associated to an improved theoretical binding affinity with respect to that of known selective G-binders. Among these hits there is a chalcone derivative; structurally very similar to the polyphenol butein; known to remarkably inhibit the telomerase activity.

  10. Not All G-Quadruplexes are Created Equally: An Investigation of the Structural Polymorphism of the c-Myc G-Quadruplex-Forming Sequence and its Interaction with the Porphyrin meso-Tetra(N-methyl-4-pyridyl)porphine

    Science.gov (United States)

    Le, Huy T.; Miller, M. Clarke; Buscaglia, Robert; Dean, William L.; Holt, Patrick A.; Chaires, Jonathan B.; Trent, John O.

    2012-01-01

    G-quadruplexes, DNA tertiary structures highly localized to functionally important sites within the human genome, have emerged as important new drug targets. The putative G-quadruplex-forming sequence (Pu27) in the NHE-III1 promoter region of the c-Myc gene is of particular interest as stabilization of this G-quadruplex with TMPyP4 has been shown to repress c-Myc transcription. In this study, we examine the Pu27 G-quadruplex-forming sequence and its interaction with TMPyP4. We report that the Pu27 sequence exists as a heterogeneous mixture of monomeric and higher-order G-quadruplex species in vitro and that this mixture can be partially resolved by size exclusion chromatography (SEC) separation. Within this ensemble of configurations, the equilibrium can be altered by modifying the buffer composition, annealing procedure, and dialysis protocol thereby affecting the distribution of G-quadruplex species formed. TMPyP4 was found to bind preferentially to higher-order G-quadruplex species suggesting the possibility of stabilization of the junctions of the c-Myc G-quadruplex multimers by porphyrin end-stacking. We also examined four modified c-Myc sequences that have been previously reported and found a narrower distribution of quadruplex configurations compared to the parent Pu27 sequence. We could not definitively conclude whether these G-quadruplex structures were selected from the original ensemble or if they are new G-quadruplex structures. Since these sequences differ considerably from the wild-type promoter sequence, it is unclear whether their structures have any actual biological relevance. Additional studies are needed to examine how the polymorphic nature of G-quadruplexes affects the interpretation of in vitro data for c-Myc and other G-quadruplexes. The findings reported here demonstrate that experimental conditions contribute significantly to G-quadruplex formation and should be carefully considered, controlled, and reported in detail. PMID:23108607

  11. Aminoglycosylation can enhance the G-quadruplex binding activity of epigallocatechin.

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    Li-Ping Bai

    Full Text Available With the aim of enhancing G-quadruplex binding activity, two new glucosaminosides (16, 18 of penta-methylated epigallocatechin were synthesized by chemical glycosylation. Subsequent ESI-TOF-MS analysis demonstrated that these two glucosaminoside derivatives exhibit much stronger binding activity to human telomeric DNA and RNA G-quadruplexes than their parent structure (i.e., methylated EGC (14 as well as natural epigallocatechin (EGC, 6. The DNA G-quadruplex binding activity of 16 and 18 is even more potent than strong G-quadruplex binder quercetin, which has a more planar structure. These two synthetic compounds also showed a higher binding strength to human telomeric RNA G-quadruplex than its DNA counterpart. Analysis of the structure-activity relationship revealed that the more basic compound, 16, has a higher binding capacity with DNA and RNA G-quadruplexes than its N-acetyl derivative, 18, suggesting the importance of the basicity of the aminoglycoside for G-quadruplex binding activity. Molecular docking simulation predicted that the aromatic ring of 16 π-stacks with the aromatic ring of guanine nucleotides, with the glucosamine moiety residing in the groove of G-quadruplex. This research indicates that glycosylation of natural products with aminosugar can significantly enhance their G-quadruplex binding activities, thus is an effective way to generate small molecules targeting G-quadruplexes in nucleic acids. In addition, this is the first report that green tea catechin can bind to nucleic acid G-quadruplex structures.

  12. Xanthene and Xanthone Derivatives as G-Quadruplex Stabilizing Ligands

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    Alessandro Altieri

    2013-10-01

    Full Text Available Following previous studies on anthraquinone and acridine-based G-quadruplex ligands, here we present a study of similar aromatic cores, with the specific aim of increasing G-quadruplex binding and selectivity with respect to duplex DNA. Synthesized compounds include two and three-side chain xanthone and xanthene derivatives, as well as a dimeric “bridged” form. ESI and FRET measurements suggest that all the studied molecules are good G-quadruplex ligands, both at telomeres and on G-quadruplex forming sequences of oncogene promoters. The dimeric compound and the three-side chain xanthone derivative have been shown to represent the best compounds emerging from the different series of ligands presented here, having also high selectivity for G-quadruplex structures with respect to duplex DNA. Molecular modeling simulations are in broad agreement with the experimental data.

  13. Mechanistic insight into the interaction of BLM helicase with intra-strand G-quadruplex structures

    DEFF Research Database (Denmark)

    Chatterjee, Sujoy; Zagelbaum, Jennifer; Savitsky, Pavel

    2014-01-01

    Bloom syndrome is an autosomal recessive disorder caused by mutations in the RecQ family helicase BLM that is associated with growth retardation and predisposition to cancer. BLM helicase has a high specificity for non-canonical G-quadruplex (G4) DNA structures, which are formed by G-rich DNA...

  14. G-quadruplex structure of an anti-proliferative DNA sequence.

    Science.gov (United States)

    Do, Ngoc Quang; Chung, Wan Jun; Truong, Thi Hong Anh; Heddi, Brahim; Phan, Anh Tuân

    2017-07-07

    AGRO100 (also known as AS1411) is a G-rich oligonucleotide that has long been established as a potent anti-cancer aptamer. However, the structure of AGRO100 remained unresolved, due to the co-existence of multiple different G-quadruplex conformations. We identified a DNA sequence named AT11, derived from AGRO100, which formed a single major G-quadruplex conformation and exhibited a similar anti-proliferative activity as AGRO100. The solution structure of AT11 revealed a four-layer G-quadruplex comprising of two propeller-type parallel-stranded subunits connected through a central linker. The stacking between the two subunits occurs at the 3΄-end of the first block and the 5΄-end of the second block. The structure of the anti-proliferative DNA sequence AT11 will allow greater understanding on the G-quadruplex folding principles and aid in structural optimization of anti-proliferative oligonucleotides. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Structural and Affinity Analyses of G-Quadruplex DNA Aptamers for Camptothecin Derivatives

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    Naoki Sugimoto

    2013-08-01

    Full Text Available We recently selected DNA aptamers that bind to camptothecin (CPT and CPT derivatives from a 70-mer oligodeoxyribonucleotide (ODN library using the Systematic Evolution of Ligands by EXponential enrichment (SELEX method. The target-binding activity of the obtained 70-mer CPT-binding DNA aptamer, termed CA-70, which contains a 16-mer guanine (G-core motif (G3TG3TG3T2G3 that forms a three-tiered G-quadruplex, was determined using fluorescence titration. In this study, truncated fragments of CA-70 that all have the G-core motif, CA-40, -20, -19, -18A, -18B, -17, and -16, were carefully analyzed. We found that CA-40 retained the target-binding activity, whereas CA-20, -19, and -18B exhibited little or no binding activities. Further, not only CA-18A but also the shorter length fragments CA-17 and -16 clearly retained the binding activity, indicating that tail strands of the G-quadruplex structure can significantly affect the target binding of G-quadruplex DNA aptamers. Further analyses using circular dichroism (CD spectroscopy and fluorescence polarization (FP assay were conducted to investigate the structure and affinity of G-quadruplex DNA aptamers.

  16. Toward the design of a catalytic metallodrug: selective cleavage of G-quadruplex telomeric DNA by an anticancer copper-acridine-ATCUN complex.

    Science.gov (United States)

    Yu, Zhen; Han, Menglu; Cowan, James A

    2015-02-02

    Telomeric DNA represents a novel target for the development of anticancer drugs. By application of a catalytic metallodrug strategy, a copper-acridine-ATCUN complex (CuGGHK-Acr) has been designed that targets G-quadruplex telomeric DNA. Both fluorescence solution assays and gel sequencing demonstrate the CuGGHK-Acr catalyst to selectively bind and cleave the G-quadruplex telomere sequence. The cleavage pathway has been mapped by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) experiments. CuGGHK-Acr promotes significant inhibition of cancer cell proliferation and shortening of telomere length. Both senescence and apoptosis are induced in the breast cancer cell line MCF7. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. The nickel(II) complex of guanidinium phenyl porphyrin, a specific G-quadruplex ligand, targets telomeres and leads to POT1 mislocalization in culture cells.

    Science.gov (United States)

    Sabater, Laurent; Nicolau-Travers, Marie-Laure; De Rache, Aurore; Prado, Enora; Dejeu, Jérôme; Bombarde, Oriane; Lacroix, Joris; Calsou, Patrick; Defrancq, Eric; Mergny, Jean-Louis; Gomez, Dennis; Pratviel, Geneviève

    2015-06-01

    With the aim of finding selective and biologically active G-quadruplex ligands, modified porphyrin with bulky cationic substituents, meso-5,10,15,20-tetrakis(4-guanidinophenyl)porphyrin tetrahydrochloride, referred to as guanidinium phenyl porphyrin, was prepared. The corresponding nickel(II) and cobalt(III) metallated porphyrins were also synthesized. Interaction with quadruplexes was examined by means of fluorescence resonance energy transfer melting and surface plasmon resonance-based assays: the three compounds proved to bind to G-quadruplex DNA in a similar and highly selective way. Guanidinium phenyl porphyrin and its nickel(II) metallated derivative exhibit moderate cytotoxicity toward cells in culture. Strikingly, the nickel porphyrin derivative was able to displace hPOT1 shelterin protein from telomeres in human cells. Nickel(II) guanidinium phenyl porphyrin, a cationic bulky porphyrin is a powerful specific G-quadruplex DNA ligand. It enters the cells and induces shelterin modification.

  18. A G-quadruplex structure within the 5'-UTR of TRF2 mRNA represses translation in human cells.

    Science.gov (United States)

    Gomez, Dennis; Guédin, Aurore; Mergny, Jean-Louis; Salles, Bernard; Riou, Jean-François; Teulade-Fichou, Marie-Paule; Calsou, Patrick

    2010-11-01

    Telomeres protect chromosome ends from being recognized as double-stranded breaks. Telomeric function is ensured by the shelterin complex in which TRF2 protein is an essential player. The G-rich strand of telomere DNA can fold into G-quadruplex (G4) structure. Small molecules stabilizing G4 structures, named G4 ligands, have been shown to alter telomeric functions in human cells. In this study, we show that a guanine-rich RNA sequence located in the 5'-UTR region of the TRF2 mRNA (hereafter 91TRF2G) is capable of forming a stable quadruplex that causes a 2.8-fold decrease in the translation of a reporter gene in human cells, as compared to a mutant 5'-UTR unable to fold into G4. We also demonstrate that several highly selective G4 ligands, the pyridine dicarboxamide derivative 360A and bisquinolinium compounds Phen-DC(3) and Phen-DC(6), are able to bind the 91TRF2G:RNA sequence and to modulate TRF2 protein translation in vitro. Since the naturally occurring 5'-UTR TRF2:RNA G4 element was used here, which is conserved in several vertebrate orthologs, the present data substantiate a potential translational mechanism mediated by a G4 RNA motif for the downregulation of TRF2 expression.

  19. Identification of G-quadruplex DNA/RNA binders: Structure-based virtual screening and biophysical characterization.

    Science.gov (United States)

    Rocca, Roberta; Moraca, Federica; Costa, Giosuè; Nadai, Matteo; Scalabrin, Matteo; Talarico, Carmine; Distinto, Simona; Maccioni, Elias; Ortuso, Francesco; Artese, Anna; Alcaro, Stefano; Richter, Sara N

    2017-05-01

    Recent findings demonstrated that, in mammalian cells, telomere DNA (Tel) is transcribed into telomeric repeat-containing RNA (TERRA), which is involved in fundamental biological processes, thus representing a promising anticancer target. For this reason, the discovery of dual (as well as selective) Tel/TERRA G-quadruplex (G4) binders could represent an innovative strategy to enhance telomerase inhibition. Initially, docking simulations of known Tel and TERRA active ligands were performed on the 3D coordinates of bimolecular G4 Tel DNA (Tel 2 ) and TERRA (TERRA 2 ). Structure-based pharmacophore models were generated on the best complexes and employed for the virtual screening of ~257,000 natural compounds. The 20 best candidates were submitted to biophysical assays, which included circular dichroism and mass spectrometry at different K + concentrations. Three hits were here identified and characterized by biophysical assays. Compound 7 acts as dual Tel 2 /TERRA 2 G4-ligand at physiological KCl concentration, while hits 15 and 17 show preferential thermal stabilization for Tel 2 DNA. The different molecular recognition against the two targets was also discussed. Our successful results pave the way to further lead optimization to achieve both increased selectivity and stabilizing effect against TERRA and Tel DNA G4s. The current study combines for the first time molecular modelling and biophysical assays applied to bimolecular DNA and RNA G4s, leading to the identification of innovative ligand chemical scaffolds with a promising anticancer profile. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Biochemical techniques for the characterization of G-quadruplex structures: EMSA, DMS footprinting, and DNA polymerase stop assay.

    Science.gov (United States)

    Sun, Daekyu; Hurley, Laurence H

    2010-01-01

    The proximal promoter region of many human growth-related genes contains a polypurine/polypyrimidine tract that serves as multiple binding sites for Sp1 or other transcription factors. These tracts often contain a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif known for the formation of an intramolecular G-quadruplex. Recent results provide strong evidence that specific G-quadruplex structures form naturally within these polypurine/polypyrimidine tracts in many human promoter regions, raising the possibility that the transcriptional control of these genes can be modulated by G-quadruplex-interactive agents. In this chapter, we describe three general biochemical methodologies, electrophoretic mobility shift assay (EMSA), dimethylsulfate (DMS) footprinting, and the DNA polymerase stop assay, which can be useful for initial characterization of G-quadruplex structures formed by G-rich sequences.

  1. Fragile X mental retardation protein recognition of G quadruplex structure per se is sufficient for high affinity binding to RNA.

    Science.gov (United States)

    Bole, Medhavi; Menon, Lakshmi; Mihailescu, Mihaela-Rita

    2008-12-01

    Fragile X syndrome, the most common form of inherited mental retardation is caused by the expansion of a CGG trinucleotide repeat in the fragile X mental retardation 1 (fmr1) gene. The abnormal expansion of the CGG repeat causes hypermethylation and subsequent silencing of the fmr1 gene, resulting in the loss of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine rich region (RGG box) to bind to messenger RNAs that form G quadruplex structures. Several studies reported that the G quadruplex RNA recognition alone is not sufficient for FMRP RGG box binding and that an additional stem and/or a G quadruplex-stem junction region may also be important in recognition. In this study we have used biophysical methods such as fluorescence, UV, CD and NMR spectroscopy to demonstrate that the recognition of the RNA G quadruplex structure per se, in the absence of a stem region, is sufficient for the FMRP high affinity and specific binding. These findings indicate that the presence of a stem structure in some of the FMRP G quadruplex forming mRNAs is not a requirement for protein recognition as previously believed, but rather for the proper formation of the correct RNA G quadruplex structure recognized by FMRP.

  2. Controlling the stoichiometry and strand polarity of a tetramolecular G-quadruplex structure by using a DNA origami frame

    Science.gov (United States)

    Rajendran, Arivazhagan; Endo, Masayuki; Hidaka, Kumi; Lan Thao Tran, Phong; Mergny, Jean-Louis; Sugiyama, Hiroshi

    2013-01-01

    Guanine-rich oligonucleotides often show a strong tendency to form supramolecular architecture, the so-called G-quadruplex structure. Because of the biological significance, it is now considered to be one of the most important conformations of DNA. Here, we describe the direct visualization and single-molecule analysis of the formation of a tetramolecular G-quadruplex in KCl solution. The conformational changes were carried out by incorporating two duplex DNAs, with G–G mismatch repeats in the middle, inside a DNA origami frame and monitoring the topology change of the strands. In the absence of KCl, incorporated duplexes had no interaction and laid parallel to each other. Addition of KCl induced the formation of a G-quadruplex structure by stably binding the duplexes to each other in the middle. Such a quadruplex formation allowed the DNA synapsis without disturbing the duplex regions of the participating sequences, and resulted in an X-shaped structure that was monitored by atomic force microscopy. Further, the G-quadruplex formation in KCl solution and its disruption in KCl-free buffer were analyzed in real-time. The orientation of the G-quadruplex is often difficult to control and investigate using traditional biochemical methods. However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex. PMID:23863846

  3. Sugar-modified G-quadruplexes: effects of LNA-, 2′F-RNA– and 2′F-ANA-guanosine chemistries on G-quadruplex structure and stability

    Science.gov (United States)

    Li, Zhe; Lech, Christopher Jacques; Phan, Anh Tuân

    2014-01-01

    G-quadruplex-forming oligonucleotides containing modified nucleotide chemistries have demonstrated promising pharmaceutical potential. In this work, we systematically investigate the effects of sugar-modified guanosines on the structure and stability of a (4+0) parallel and a (3+1) hybrid G-quadruplex using over 60 modified sequences containing a single-position substitution of 2′-O-4′-C-methylene-guanosine (LNAG), 2′-deoxy-2′-fluoro-riboguanosine (FG) or 2′-deoxy-2′-fluoro-arabinoguanosine (FANAG). Our results are summarized in two parts: (I) Generally, LNAG substitutions into ‘anti’ position guanines within a guanine-tetrad lead to a more stable G-quadruplex, while substitutions into ‘syn’ positions disrupt the native G-quadruplex conformation. However, some interesting exceptions to this trend are observed. We discover that a LNAG modification upstream of a short propeller loop hinders G-quadruplex formation. (II) A single substitution of either FG or FANAG into a ‘syn’ position is powerful enough to perturb the (3+1) G-quadruplex. Substitution of either FG or FANAG into any ‘anti’ position is well tolerated in the two G-quadruplex scaffolds. FANAG substitutions to ‘anti’ positions are better tolerated than their FG counterparts. In both scaffolds, FANAG substitutions to the central tetrad layer are observed to be the most stabilizing. The observations reported herein on the effects of LNAG, FG and FANAG modifications on G-quadruplex structure and stability will enable the future design of pharmaceutically relevant oligonucleotides. PMID:24371274

  4. G-quadruplex structures within the 3' UTR of LINE-1 elements stimulate retrotransposition.

    Science.gov (United States)

    Sahakyan, Aleksandr B; Murat, Pierre; Mayer, Clemens; Balasubramanian, Shankar

    2017-03-01

    Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in higher eukaryotes that have a significant role in shaping genomes, owing to their abundance. Here we report that guanine-rich sequences in the 3' untranslated regions (UTRs) of hominoid-specific LINE-1 elements are coupled with retrotransposon speciation and contribute to retrotransposition through the formation of G-quadruplex (G4) structures. We demonstrate that stabilization of the G4 motif of a human-specific LINE-1 element by small-molecule ligands stimulates retrotransposition.

  5. Tetrazolylpyrene unnatural nucleoside as a human telomeric multimeric G-quadruplex selective switch-on fluorescent sensor.

    Science.gov (United States)

    Bag, Subhendu Sekhar; Pradhan, Manoj Kumar; Talukdar, Sangita

    2017-12-13

    We report herein the specific sensing of dimeric H45 G-quadruplex DNA via a fluorescence light-up response using fluorescent tetrazolylpyrene nucleoside ( TzPy B Do ) as a probe. The strong binding of the probe via an intercalative stacking interaction inside the connecting loop of two G-quadruplex units of H45 and the discrimination to other monomeric and long DNA duplexes are accompanied by a drastic enhancement of the emission intensity without compromising the conformation and stability.

  6. Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome.

    Science.gov (United States)

    De Nicola, Beatrice; Lech, Christopher J; Heddi, Brahim; Regmi, Sagar; Frasson, Ilaria; Perrone, Rosalba; Richter, Sara N; Phan, Anh Tuân

    2016-07-27

    The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. General cell-binding activity of intramolecular G-quadruplexes with parallel structure.

    Science.gov (United States)

    Chang, Tianjun; Qi, Cui; Meng, Jie; Zhang, Nan; Bing, Tao; Yang, Xianda; Cao, Zehui; Shangguan, Dihua

    2013-01-01

    G-quadruplexes (G4s) are four-stranded nucleic acid structures adopted by some repetitive guanine-rich sequences. Putative G-quadruplex-forming sequences (PQSs) are highly prevalent in human genome. Recently some G4s have been reported to have cancer-selective antiproliferative activity. A G4 DNA, AS1411, is currently in phase II clinical trials as an anticancer agent, which is reported to bind tumor cells by targeting surface nucleolin. AS1411 also has been extensively investigated as a target-recognition element for cancer cell specific drug delivery or cancer cell imaging. Here we show that, in addition to AS1411, intramolecular G4s with parallel structure (including PQSs in genes) have general binding activity to many cell lines with different affinity. The binding of these G4s compete with each other, and their targets are certain cellular surface proteins. The tested G4s exhibit enhanced cellular uptake than non-G4 sequences. This uptake may be through the endosome/lysosome pathway, but it is independent of cellular binding of the G4s. The tested G4s also show selective antiproliferative activity that is independent of their cellular binding. Our findings provide new insight into the molecular recognition of G4s by cells; offer new clues for understanding the functions of G4s in vivo, and may extend the potential applications of G4s.

  8. General cell-binding activity of intramolecular G-quadruplexes with parallel structure.

    Directory of Open Access Journals (Sweden)

    Tianjun Chang

    Full Text Available G-quadruplexes (G4s are four-stranded nucleic acid structures adopted by some repetitive guanine-rich sequences. Putative G-quadruplex-forming sequences (PQSs are highly prevalent in human genome. Recently some G4s have been reported to have cancer-selective antiproliferative activity. A G4 DNA, AS1411, is currently in phase II clinical trials as an anticancer agent, which is reported to bind tumor cells by targeting surface nucleolin. AS1411 also has been extensively investigated as a target-recognition element for cancer cell specific drug delivery or cancer cell imaging. Here we show that, in addition to AS1411, intramolecular G4s with parallel structure (including PQSs in genes have general binding activity to many cell lines with different affinity. The binding of these G4s compete with each other, and their targets are certain cellular surface proteins. The tested G4s exhibit enhanced cellular uptake than non-G4 sequences. This uptake may be through the endosome/lysosome pathway, but it is independent of cellular binding of the G4s. The tested G4s also show selective antiproliferative activity that is independent of their cellular binding. Our findings provide new insight into the molecular recognition of G4s by cells; offer new clues for understanding the functions of G4s in vivo, and may extend the potential applications of G4s.

  9. Structural Analysis of G-Quadruplex Formation at the Human MEST Promoter.

    Directory of Open Access Journals (Sweden)

    Aaron J Stevens

    Full Text Available The promoter region of the imprinted gene MEST contains several motifs capable of forming G-quadruplex (G4 structures, which appear to contribute to consistent allelic dropout during polymerase chain reaction (PCR analysis of this region. Here, we extend our previous analysis of MEST G4 structures by applying fluorescent footprinting techniques to assess non B-DNA structure and topology in dsDNA from the full MEST promoter region, under conditions that mimic PCR. We demonstrate that the buffer used for PCR provides an extremely favourable milieu for G4 formation, and that cytosine methylation helps maintain G4 structures during PCR. Additionally, we demonstrate G4 formation at motifs not previously identified through bioinformatic analysis of the MEST promoter, and provide nucleotide level resolution for topological reconstruction of these structures. These observations increase our understanding of the mechanisms through which methylation and G4 contribute towards allelic drop-out during PCR.

  10. Structural and biophysical insight into dual site binding of the protoberberine alkaloid palmatine to parallel G-quadruplex DNA using NMR, fluorescence and circular dichroism spectroscopy.

    Science.gov (United States)

    Padmapriya, Kumar; Barthwal, Ritu

    2018-02-20

    Plant derived small molecules, which interact with and stabilize G-quadruplex DNA, act as inhibitors of telomere elongation and oncogene expression in humans. The inhibition of telomerase enzyme has immense potential since it is over expressed in most cancer cells. Interaction of palmatine, an antitumor alkaloid, to parallel G-quadruplex DNA, [d(TTGGGGT)] 4 and [d(TTAGGGT)] 4 , has been investigated using Nuclear Magnetic Resonance (NMR), fluorescence and Circular Dichroism (CD) spectroscopy. Titrations were monitored by recording 1 H and 31 P NMR spectra and solution structure of palmatine-[d(TTGGGGT)] 4 complex was obtained by restrained Molecular Dynamics (rMD) simulations using distance restraints from 2D NOESY spectra. Thermal stabilization of DNA was determined by CD, 1 H NMR and Differential Scanning Calorimetry (DSC). Binding of palmatine induces 98% enhancement of fluorescence accompanied by blue shift ∼8 nm. CD spectral bands of DNA show minor changes. Diffusion NMR studies confirm formation of a stable complex. Proton NMR signals of palmatine shift upfield upon binding and NOE cross peaks of H10, H3, H28, 5OCH 3 protons with T2, A3/G3, G6 and T7 residues reveal dual recognition sites in both G-quadruplex DNA sequences, resulting in thermal stabilization of G-quadruplex by ∼13-17 °C. Restrained molecular dynamics simulations using NOE distance restraints for 2:1 palmatine-[d(TTGGGGT)] 4 complex reveal end-stacking of palmatine at G6pT7 step and groove binding along T2pG3 step. Binding to [d(TTAGGGT)] 4 takes place at T2pA3pG4 and G6pT7 steps. Structural features of molecular recognition of two different G-quadruplex DNA sequences by palmatine have relevance in rational drug development for anti-cancer therapy. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  11. Use of alternative alkali chlorides in RT and PCR of polynucleotides containing G quadruplex structures.

    Science.gov (United States)

    Ramos-Alemán, Fabiola; González-Jasso, Eva; Pless, Reynaldo C

    2017-12-02

    Several alkali chlorides were compared for their use in reverse transcription (RT) and PCR of different types of nucleic acid templates. On a test region of biological DNA incapable of forming G quadruplex (G4) structures, Taq DNA polymerase showed similar PCR performance with 50 mM KCl, CsCl, LiCl, and NaCl. In contrast, on a synthetic model polydeoxyribonucleotide prone to G4 formation, good PCR amplification was obtained with 50 mM CsCl, but little or none with LiCl or KCl. Similarly, in RT of a G4-prone model polyribonucleotide, MMLV reverse transcriptase produced a good yield with 50 mM CsCl, mediocre yields with LiCl or without added alkali chloride, and a poor yield with 50 mM KCl. The full RT-PCR assay starting from the G4-prone polyribonucleotide, showed good results with CsCl in both stages, poor results with LiCl, and no product formation with KCl. The model polynucleotides showed fast G quadruplex formation under PCR or RT conditions with 50 mM KCl, but not with CsCl or LiCl. The results argue for the use of CsCl instead of KCl for RT and PCR of G4-prone sequences. No advantage was observed when using the 7-deaza type nucleotide analog c 7 dGTP in PCR amplification of the G4-prone polydeoxyribonucleotide. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. FMRP interacts with G-quadruplex structures in the 3'-UTR of its dendritic target Shank1 mRNA.

    Science.gov (United States)

    Zhang, Yang; Gaetano, Christian M; Williams, Kathryn R; Bassell, Gary J; Mihailescu, Mihaela Rita

    2014-01-01

    Fragile X syndrome (FXS), the most common cause of inherited intellectual disability, is caused by the loss of expression of the fragile X mental retardation protein (FMRP). FMRP, which regulates the transport and translation of specific mRNAs, uses its RGG box domain to bind mRNA targets that form G-quadruplex structures. One of the FMRP in vivo targets, Shank1 mRNA, encodes the master scaffold proteins of the postsynaptic density (PSD) which regulate the size and shape of dendritic spines because of their capacity to interact with many different PSD components. Due to their effect on spine morphology, altered translational regulation of Shank1 transcripts may contribute to the FXS pathology. We hypothesized that the FMRP interactions with Shank1 mRNA are mediated by the recognition of the G quadruplex structure, which has not been previously demonstrated. In this study we used biophysical techniques to analyze the Shank1 mRNA 3'-UTR and its interactions with FMRP and its phosphorylated mimic FMRP S500D. We found that the Shank1 mRNA 3 ' -UTR adopts two very stable intramolecular G-quadruplexes which are bound specifically and with high affinity by FMRP both in vitro and in vivo. These results suggest a role of G-quadruplex RNA motif as a structural element in the common mechanism of FMRP regulation of its dendritic mRNA targets.

  13. G-quadruplex ligand-induced DNA damage response coupled with telomere dysfunction and replication stress in glioma stem cells

    OpenAIRE

    Hasegawa, Daiki; Okabe, Sachiko; Okamoto, Keiji; Nakano, Ichiro; Shin-ya, Kazuo; Seimiya, Hiroyuki

    2016-01-01

    Glioblastoma (GBM) is an invariably fatal brain tumor in which a small subpopulation of self-renewable glioma stem cells (GSCs) contributes to tumor propagation and relapse. Targeting GSCs could therefore have a significant clinical impact for GBM. Telomestatin is a naturally-occurring compound that preferentially impairs GSC growth by perturbing transcription and inducing a DNA damage response. Telomestatin stabilizes G-quadruplexes (G4s), which are guanine-rich four-strand nucleic acid stru...

  14. Chelerythrine down regulates expression of VEGFA, BCL2 and KRAS by arresting G-Quadruplex structures at their promoter regions

    Science.gov (United States)

    Jana, Jagannath; Mondal, Soma; Bhattacharjee, Payel; Sengupta, Pallabi; Roychowdhury, Tanaya; Saha, Pranay; Kundu, Pallob; Chatterjee, Subhrangsu

    2017-01-01

    A putative anticancer plant alkaloid, Chelerythrine binds to G-quadruplexes at promoters of VEGFA, BCL2 and KRAS genes and down regulates their expression. The association of Chelerythrine to G-quadruplex at the promoters of these oncogenes were monitored using UV absorption spectroscopy, fluorescence anisotropy, circular dichroism spectroscopy, CD melting, isothermal titration calorimetry, molecular dynamics simulation and quantitative RT-PCR technique. The pronounced hypochromism accompanied by red shifts in UV absorption spectroscopy in conjunction with ethidium bromide displacement assay indicates end stacking mode of interaction of Chelerythrine with the corresponding G-quadruplex structures. An increase in fluorescence anisotropy and CD melting temperature of Chelerythrine-quadruplex complex revealed the formation of stable Chelerythrine-quadruplex complex. Isothermal titration calorimetry data confirmed that Chelerythrine-quadruplex complex formation is thermodynamically favourable. Results of quantative RT-PCR experiment in combination with luciferase assay showed that Chelerythrine treatment to MCF7 breast cancer cells effectively down regulated transcript level of all three genes, suggesting that Chelerythrine efficiently binds to in cellulo quadruplex motifs. MD simulation provides the molecular picture showing interaction between Chelerythrine and G-quadruplex. Binding of Chelerythrine with BCL2, VEGFA and KRAS genes involved in evasion, angiogenesis and self sufficiency of cancer cells provides a new insight for the development of future therapeutics against cancer.

  15. Escherichia coli DNA polymerase I can disrupt G-quadruplex structures during DNA replication.

    Science.gov (United States)

    Teng, Fang-Yuan; Hou, Xi-Miao; Fan, San-Hong; Rety, Stephane; Dou, Shuo-Xing; Xi, Xu-Guang

    2017-12-01

    Non-canonical four-stranded G-quadruplex (G4) DNA structures can form in G-rich sequences that are widely distributed throughout the genome. The presence of G4 structures can impair DNA replication by hindering the progress of replicative polymerases (Pols), and failure to resolve these structures can lead to genetic instability. In the present study, we combined different approaches to address the question of whether and how Escherichia coli Pol I resolves G4 obstacles during DNA replication and/or repair. We found that E. coli Pol I-catalyzed DNA synthesis could be arrested by G4 structures at low protein concentrations and the degree of inhibition was strongly dependent on the stability of the G4 structures. Interestingly, at high protein concentrations, E. coli Pol I was able to overcome some kinds of G4 obstacles without the involvement of other molecules and could achieve complete replication of G4 DNA. Mechanistic studies suggested that multiple Pol I proteins might be implicated in G4 unfolding, and the disruption of G4 structures requires energy derived from dNTP hydrolysis. The present work not only reveals an unrealized function of E. coli Pol I, but also presents a possible mechanism by which G4 structures can be resolved during DNA replication and/or repair in E. coli. © 2017 Federation of European Biochemical Societies.

  16. Mechanistic insight into the interaction of BLM helicase with intra-strand G-quadruplex structures

    Science.gov (United States)

    Chatterjee, Sujoy; Zagelbaum, Jennifer; Savitsky, Pavel; Sturzenegger, Andreas; Huttner, Diana; Janscak, Pavel; Hickson, Ian D.; Gileadi, Opher; Rothenberg, Eli

    2014-11-01

    Bloom syndrome is an autosomal recessive disorder caused by mutations in the RecQ family helicase BLM that is associated with growth retardation and predisposition to cancer. BLM helicase has a high specificity for non-canonical G-quadruplex (G4) DNA structures, which are formed by G-rich DNA strands and play an important role in the maintenance of genomic integrity. Here we used single-molecule FRET to define the mechanism of interaction of BLM helicase with intra-stranded G4 structures. We show that the activity of BLM is substrate dependent, and highly regulated by a short-strand DNA (ssDNA) segment that separates the G4 motif from double-stranded DNA. We demonstrate cooperativity between the RQC and HRDC domains of BLM during binding and unfolding of the G4 structure, where the RQC domain interaction with G4 is stabilized by HRDC binding to ssDNA. We present a model that proposes a unique role for G4 structures in modulating the activity of DNA processing enzymes.

  17. DSC Deconvolution of the Structural Complexity of c-MYC P1 Promoter G-Quadruplexes

    Science.gov (United States)

    Dettler, Jamie M.; Buscaglia, Robert; Le, Vu H.; Lewis, Edwin A.

    2011-01-01

    We completed a biophysical characterization of the c-MYC proto-oncogene P1 promoter quadruplex and its interaction with a cationic porphyrin, 5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin (TMPyP4), using differential scanning calorimetry, isothermal titration calorimetry, and circular dichroism spectroscopy. We examined three different 24-mer oligonucleotides, including the wild-type (WT) sequence found in the c-MYC P1 promoter and two mutant G→T sequences that are known to fold into single 1:2:1 and 1:6:1 loop isomer quadruplexes. Biophysical experiments were performed on all three oligonucleotide sequences at two different ionic strengths (30 mM [K+] and 130 mM [K+]). Differential scanning calorimetry experiments demonstrated that the WT quadruplex consists of a mixture of at least two different folded conformers at both ionic strengths, whereas both mutant sequences exhibit a single two-state melting transition at both ionic strengths. Isothermal titration calorimetry experiments demonstrated that both mutant sequences bind 4 mols of TMPyP4 to 1 mol of DNA, in similarity to the WT sequence. The circular dichroism spectroscopy signatures for all three oligonucleotides at both ionic strengths are consistent with an intramolecular parallel stranded G-quadruplex structure, and no change in quadruplex structure is observed upon addition of saturating amounts of TMPyP4 (i.e., 4:1 TMPyP4/DNA). PMID:21402034

  18. Structural and functional analysis of DNA sequences with potential for forming G-quadruplex

    OpenAIRE

    Luciana Souto Mofatto

    2013-01-01

    Resumo: Os G-quadruplexes são estruturas secundárias de DNA altamente organizadas, constituídas por sequências ricas em guaninas capazes de formar tétrades ligadas por pontes de hidrogênio. Essas sequências são capazes de modular a transcrição gênica e o splicing alternativo de éxons. Além disso, estudos também mostraram que os G-quadruplexes estão presentes na região promotora de oncogenes (como c-MYC) e nas regiões terminais dos telômeros, indicando que o G-quadruplex pode ser um possível a...

  19. Specialization among Iron-Sulfur Cluster Helicases to Resolve G-quadruplex DNA Structures That Threaten Genomic Stability*

    Science.gov (United States)

    Bharti, Sanjay Kumar; Sommers, Joshua A.; George, Fourbears; Kuper, Jochen; Hamon, Florian; Shin-ya, Kazuo; Teulade-Fichou, Marie-Paule; Kisker, Caroline; Brosh, Robert M.

    2013-01-01

    G-quadruplex (G4) DNA, an alternate structure formed by Hoogsteen hydrogen bonds between guanines in G-rich sequences, threatens genomic stability by perturbing normal DNA transactions including replication, repair, and transcription. A variety of G4 topologies (intra- and intermolecular) can form in vitro, but the molecular architecture and cellular factors influencing G4 landscape in vivo are not clear. Helicases that unwind structured DNA molecules are emerging as an important class of G4-resolving enzymes. The BRCA1-associated FANCJ helicase is among those helicases able to unwind G4 DNA in vitro, and FANCJ mutations are associated with breast cancer and linked to Fanconi anemia. FANCJ belongs to a conserved iron-sulfur (Fe S) cluster family of helicases important for genomic stability including XPD (nucleotide excision repair), DDX11 (sister chromatid cohesion), and RTEL (telomere metabolism), genetically linked to xeroderma pigmentosum/Cockayne syndrome, Warsaw breakage syndrome, and dyskeratosis congenita, respectively. To elucidate the role of FANCJ in genomic stability, its molecular functions in G4 metabolism were examined. FANCJ efficiently unwound in a kinetic and ATPase-dependent manner entropically favored unimolecular G4 DNA, whereas other Fe-S helicases tested did not. The G4-specific ligands Phen-DC3 or Phen-DC6 inhibited FANCJ helicase on unimolecular G4 ∼1000-fold better than bi- or tetramolecular G4 DNA. The G4 ligand telomestatin induced DNA damage in human cells deficient in FANCJ but not DDX11 or XPD. These findings suggest FANCJ is a specialized Fe-S cluster helicase that preserves chromosomal stability by unwinding unimolecular G4 DNA likely to form in transiently unwound single-stranded genomic regions. PMID:23935105

  20. Specialization among iron-sulfur cluster helicases to resolve G-quadruplex DNA structures that threaten genomic stability.

    Science.gov (United States)

    Bharti, Sanjay Kumar; Sommers, Joshua A; George, Fourbears; Kuper, Jochen; Hamon, Florian; Shin-ya, Kazuo; Teulade-Fichou, Marie-Paule; Kisker, Caroline; Brosh, Robert M

    2013-09-27

    G-quadruplex (G4) DNA, an alternate structure formed by Hoogsteen hydrogen bonds between guanines in G-rich sequences, threatens genomic stability by perturbing normal DNA transactions including replication, repair, and transcription. A variety of G4 topologies (intra- and intermolecular) can form in vitro, but the molecular architecture and cellular factors influencing G4 landscape in vivo are not clear. Helicases that unwind structured DNA molecules are emerging as an important class of G4-resolving enzymes. The BRCA1-associated FANCJ helicase is among those helicases able to unwind G4 DNA in vitro, and FANCJ mutations are associated with breast cancer and linked to Fanconi anemia. FANCJ belongs to a conserved iron-sulfur (Fe S) cluster family of helicases important for genomic stability including XPD (nucleotide excision repair), DDX11 (sister chromatid cohesion), and RTEL (telomere metabolism), genetically linked to xeroderma pigmentosum/Cockayne syndrome, Warsaw breakage syndrome, and dyskeratosis congenita, respectively. To elucidate the role of FANCJ in genomic stability, its molecular functions in G4 metabolism were examined. FANCJ efficiently unwound in a kinetic and ATPase-dependent manner entropically favored unimolecular G4 DNA, whereas other Fe-S helicases tested did not. The G4-specific ligands Phen-DC3 or Phen-DC6 inhibited FANCJ helicase on unimolecular G4 ∼1000-fold better than bi- or tetramolecular G4 DNA. The G4 ligand telomestatin induced DNA damage in human cells deficient in FANCJ but not DDX11 or XPD. These findings suggest FANCJ is a specialized Fe-S cluster helicase that preserves chromosomal stability by unwinding unimolecular G4 DNA likely to form in transiently unwound single-stranded genomic regions.

  1. Probe development for detection of TERRA 1 intramolecular G-quadruplex formation using a fluorescent adenosine derivative.

    Science.gov (United States)

    Kim, In Sun; Seo, Young Jun

    2014-03-15

    We developed a probing system to detect the intramolecular G-quadruplex of telomeric repeat-containing RNA (TERRA 1). We used a fluorescent adenosine derivative rA(py) as a fluorophore and incorporated it into the dangling position of the parallel-type G-quadruplex sequence of TERRA 1. The rA(py)-modified G-quadruplex structure exhibited a strong fluorescence emission signal, while the emission signals of the single-strand and duplex structures were much lower. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Macrocyclic G-quadruplex ligands

    DEFF Research Database (Denmark)

    Nielsen, M C; Ulven, Trond

    2010-01-01

    are macrocyclic structures which have been modeled after the natural product telomestatin or from porphyrin-based ligands discovered in the late 1990s. These two structural classes of G-quadruplex ligands are reviewed here with special attention to selectivity and structure-activity relationships, and with focus...

  3. Sedimentation Velocity Ultracentrifugation Analysis for Hydrodynamic Characterization of G-Quadruplex Structures

    Science.gov (United States)

    Garbett, Nichola C.; Mekmaysy, Chongkham S.; Chaires, Jonathan B.

    2010-01-01

    Summary Analytical ultracentrifugation (AUC) is a powerful technique for the characterization of hydrodynamic and thermodynamic properties. The intent of this article is to demonstrate the utility of sedimentation velocity (SV) studies to obtain hydrodynamic information for G-quadruplex systems and to provide insights into one part of this process, namely, data analysis of existing SV data. An array of data analysis software is available, mostly written and continually developed by established researchers in the AUC field, with particularly rapid advances in the analysis of SV data. Each program has its own learning curve and this article is intended as a resource in the data analysis process for beginning researchers in the field. We discuss the application of three of the most commonly used data analysis programs, DCDT+, Sedfit and SedAnal, to the interpretation of SV data obtained in our laboratory on two G-quadruplex systems. PMID:20012418

  4. The study of binding of methyl tert-butyl ether to human telomeric G-quadruplex and calf thymus DNA by gas chromatography, a thermodynamic discussion.

    Science.gov (United States)

    Ghasemi, Sahar; Ahmadi, Farhad

    2014-11-15

    Methyl-tert-butyl ether (MTBE) is widely used as an antiknock additive for increasing octane number of gasoline. Recently, the in vivo studies demonstrated that MTBE has genotoxic potential and able to form adducts with DNA. In the work, the interactions of MTBE with calf thymus DNA (ct-DNA) and the Na(+) form of G-quadruplex DNA (wtTel22) were studied by using of head space-solid phase microextraction technique coupled to gas chromatography. The binding equilibrium constants were measured through the equilibriums of a four phase system. In addition, the MTBE Henry's law constants for two different buffers in the temperature range of 283-303K were measured. Thermodynamic studies revealed that the complexation of MTBE to both DNAs is enthalpy favored and entropy disfavored. The thermodynamic results revealed that MTBE may have interaction with ct-DNA via the minor groove of DNA. Also, MTBE may be complexed into the basket of G-quadruplex structure. In addition, the low difference in the binding constants of MTBE for both different DNA targets may confirm that MTBE is poorly selective for different conformations of DNA. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Structure-based design of platinum(II) complexes as c-myc oncogene down-regulators and luminescent probes for G-quadruplex DNA.

    Science.gov (United States)

    Wang, Ping; Leung, Chung-Hang; Ma, Dik-Lung; Yan, Siu-Cheong; Che, Chi-Ming

    2010-06-18

    A series of platinum(II) complexes with tridentate ligands was synthesized and their interactions with G-quadruplex DNA within the c-myc gene promoter were evaluated. Complex 1, which has a flat planar 2,6-bis(benzimidazol-2-yl)pyridine (bzimpy) scaffold, was found to stabilize the c-myc G-quadruplex structure in a cell-free system. An in silico G-quadruplex DNA model has been constructed for structure-based virtual screening to develop new Pt(II)-based complexes with superior inhibitory activities. By using complex 1 as the initial structure for hit-to-lead optimization, bzimpy and related 2,6-bis(pyrazol-3-yl)pyridine (dPzPy) scaffolds containing amine side-chains emerge as the top candidates. Six of the top-scoring complexes were synthesized and their interactions with c-myc G-quadruplex DNA have been investigated. The results revealed that all of the complexes have the ability to stabilize the c-myc G-quadruplex. Complex 3 a ([Pt(II)L2R](+); L2=2,6-bis[1-(3-piperidinepropyl)-1H-enzo[d]imidazol-2-yl]pyridine, R=Cl) displayed the strongest inhibition in a cell-free system (IC(50)=2.2 microM) and was 3.3-fold more potent than that of 1. Complexes 3 a and 4 a ([Pt(II)L3R](+); L3=2,6-bis[1-(3-morpholinopropyl)-1H-pyrazol-3-yl]pyridine, R=Cl) were found to effectively inhibit c-myc gene expression in human hepatocarcinoma cells with IC(50) values of approximately 17 microM, whereas initial hit 1 displayed no significant effect on gene expression at concentrations up to 50 microM. Complexes 3 a and 4 a have a strong preference for G-quadruplex DNA over duplex DNA, as revealed by competition dialysis experiments and absorption titration; 3 a and 4 a bind G-quadruplex DNA with binding constants (K) of approximately 10(6)-10(7) dm(3) mol(-1), which are at least an order of magnitude higher than the K values for duplex DNA. NMR spectroscopic titration experiments and molecular modeling showed that 4 a binds c-myc G-quadruplex DNA through an external end-stacking mode at

  6. Inverting the G-Tetrad Polarity of a G-Quadruplex by Using Xanthine and 8-Oxoguanine.

    Science.gov (United States)

    Cheong, Vee Vee; Lech, Christopher Jacques; Heddi, Brahim; Phan, Anh Tuân

    2016-01-04

    G-quadruplexes are four-stranded nucleic acid structures that are built from consecutively stacked guanine tetrad (G-tetrad) assemblies. The simultaneous incorporation of two guanine base lesions, xanthine (X) and 8-oxoguanine (O), within a single G-tetrad of a G-quadruplex was recently shown to lead to the formation of a stable G⋅G⋅X⋅O tetrad. Herein, a judicious introduction of X and O into a human telomeric G-quadruplex-forming sequence is shown to reverse the hydrogen-bond polarity of the modified G-tetrad while preserving the original folding topology. The control exerted over G-tetrad polarity by joint X⋅O modification will be valuable for the design and programming of G-quadruplex structures and their properties. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Clerocidin-mediated DNA footprinting discriminates among different G-quadruplex conformations and detects tetraplex folding in a duplex environment.

    Science.gov (United States)

    Nadai, Matteo; Sattin, Giovanna; Palù, Giorgio; Palumbo, Manlio; Richter, Sara N

    2013-10-01

    G-quadruplexes are polymorphic non-canonical nucleic acid conformations involved both in physiological and pathological processes. Given the high degree of folding heterogeneity and comparable conformational stabilities, different G-quadruplex forms can occur simultaneously, hence rendering the use of basic instrumental methods for structure determination, like X-ray diffraction or NMR, hardly useful. Footprinting techniques represent valuable and relatively rapid alternative to characterize DNA folding. The natural diterpenoid clerocidin is an alkylating agent that specifically reacts at single-stranded DNA regions, with different mechanisms depending on the exposed nucleotide. Clerocidin was used to footprint G-quadruplex structures formed by telomeric and oncogene promoter sequences (c-myc, bcl-2, c-kit2), and by the thrombin binding aptamer. The easy modulability of CL reactivity towards DNA bases permitted to discriminate fully and partially protected sites, highlights stretched portions of the G-quadruplex conformation, and discriminate among topologies adopted by one sequence in different environmental conditions. Importantly, CL displayed the unique property to allow detection of G-quadruplex folding within a duplex context. CL is a finely performing new tool to unveil G-quadruplex arrangements in DNA sequences under genomically relevant conditions. Nucleic acid G-quadruplex structures are an emerging research field because of the recent indication of their involvement in a series of key biological functions, in particular in regulation of proliferation-associated gene expression. The use of clerocidin as footprinting agent to identify G-quadruplex structures under genomically relevant conditions may allow detection of new G-quadruplex-based regulatory regions. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Seven essential questions on G-quadruplexes.

    Science.gov (United States)

    König, Sebastian L B; Evans, Amanda C; Huppert, Julian L

    2010-08-01

    The helical duplex architecture of DNA was discovered by Francis Crick and James Watson in 1951 and is well known and understood. However, nucleic acids can also adopt alternative structural conformations that are less familiar, although no less biologically relevant, such as the G-quadruplex. G-quadruplexes continue to be the subject of a rapidly expanding area of research, owing to their significant potential as therapeutic targets and their unique biophysical properties. This review begins by focusing on G-quadruplex structure, elucidating the intermolecular and intramolecular interactions underlying its formation and highlighting several substructural variants. A variety of methods used to characterize these structures are also outlined. The current state of G-quadruplex research is then addressed by proffering seven pertinent questions for discussion. This review concludes with an overview of possible directions for future research trajectories in this exciting and relevant field.

  9. The NEIL glycosylases remove oxidized guanine lesions from telomeric and promoter quadruplex DNA structures

    OpenAIRE

    Zhou, Jia; Fleming, Aaron M.; Averill, April M.; Burrows, Cynthia J.; Wallace, Susan S.

    2015-01-01

    G-quadruplex is a four-stranded G-rich DNA structure that is highly susceptible to oxidation. Despite the important roles that G-quadruplexes play in telomere biology and gene transcription, neither the impact of guanine lesions on the stability of quadruplexes nor their repair are well understood. Here, we show that the oxidized guanine lesions 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) reduce the thermostability and alter the folding of telomer...

  10. Circular Dichroism of DNA G-Quadruplexes: Combining Modeling and Spectroscopy To Unravel Complex Structures.

    Science.gov (United States)

    Gattuso, Hugo; Spinello, Angelo; Terenzi, Alessio; Assfeld, Xavier; Barone, Giampaolo; Monari, Antonio

    2016-03-31

    We report on the comparison between the computational and experimental determination of electronic circular dichroism spectra of different guanine quadruplexes obtained from human telomeric sequences. In particular the difference between parallel, antiparallel, and hybrid structures is evidenced, as well as the induction of transitions between the polymorphs depending on the solution environment. Extensive molecular dynamics simulations (MD) are used to probe the conformational space of the different quadruplexes, and subsequently state-of-the-art hybrid quantum mechanics/molecular mechanics (QM/MM) techniques coupled with excitonic semiempirical Hamiltonian are used to simulate the macromolecular induced circular dichroism. The coupling of spectroscopy and molecular simulation allows an efficient one-to-one mapping between structures and optical properties, offering a way to disentangle the rich, yet complicated, quantity of information embedded in circular dichroism spectra. We show that our methodology is robust and efficient and allows us to take into account subtle conformational changes. As such, it could be used as an efficient tool to investigate structural modification upon DNA/drug interactions.

  11. Synthesis, Binding and Antiviral Properties of Potent Core-Extended Naphthalene Diimides Targeting the HIV-1 Long Terminal Repeat Promoter G-Quadruplexes.

    Science.gov (United States)

    Perrone, Rosalba; Doria, Filippo; Butovskaya, Elena; Frasson, Ilaria; Botti, Silvia; Scalabrin, Matteo; Lago, Sara; Grande, Vincenzo; Nadai, Matteo; Freccero, Mauro; Richter, Sara N

    2015-12-24

    We have previously reported that stabilization of the G-quadruplex structures in the HIV-1 long terminal repeat (LTR) promoter suppresses viral transcription. Here we sought to develop new G-quadruplex ligands to be exploited as antiviral compounds by enhancing binding toward the viral G-quadruplex structures. We synthesized naphthalene diimide derivatives with a lateral expansion of the aromatic core. The new compounds were able to bind/stabilize the G-quadruplex to a high extent, and some of them displayed clear-cut selectivity toward the viral G-quadruplexes with respect to the human telomeric G-quadruplexes. This feature translated into low nanomolar anti-HIV-1 activity toward two viral strains and encouraging selectivity indexes. The selectivity depended on specific recognition of LTR loop residues; the mechanism of action was ascribed to inhibition of LTR promoter activity in cells. This is the first example of G-quadruplex ligands that show increased selectivity toward the viral G-quadruplexes and display remarkable antiviral activity.

  12. Novel multi-targeting anthra[2,3-b]thiophene-5,10-diones with guanidine-containing side chains: interaction with telomeric G-quadruplex, inhibition of telomerase and topoisomerase I and cytotoxic properties.

    Science.gov (United States)

    Ilyinsky, Nikolay S; Shchyolkina, Anna K; Borisova, Olga F; Mamaeva, Olga K; Zvereva, Maria I; Azhibek, Dulat M; Livshits, Mikhail A; Mitkevich, Vladimir A; Balzarini, Jan; Sinkevich, Yuri B; Luzikov, Yuri N; Dezhenkova, Lybov G; Kolotova, Ekaterina S; Shtil, Alexander A; Shchekotikhin, Andrey E; Kaluzhny, Dmitry N

    2014-10-06

    Novel generations of antitumor anthraquinones are expected to be advantageous over the conventional chemotherapeutic agents. Previous structure-activity relationship studies demonstrated an importance of the positively charged side chains conjugated to anthra[2,3-b]thiophene-5,10-dione scaffolds. Exploring a role of individual side chain moieties in binding to the duplex and G-quadruplex DNA, modulation of telomerase and topoisomerase I activities, intracellular accumulation and cytostatic potency, we herein analyzed a series of reported and newly synthesized guanidine-containing derivatives of anthra[2,3-b]thiophene-5,10-dione. We found that the number of cationic side chains (namely, two) is critical for a tight interaction with human telomeric G-quadruplex (TelQ). Along with a larger drug-TelQ association constant, the telomerase attenuation by anthrathiophenediones with two basic groups in the side chains was more pronounced than by the analogs bearing one basic group. For mono-guanidinated compounds the substituent with the amino group in the side chain provided better TelQ affinity than the methylamine residue. The intracellular uptake of the mono-guanidino derivative with two side chains was >2-fold higher than the respective value for the bis(guanidino) derivative. This difference can explain a lower antiproliferative potency of bis(guanidine) containing compounds. Thus, the modifications of side chains of anthra[2,3-b]thiophene-5,10-dione differently modulated drug-target interactions and cellular effects. Nevertheless, the selected compound 11-(3-aminopropylamino)-4-(2-guanidinoethylamino)anthra[2,3-b]thiophene-5,10-dione 13 demonstrated a high affinity to TelQ and the ability to stabilize the quadruplex structure. These properties were paralleled by reasonable potency of 13 as a telomerase/topoisomerase I inhibitor and an antiproliferative agent. These results indicate that the structural elements of anthra[2,3-b]thiophene-5,10-dione derivatives can be

  13. Targeting human telomeric G-quadruplex DNA and inhibition of telomerase activity with [(dmb2Ru(obipRu(dmb2](4+.

    Directory of Open Access Journals (Sweden)

    Shuo Shi

    Full Text Available Inhibition of telomerase by inducing/stabilizing G-quadruplex formation is a promising strategy to design new anticancer drugs. We synthesized and characterized a new dinuclear complex [(dmb2Ru(obipRu(dmb2](4+ (dmb = 4,4'-dimethyl-2,2'-bipyridine, obip = (2-(2-pyridylimidazo[4,5-f][1,10]phenanthroline with high affinity for both antiparallel and mixed parallel / antiparallel G-quadruplex DNA. This complex can promote the formation and stabilize G-quadruplex DNA. Dialysis and TRAP experiments indicated that [(dmb2Ru(obipRu(dmb2](4+ acted as an excellent telomerase inhibitor due to its obvious selectivity for G-quadruplex DNA rather than double stranded DNA. In vitro co-culture experiments implied that [(dmb2Ru(obipRu(dmb2](4+ inhibited telomerase activity and hindered cancer cell proliferation without side effects to normal fibroblast cells. TUNEL assay indicated that inhibition of telomerase activity induced DNA cleavage further apoptosis in cancer cells. Therefore, Ru(II complex represents an exciting opportunity for anticancer drug design by specifically targeting cancer cell G-quadruplexes DNA.

  14. FMRP interacts with G-quadruplex structures in the 3’-UTR of its dendritic target Shank1 mRNA

    Science.gov (United States)

    Zhang, Yang; Gaetano, Christian M; Williams, Kathryn R; Bassell, Gary J; Mihailescu, Mihaela Rita

    2014-01-01

    ABSTRACT Fragile X syndrome (FXS), the most common cause of inherited intellectual disability, is caused by the loss of expression of the fragile X mental retardation protein (FMRP). FMRP, which regulates the transport and translation of specific mRNAs, uses its RGG box domain to bind mRNA targets that form G-quadruplex structures. One of the FMRP in vivo targets, Shank1 mRNA, encodes the master scaffold proteins of the postsynaptic density (PSD) which regulate the size and shape of dendritic spines because of their capacity to interact with many different PSD components. Due to their effect on spine morphology, altered translational regulation of Shank1 transcripts may contribute to the FXS pathology. We hypothesized that the FMRP interactions with Shank1 mRNA are mediated by the recognition of the G quadruplex structure, which has not been previously demonstrated. In this study we used biophysical techniques to analyze the Shank1 mRNA 3’-UTR and its interactions with FMRP and its phosphorylated mimic FMRP S500D. We found that the Shank1 mRNA 3 ′ -UTR adopts two very stable intramolecular G-quadruplexes which are bound specifically and with high affinity by FMRP both in vitro and in vivo. These results suggest a role of G-quadruplex RNA motif as a structural element in the common mechanism of FMRP regulation of its dendritic mRNA targets. PMID:25692235

  15. Putative DNA G-quadruplex formation within the promoters of Plasmodium falciparum var genes

    Directory of Open Access Journals (Sweden)

    Rowe J

    2009-08-01

    Full Text Available Abstract Background Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro. Results We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes. The var gene family encodes PfEMP1, the parasite's major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q to form stable G-quadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q. Conclusion This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family.

  16. Formation of highly ordered multimers in G-quadruplexes.

    Science.gov (United States)

    Tóthová, Petra; Krafčíková, Petra; Víglaský, Viktor

    2014-11-18

    G-Rich DNA and RNA have a higher propensity to form G-quadruplex structures, but the presence of G-runs alone is not sufficient to prove that such sequences can form stable G-quadruplexes. While G-rich sequences are essential for G-quadruplex formation, not all G-rich sequences have the propensity to form G-quadruplex structures. In addition, monovalent metal ions, dehydrating agents, and loop sequences connecting the G-runs also play important roles in the topology of G-quadruplex folding. To date, no quantitative analysis of the CD spectra of G-quadruplexes in confrontation with the electrophoretic results has been performed. Therefore, in this study, we use information gained through the analysis of a series of well-known G-quadruplex-forming sequences to evaluate other less-studied sets of aptameric sequences. A simple and cost-effective methodology that can verify the formation of G-quadruplex motifs from oligomeric DNA sequences and a technique to determine the molecularity of these structures are also described. This methodology could be of great use in the prediction of G-quadruplex assembly, and the basic principles of our techniques can be extrapolated for any G-rich DNA sequences. This study also presents a model that can predict the multimerization of G-quadruplexes; the predictions offered by this model are shown to match the results obtained using circular dichroism.

  17. G quadruplex RNA structures in PSD-95 mRNA: potential regulators of miR-125a seed binding site accessibility.

    Science.gov (United States)

    Stefanovic, Snezana; Bassell, Gary J; Mihailescu, Mihaela Rita

    2015-01-01

    Fragile X syndrome (FXS) is the most common inherited form of intellectual disability caused by the CGG trinucleotide expansion in the 3'-untranslated region of the FMR1 gene on the X chromosome, that silences the expression of the Fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G-rich region within the PSD-95 mRNA which encodes for the postsynaptic density protein 95 (PSD-95), and together with the microRNA miR-125a, to play an important role in the reversible inhibition of the PSD-95 mRNA translation in neurons. The loss of FMRP in Fmr1 KO mice disables this translation control in the production of the PSD-95 protein. Interestingly, the miR-125a binding site on PSD-95 mRNA is embedded in the G-rich region bound by FMRP and postulated to adopt one or more G quadruplex structures. In this study, we have used different biophysical techniques to validate and characterize the formation of parallel G quadruplex structures and binding of miR-125a to its complementary sequence located within the 3' UTR of PSD-95 mRNA. Our results indicate that the PSD-95 mRNA G-rich region folds into alternate G quadruplex conformations that coexist in equilibrium. miR-125a forms a stable complex with PSD-95 mRNA, as evident by characteristic Watson-Crick base-pairing that coexists with one of the G quadruplex forms, suggesting a novel mechanism for G quadruplex structures to regulate the access of miR-125a to its binding site. © 2014 Stefanovic et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  18. Insight into the Complexity of the i-Motif and G-Quadruplex DNA Structures Formed in the KRAS Promoter and Subsequent Drug-Induced Gene Repression.

    Science.gov (United States)

    Kaiser, Christine E; Van Ert, Natalie A; Agrawal, Prashansa; Chawla, Reena; Yang, Danzhou; Hurley, Laurence H

    2017-06-28

    Activating KRAS mutations frequently occur in pancreatic, colorectal, and lung adenocarcinomas. While many attempts have been made to target oncogenic KRAS, no clinically useful therapies currently exist. Most efforts to target KRAS have focused on inhibiting the mutant protein; a less explored approach involves targeting KRAS at the transcriptional level. The promoter element of the KRAS gene contains a GC-rich nuclease hypersensitive site with three potential DNA secondary structure-forming regions. These are referred to as the Near-, Mid-, and Far-regions, on the basis of their proximity to the transcription start site. As a result of transcription-induced negative superhelicity, these regions can open up to form unique DNA secondary structures: G-quadruplexes on the G-rich strand and i-motifs on the C-rich strand. While the G-quadruplexes have been well characterized, the i-motifs have not been investigated as thoroughly. Here we show that the i-motif that forms in the C-rich Mid-region is the most stable and exists in a dynamic equilibrium with a hybrid i-motif/hairpin species and an unfolded hairpin species. The transcription factor heterogeneous nuclear ribonucleoprotein K (hnRNP K) was found to bind selectively to the i-motif species and to positively modulate KRAS transcription. Additionally, we identified a benzophenanthridine alkaloid that dissipates the hairpin species and destabilizes the interaction of hnRNP K with the Mid-region i-motif. This same compound stabilizes the three existing KRAS G-quadruplexes. The combined effect of the compound on the Mid-region i-motif and the G-quadruplexes leads to downregulation of KRAS gene expression. This dual i-motif/G-quadruplex-interactive compound presents a new mechanism to modulate gene expression.

  19. New insights from molecular dynamic simulation studies of the multiple binding modes of a ligand with G-quadruplex DNA

    Science.gov (United States)

    Hou, Jin-Qiang; Chen, Shuo-Bin; Tan, Jia-Heng; Luo, Hai-Bin; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu

    2012-12-01

    G-quadruplexes are higher-order DNA and RNA structures formed from guanine-rich sequences. These structures have recently emerged as a new class of potential molecular targets for anticancer drugs. An understanding of the three-dimensional interactions between small molecular ligands and their G-quadruplex targets in solution is crucial for rational drug design and the effective optimization of G-quadruplex ligands. Thus far, rational ligand design has been focused mainly on the G-quartet platform. It should be noted that small molecules can also bind to loop nucleotides, as observed in crystallography studies. Hence, it would be interesting to elucidate the mechanism underlying how ligands in distinct binding modes influence the flexibility of G-quadruplex. In the present study, based on a crystal structure analysis, the models of a tetra-substituted naphthalene diimide ligand bound to a telomeric G-quadruplex with different modes were built and simulated with a molecular dynamics simulation method. Based on a series of computational analyses, the structures, dynamics, and interactions of ligand-quadruplex complexes were studied. Our results suggest that the binding of the ligand to the loop is viable in aqueous solutions but dependent on the particular arrangement of the loop. The binding of the ligand to the loop enhances the flexibility of the G-quadruplex, while the binding of the ligand simultaneously to both the quartet and the loop diminishes its flexibility. These results add to our understanding of the effect of a ligand with different binding modes on G-quadruplex flexibility. Such an understanding will aid in the rational design of more selective and effective G-quadruplex binding ligands.

  20. A Selective G-Quadruplex DNA-Stabilizing Ligand Based on a Cyclic Naphthalene Diimide Derivative

    Directory of Open Access Journals (Sweden)

    Md. Monirul Islam

    2015-06-01

    Full Text Available A cyclic naphthalene diimide (cyclic NDI, 1, carrying a benzene moiety as linker chain, was synthesized and its interaction with G-quadruplex DNAs of a-core and a-coreTT as a human telomeric DNA, c-kit and c-myc as DNA sequence at promoter region, or thrombin-binding aptamer (TBA studied based on UV-VIS and circular dichroism (CD spectroscopic techniques, thermal melting temperature measurement, and FRET-melting assay. The circular dichroism spectra showed that 1 induced the formation of different types of G-quadruplex DNA structure. Compound 1 bound to these G-quadruplexes with affinities in the range of 106–107 M−1 order and a 2:1 stoichiometry. Compound 1 showed 270-fold higher selectivity for a-core than dsDNA with a preferable a-core binding than a-coreTT, c-kit, c-myc and TBA in the presence of K+, which is supported by thermal melting studies. The FRET-melting assay also showed that 1 bound preferentially to human telomeric DNA. Compound 1 showed potent inhibition against telomerase activity with an IC50 value of 0.9 μM and preferable binding to G-quadruplexes DNA than our previously published cyclic NDI derivative 3 carrying a benzene moiety as longer linker chain.

  1. Fragile X Mental Retardation Protein Interactions with a G quadruplex structure in the 3′-Untranslated Region of NR2B mRNA

    Science.gov (United States)

    Stefanovic, Snezana; DeMarco, Brett A.; Underwood, Ayana; Williams, Kathryn R.; Bassell, Gary J.; Mihailescu, Mihaela Rita

    2015-01-01

    Fragile X syndrome, the most common cause of inherited intellectual disability, is caused by a trinucleotide CGG expansion in the 5′-untranslated region of the FMR1 gene, which leads to the loss of expression of the fragile X mental retardation protein (FMRP). FMRP, an RNA-binding protein that regulates the translation of specific mRNAs, has been shown to bind a subset of its mRNA targets by recognizing G quadruplex structures. It has been suggested that FMRP controls the local protein synthesis of several protein components of the Post Synaptic Density (PSD) in response to specific cellular needs. We have previously shown that the interactions between FMRP and mRNAs of the PSD scaffold proteins PSD-95 and Shank1 are mediated via stable G-quadruplex structures formed within the 3′-untranslated regions of these mRNAs. In this study we used biophysical methods to show that a comparable G quadruplex structure forms in the 3′-untranslated region of the glutamate receptor subunit NR2B mRNA encoding for a subunit of N-methyl-D-aspartate (NMDA) receptors that is recognized specifically by FMRP, suggesting a common theme for FMRP recognition of its dendritic mRNA targets. PMID:26412477

  2. Fragile X mental retardation protein interactions with a G quadruplex structure in the 3'-untranslated region of NR2B mRNA.

    Science.gov (United States)

    Stefanovic, Snezana; DeMarco, Brett A; Underwood, Ayana; Williams, Kathryn R; Bassell, Gary J; Mihailescu, Mihaela Rita

    2015-12-01

    Fragile X syndrome, the most common cause of inherited intellectual disability, is caused by a trinucleotide CGG expansion in the 5'-untranslated region of the FMR1 gene, which leads to the loss of expression of the fragile X mental retardation protein (FMRP). FMRP, an RNA-binding protein that regulates the translation of specific mRNAs, has been shown to bind a subset of its mRNA targets by recognizing G quadruplex structures. It has been suggested that FMRP controls the local protein synthesis of several protein components of the post synaptic density (PSD) in response to specific cellular needs. We have previously shown that the interactions between FMRP and mRNAs of the PSD scaffold proteins PSD-95 and Shank1 are mediated via stable G-quadruplex structures formed within the 3'-untranslated regions of these mRNAs. In this study we used biophysical methods to show that a comparable G quadruplex structure forms in the 3'-untranslated region of the glutamate receptor subunit NR2B mRNA encoding for a subunit of N-methyl-d-aspartate (NMDA) receptors that is recognized specifically by FMRP, suggesting a common theme for FMRP recognition of its dendritic mRNA targets.

  3. Architecture based on the integration of intermolecular G-quadruplex structure with sticky-end pairing and colorimetric detection of DNA hybridization.

    Science.gov (United States)

    Li, Hongbo; Wu, Zai-Sheng; Shen, Zhifa; Shen, Guoli; Yu, Ruqin

    2014-02-21

    An interesting discovery is reported in that G-rich hairpin-based recognition probes can self-assemble into a nano-architecture based on the integration of an intermolecular G-quadruplex structure with the sticky-end pairing effect in the presence of target DNAs. Moreover, GNPs modified with partly complementary DNAs can intensively aggregate by hybridization-based intercalation between intermolecular G-quadruplexes, indicating an inspiring assembly mechanism and a powerful colorimetric DNA detection. The proposed intermolecular G-quadruplex-integrated sticky-end pairing assembly (called GISA)-based colorimetric system allows a specific and quantitative assay of p53 DNA with a linear range of more than two orders of magnitude and a detection limit of 0.2 nM, suggesting a considerably improved analytical performance. And more to the point, the discrimination of single-base mismatched target DNAs can be easily conducted via visual observation. The successful development of the present colorimetric system, especially the GISA-based aggregation mechanism of GNPs is different from traditional approaches, and offers a critical insight into the dependence of the GNP aggregation on the structural properties of oligonucleotides, opening a good way to design colorimetric sensing probes and DNA nanostructure.

  4. Mechanism of the Antiproliferative Activity of Some Naphthalene Diimide G-Quadruplex Ligands

    Science.gov (United States)

    Hampel, Sonja M.; Pepe, Antonella; Greulich-Bode, Karin M.; Malhotra, Sanjay V.; Reszka, Anthony P.; Veith, Sebastian; Boukamp, Petra

    2013-01-01

    G-quadruplexes are higher-order nucleic acid structures that can form in G-rich telomeres and promoter regions of oncogenes. Telomeric quadruplex stabilization by small molecules can lead to telomere uncapping, followed by DNA damage response and senescence, as well as chromosomal fusions leading to deregulation of mitosis, followed by apoptosis and downregulation of oncogene expression. We report here on investigations into the mechanism of action of tetra-substituted naphthalene diimide ligands on the basis of cell biologic data together with a National Cancer Institute COMPARE study. We conclude that four principal mechanisms of action are implicated for these compounds: 1) telomere uncapping with subsequent DNA damage response and senescence; 2) inhibition of transcription/translation of oncogenes; 3) genomic instability through telomeric DNA end fusions, resulting in mitotic catastrophe and apoptosis; and 4) induction of chromosomal instability by telomere aggregate formation. PMID:23188717

  5. Selective Binding of Distamycin A Derivative to G-Quadruplex Structure [d(TGGGGT]4

    Directory of Open Access Journals (Sweden)

    Bruno Pagano

    2010-01-01

    are entropically driven processes with a small favourable enthalpic contribution. Interestingly, the structural modifications of compound 1 decrease the affinity of the ligand toward the duplex, enhancing the selectivity.

  6. Responsive DNA G-quadruplex micelles

    NARCIS (Netherlands)

    Cozzoli, Liliana; Gjonaj, Lorina; Stuart, Marc C A; Poolman, Bert; Roelfes, Gerard

    2018-01-01

    A novel and versatile design of DNA-lipid conjugates is presented. The assembly of the DNA headgroups into G-quadruplex structures is essential for the formation of micelles and their stability. By hybridization with a complementary oligonucleotide the micelles were destabilized, resulting in cargo

  7. Macrocyclic naphthalene diimides as G-quadruplex binders.

    Science.gov (United States)

    Marchetti, Chiara; Minarini, Anna; Tumiatti, Vincenzo; Moraca, Federica; Parrotta, Lucia; Alcaro, Stefano; Rigo, Riccardo; Sissi, Claudia; Gunaratnam, Mekala; Ohnmacht, Stephan A; Neidle, Stephen; Milelli, Andrea

    2015-07-01

    The synthesis, biological and molecular modeling evaluation of a series of macrocyclic naphthalene diimides is reported. The present investigation expands on the study of structure-activity relationships of prototype compound 2 by constraining the molecule into a macrocyclic structure with the aim of improving its G-quadruplex binding activity and selectivity. The new derivatives, compounds 4-7 carry spermidine- and spermine-like linkers while in compound 8 the inner basic nitrogen atoms of spermine have been replaced with oxygen atoms. The design strategy has led to potent compounds stabilizing both human telomeric (F21T) and c-KIT2 quadruplex sequences, and high selectivity for quadruplex in comparison to duplex DNA. Antiproliferative effects of the new derivatives 4-8 have been evaluated in a panel of cancer cell lines and all the tested compounds showed activity in the low micromolar or sub-micromolar range of concentrations. In order to rationalize the molecular basis of the DNA G-quadruplex versus duplex recognition preference, docking and molecular dynamics studies have been performed. The computational results support the observation that the main driving force in the recognition is due to electrostatic factors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. A single-molecule force-spectroscopic study on stabilization of G-quadruplex DNA by a telomerase inhibitor.

    Science.gov (United States)

    Funayama, Ryoto; Nakahara, Yoshio; Kado, Shinpei; Tanaka, Mutsuo; Kimura, Keiichi

    2014-08-21

    Single-molecule force spectroscopy was carried out using AFM force measurements for the purpose of direct observation of the stabilization of G-quadruplex DNA by a telomerase inhibitor, which is 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin tetrakis(p-toluenesulfonate) (TMPyP). In AFM force measurements, we used an AFM tip and an Au substrate modified chemically with terminal-biotinylated telomere DNA and streptavidin, respectively. The telomere DNA was fully stretched by the AFM tip based on the bridge formation between the AFM tip and the Au substrate through the streptavidin-biotin interaction. The force-extension curves, which reflected the stretching of a single DNA molecule, were distinguished from all of the curves, judging from the rupture force and the contour length. The selected curves were analyzed using a worm-like chain model, and one of the fitting parameters, persistence length (lp), was used as an index for the stabilization of the G-quadruplex structure. Consequently, the lp value was significantly increased by the addition of TMPyP under the experimental conditions where the G-quadruplex structure could be formed. On the other hand, the value was hardly changed by the addition of TMPyP under the conditions except the above. Furthermore, the methodology developed and demonstrated in this work was applied to evaluate the stabilization of G-quadruplex DNA by other telomerase inhibitors such as ethidium bromide and p-xylene-bis(N-pyridinium bromide).

  9. Structural Insight into the interaction of Flavonoids with Human Telomeric Sequence

    Science.gov (United States)

    Tawani, Arpita; Kumar, Amit

    2015-01-01

    Flavonoids are a group of naturally available compounds that are an attractive source for drug discovery. Their potential to act as anti-tumourigenic and anti-proliferative agents has been reported previously but is not yet fully understood. Targeting human telomeric G-quadruplex DNA could be one of the mechanisms by which these flavonoids exert anticancer activity. We have performed detailed biophysical studies for the interaction of four representative flavonoids, Luteolin, Quercetin, Rutin and Genistein, with the human telomeric G-quadruplex sequence tetramolecular d-(T2AG3T) (Tel7). In addition, we used NMR spectroscopy to derive the first model for the complex formed between Quercetin and G-quadruplex sequence. The model showed that Quercetin stabilises the G-quadruplex structure and does not open the G-tetrad. It interacts with the telomeric sequence through π-stacking at two sites: between T1pT2 and between G6pT7. Based on our findings, we suggest that Quercetin could be a potent candidate for targeting the telomere and thus, act as a potent anti-cancer agent. PMID:26627543

  10. Effect of Locked-Nucleic Acid on a Biologically Active G-Quadruplex. A Structure-Activity Relationship of the Thrombin Aptamer

    Directory of Open Access Journals (Sweden)

    Michael B. Jarstfer

    2008-03-01

    Full Text Available Here we tested the ability to augment the biological activity of the thrombin aptamer, d(GGTTGGTGTGGTTGG, by using locked nucleic acid (LNA to influence its G-quadruplex structure. Compared to un-substituted control aptamer, LNA-containing aptamers displayed varying degrees of thrombin inhibition. Aptamers with LNA substituted in either positions G5, T7, or G8 showed decreased thrombin inhibition, whereas LNA at position G2 displayed activity comparable to un-substituted control aptamer. Interestingly, the thermal stability of the substituted aptamers does not correlate to activity – the more stable aptamers with LNA in position G5, T7, or G8 showed the least thrombin inhibition, while a less stable aptamer with LNA at G2 was as active as the un-substituted aptamer. These results suggest that LNA substitution at sites G5, T7, and G8 directly perturbs aptamer-thrombin affinity. This further implies that for the thrombin aptamer, activity is not dictated solely by the stability of the G-quadruplex structure, but by specific interactions between the central TGT loop and thrombin and that LNA can be tolerated in a biologically active nucleic acid structure albeit in a position dependent fashion.

  11. Exploration of the selective recognition of the G-quadruplex in the N-myc oncogene by electrospray ionization mass spectrometry.

    Science.gov (United States)

    Li, Fangyuan; Chen, Han; Zhou, Jiang; Yuan, Gu

    2015-02-15

    The N-myc gene is a member of the MYC family and its amplification is highly correlated with the pathophysiology of cancers. The G-rich sequence, d(AG3CG3AG3AG3A), in the first intron of N-myc can form a G-quadruplex structure. Small molecules binding to it with high affinity and selectivity may provide a potential approach to modulate the expression of the N-myc gene. Electrospray ionization (ESI) mass spectrometry was used to analyze the G-quadruplex formation of the d(AG3CG3AG3AG3A) sequence, and to evaluate the binding affinities and selectivities of natural small molecules with the N-myc G-quadruplex. Enniatin B was found to have the highest binding affinity with this G-quadruplex within the 12 small molecules. Moreover, it also showed a biased selectivity toward the N-myc G-quadruplex compared with the other five G-quadruplexes derived from C-myc, Bcl2, Chl1, c-kit promoters and telomere G-rich sequences. In this study, we found a natural small molecule, enniatin B, which could bind to the G-quadruplex of the d(AG3CG3AG3AG3A) sequence from the first intron of the N-myc gene with high affinity and selectivity, which may lead to a potential modulation of the N-myc gene. Copyright © 2014 John Wiley & Sons, Ltd.

  12. A G-quadruplex motif in an envelope gene promoter regulates transcription and virion secretion in HBV genotype B.

    Science.gov (United States)

    Biswas, Banhi; Kandpal, Manish; Vivekanandan, Perumal

    2017-09-13

    HBV genotypes differ in pathogenicity. In addition, genotype-specific differences in the regulation of transcription and virus replication exist in HBV, but the underlying mechanisms are unknown. Here, we show the presence of a G-quadruplex motif in the promoter of the preS2/S gene; this G-quadruplex is highly conserved only in HBV genotype B but not in other HBV genotypes. We demonstrate that this G-quadruplex motif forms a hybrid intramolecular G-quadruplex structure. Interestingly, mutations disrupting the G-quadruplex in HBV genotype B reduced the preS2/S promoter activity, leading to reduced hepatitis B surface antigen (HBsAg) levels. G-quadruplex ligands stabilized the G-quadruplex in genotype B and enhanced the preS2/S promoter activity. Furthermore, mutations disrupting the G-quadruplex in the full-length HBV genotype B constructs were associated with impaired virion secretion. In contrast to typical G-quadruplexes within promoters which are negative regulators of transcription the G-quadruplex in the preS2/S promoter of HBV represents an unconventional positive regulatory element. Our findings highlight (a) G-quadruplex mediated enhancement of transcription and virion secretion in HBV and (b) a yet unknown role for DNA secondary structures in complex genotype-specific regulatory mechanisms in virus genomes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Macrocyclic Pyridyl Polyoxazoles: Structure-Activity Studies of the Aminoalkyl Side-Chain on G-Quadruplex Stabilization and Cytotoxic Activity

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    Joseph E. Rice

    2013-09-01

    Full Text Available Pyridyl polyoxazoles are 24-membered macrocyclic lactams comprised of a pyridine, four oxazoles and a phenyl ring. A derivative having a 2-(dimethylaminoethyl chain attached to the 5-position of the phenyl ring was recently identified as a selective G-quadruplex stabilizer with excellent cytotoxic activity, and good in vivo anticancer activity against a human breast cancer xenograft in mice. Here we detail the synthesis of eight new dimethylamino-substituted pyridyl polyoxazoles in which the point of attachment to the macrocycle, as well as the distance between the amine and the macrocycle are varied. Each compound was evaluated for selective G-quadruplex stabilization and cytotoxic activity. The more active analogs have the amine either directly attached to, or separated from the phenyl ring by two methylene groups. There is a correlation between those macrocycles that are effective ligands for the stabilization of G-quadruplex DNA (DTtran 15.5–24.6 °C and cytotoxicity as observed in the human tumor cell lines, RPMI 8402 (IC50 0.06–0.50 μM and KB3-1 (IC50 0.03–0.07 μM. These are highly selective G-quadruplex stabilizers, which should prove especially useful for evaluating both in vitro and in vivo mechanism(s of biological activity associated with G-quaqdruplex ligands.

  14. High-resolution three-dimensional NMR structure of theKRASproto-oncogene promoter reveals key features of a G-quadruplex involved in transcriptional regulation.

    Science.gov (United States)

    Kerkour, Abdelaziz; Marquevielle, Julien; Ivashchenko, Stefaniia; Yatsunyk, Liliya A; Mergny, Jean-Louis; Salgado, Gilmar F

    2017-05-12

    Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human KRAS proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5'-AGGGCGGTGTGGGAATAGGGAA-3') and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The NEH region therefore has been proposed as a target for new drugs that control KRAS transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Detection of G-quadruplex DNA in mammalian cells

    NARCIS (Netherlands)

    Henderson, Alexander; Wu, Yuliang; Huang, Yu Chuan; Chavez, Elizabeth A.; Platt, Jesse; Johnson, F. Brad; Brosh, Robert M.; Sen, Dipankar; Lansdorp, Peter M.

    It has been proposed that guanine-rich DNA forms four-stranded structures in vivo called G-quadruplexes or G4 DNA. G4 DNA has been implicated in several biological processes, but tools to study G4 DNA structures in cells are limited. Here we report the development of novel murine monoclonal

  16. The NEIL glycosylases remove oxidized guanine lesions from telomeric and promoter quadruplex DNA structures.

    Science.gov (United States)

    Zhou, Jia; Fleming, Aaron M; Averill, April M; Burrows, Cynthia J; Wallace, Susan S

    2015-04-30

    G-quadruplex is a four-stranded G-rich DNA structure that is highly susceptible to oxidation. Despite the important roles that G-quadruplexes play in telomere biology and gene transcription, neither the impact of guanine lesions on the stability of quadruplexes nor their repair are well understood. Here, we show that the oxidized guanine lesions 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) reduce the thermostability and alter the folding of telomeric quadruplexes in a location-dependent manner. Also, the NEIL1 and NEIL3 DNA glycosylases can remove hydantoin lesions but none of the glycosylases, including OGG1, are able to remove 8-oxoG from telomeric quadruplexes. Interestingly, a hydantoin lesion at the site most prone to oxidation in quadruplex DNA is not efficiently removed by NEIL1 or NEIL3. However, NEIL1, NEIL2 and NEIL3 remove hydantoins from telomeric quadruplexes formed by five TTAGGG repeats much more rapidly than the commonly studied four-repeat quadruplex structures. We also show that APE1 cleaves furan in selected positions in Na(+)-coordinated telomeric quadruplexes. In promoter G-quadruplex DNA, the NEIL glycosylases primarily remove Gh from Na(+)-coordinated antiparallel quadruplexes but not K(+)-coordinated parallel quadruplexes containing VEGF or c-MYC promoter sequences. Thus, the NEIL DNA glycosylases may be involved in both telomere maintenance and in gene regulation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Tracking the Dynamic Folding and Unfolding of RNA G-Quadruplexes in Live Cells.

    Science.gov (United States)

    Chen, Xiu-Cai; Chen, Shuo-Bin; Dai, Jing; Yuan, Jia-Hao; Ou, Tian-Miao; Huang, Zhi-Shu; Tan, Jia-Heng

    2018-02-17

    Due to the absence of methods for tracking RNA G-quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G-quadruplexes is so far limited. Here we reported a new red-emitting fluorescent probe, QUMA-1, for a selective, continuous and real-time visualization of RNA G-quadruplexes in live cells. The applications of QUMA-1 in several previously intractable applications, including live-cell imaging of the dynamic folding, unfolding and movement of RNA G-quadruplexes and visualizing the unwinding of RNA G-quadruplexes by RNA helicase have been demonstrated. Notably, our real-time results revealed the complexity of the dynamics of RNA G-quadruplexes in live cells. We anticipate that the further application of QUMA-1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G-quadruplexes will uncover more information about the biological roles of RNA G-quadruplexes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. G-quadruplex forming structural motifs in the genome of Deinococcus radiodurans and their regulatory roles in promoter functions.

    Science.gov (United States)

    Kota, Swathi; Dhamodharan, V; Pradeepkumar, P I; Misra, Hari S

    2015-11-01

    Deinococcus radiodurans displays compromised radioresistance in the presence of guanine quadruplex (G4)-binding drugs (G4 drugs). Genome-wide scanning showed islands of guanine runs (G-motif) in the upstream regions of coding sequences as well as in the structural regions of many genes, indicating a role for G4 DNA in the regulation of genome functions in this bacterium. G-motifs present upstream to some of the DNA damage-responsive genes like lexA, pprI, recF, recQ, mutL and radA were synthesized, and the formation of G4 DNA structures was probed in vitro. The G-motifs present at the 67th position upstream to recQ and at the 121st position upstream to mutL produced parallel and mixed G4 DNA structures, respectively. Expression of β-galactosidase under recQ and mutL promoters containing respective G-motifs was inhibited by G4 drugs under normal growth conditions in D. radiodurans. However, when such cells were exposed to γ radiation, mutL promoter activity was stimulated while recQ promoter activity was inhibited in the presence of G4 drugs. Deletion of the G-motif from the recQ promoter could relax it from G4 drug repression. D. radiodurans cells treated with G4 drug showed reduction in recQ expression and γ radiation resistance, indicating an involvement of G4 DNA in the radioresistance of this bacterium. These results suggest that G-motifs from D. radiodurans genome form different types of G4 DNA structures at least in vitro, and the recQ and mutL promoters seem to be differentially regulated at the levels of G4 DNA structures.

  19. Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe.

    Science.gov (United States)

    Zhang, Suge; Sun, Hongxia; Chen, Hongbo; Li, Qian; Guan, Aijiao; Wang, Lixia; Shi, Yunhua; Xu, Shujuan; Liu, Meirong; Tang, Yalin

    2018-02-02

    Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking. Formation of the nucleolar G-quadruplex structures was confirmed by using circular dichroism (CD) spectroscopy. The binding affinity and selectivity of Thioflavin T (ThT) towards various DNA/RNA motifs in solution and gel system were measured by using fluorescence spectroscopy and polyacrylamide gel electrophoresis (PAGE), respectively. G-quadruplex imaging in live cells was directly captured by using confocal laser scanning microscopy (CLSM). Formation of the rDNA and rRNA G-quadruplex structures is demonstrated in vitro. ThT is found to show much higher affinity and selectivity towards these G-quadruplex structures versus other nucleic acid motifs either in solution or in gel system. The nucleolar G-quadruplexes in living cells are visualized by using ThT as a fluorescent probe. G-quadruplex-ligand treatments in live cells lead to sharp decrease of ThT signal. The natural existence of the G-quadruplexes structure in the nucleoli of living cells is directly visualized by using ThT as an indicator. The research provides substantive evidence for formation of the rRNA G-quadruplex structures, and also offers an effective probe for direct visualization of the nucleolar G-quadruplexes in living cells. Copyright © 2018. Published by Elsevier B.V.

  20. Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci

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    Aaron J. Stevens

    2017-03-01

    Full Text Available Loss of one allele during polymerase chain reaction (PCR amplification of DNA, known as allelic dropout, can be caused by a variety of mechanisms. Allelic dropout during PCR may have profound implications for molecular diagnostic and research procedures that depend on PCR and assume biallelic amplification has occurred. Complete allelic dropout due to the combined effects of cytosine methylation and G-quadruplex formation was previously described for a differentially methylated region of the human imprinted gene, MEST. We now demonstrate that this parent-of-origin specific allelic dropout can potentially occur at several other genomic regions that display genomic imprinting and have propensity for G-quadruplex formation, including AIM1, BLCAP, DNMT1, PLAGL1, KCNQ1, and GRB10. These findings demonstrate that systematic allelic dropout during PCR is a general phenomenon for regions of the genome where differential allelic methylation and G-quadruplex motifs coincide, and suggest that great care must be taken to ensure biallelic amplification is occurring in such situations.

  1. Putative HIV and SIV G-Quadruplex Sequences in Coding and Noncoding Regions Can Form G-Quadruplexes

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    Petra Krafčíková

    2017-01-01

    Full Text Available The HIV virus is one of the most studied viruses in the world. This is especially true in terms of gene sequencing, and to date more than 9 thousand genomic sequences of HIV isolates have been sequenced and analyzed. In this study, a series of DNA sequences, which have the potential to form G-quadruplex structures, is analyzed. Several such sequences were found in various coding and noncoding virus domains, including the U3 LTR, tat, rev, env, and vpx regions. Interestingly, a homological sequence to the already well-known HIV integrase aptamer was identified in the minus-strand. The sequences derived from original isolates were analyzed using standard spectral and electrophoretic methods. In addition, a recently developed methodology is applied which uses induced circular dichroism spectral profiles of G-quadruplex-ligand (Thiazole Orange complexes to determine if G-rich sequences can adopt G-quadruplex structure. Targeting the G-quadruplexes or peptide domains corresponding to the G-rich coding sequence in HIV offers researchers attractive therapeutic targets which would be of particular use in the development of novel antiviral therapies. The analysis of G-rich regions can provide researchers with a path to find specific targets which could be of interest for specific types of virus.

  2. Guanine-vacancy–bearing G-quadruplexes responsive to guanine derivatives

    Science.gov (United States)

    Li, Xin-min; Zheng, Ke-wei; Zhang, Jia-yu; Liu, Hong-he; He, Yi-de; Yuan, Bi-feng; Hao, Yu-hua; Tan, Zheng

    2015-01-01

    G-quadruplex structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and nanodevices. G-quadruplexes are normally composed of four Gn (n ≥ 3) tracts assembled into a core of multiple stacked G-quartet layers. By dimethyl sulfate footprinting, circular dichroism spectroscopy, thermal melting, and photo-cross-linking, here we describe a unique type of intramolecular G-quadruplex that forms with one G2 and three G3 tracts and bears a guanine vacancy (G-vacancy) in one of the G-quartet layers. The G-vacancy can be filled up by a guanine base from GTP or GMP to complete an intact G-quartet by Hoogsteen hydrogen bonding, resulting in significant G-quadruplex stabilization that can effectively alter DNA replication in vitro at physiological concentration of GTP and Mg2+. A bioinformatic survey shows motifs of such G-quadruplexes are evolutionally selected in genes with unique distribution pattern in both eukaryotic and prokaryotic organisms, implying such G-vacancy–bearing G-quadruplexes are present and play a role in gene regulation. Because guanine derivatives are natural metabolites in cells, the formation of such G-quadruplexes and guanine fill-in (G-fill-in) may grant an environment-responsive regulation in cellular processes. Our findings thus not only expand the sequence definition of G-quadruplex formation, but more importantly, reveal a structural and functional property not seen in the standard canonical G-quadruplexes. PMID:26553979

  3. Chromatin structure in telomere dynamics

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    Alessandra eGalati

    2013-03-01

    Full Text Available The establishment of a specific nucleoprotein structure, the telomere, is required to ensure the protection of chromosome ends from being recognized as DNA damage sites. Telomere shortening below a critical length triggers a DNA damage response that leads to replicative senescence. In normal human somatic cells, characterized by telomere shortening with each cell division, telomere uncapping is a regulated process associated with cell turnover. Nevertheless, telomere dysfunction has also been associated with genomic instability, cell transformation and cancer. Despite the essential role telomeres play in chromosome protection and in tumorigenesis, our knowledge of the chromatin structure involved in telomere maintenance is still limited. Here we review the recent findings on chromatin modifications associated with the dynamic changes of telomeres from protected to de-protected state and their role in telomere functions.

  4. Synthesis and Molecular Modeling of Thermally Stable DNA G-Quadruplexes with Anthraquinone Insertions

    DEFF Research Database (Denmark)

    Gouda, Alaa S.; Amine, Mahasen S.; Pedersen, Erik Bjerregaard

    2017-01-01

    anthraquinone-modified quadruplexes revealed no change of the antiparallel structure when compared with the wild type under potassium buffer conditions. The significantly increased thermostabilities were interpreted by molecular modeling of anthraquinone-modified G-quadruplexes....

  5. Naphthalene diimide scaffolds with dual reversible and covalent interaction properties towards G-quadruplex.

    Science.gov (United States)

    Nadai, Matteo; Doria, Filippo; Di Antonio, Marco; Sattin, Giovanna; Germani, Luca; Percivalle, Claudia; Palumbo, Manlio; Richter, Sara N; Freccero, Mauro

    2011-08-01

    Selective recognition and alkylation of G-quadruplex oligonucleotides has been achieved by substituted naphathalene diimides (NDIs) conjugated to engineered phenol moieties by alkyl-amido spacers with tunable length and conformational mobility. FRET-melting assays, circular dichroism titrations and gel electrophoresis analysis have been carried out to evaluate both reversible stabilization and alkylation of the G-quadruplex. The NDIs conjugated to a quinone methide precursor (NDI-QMP) and a phenol moiety by the shortest alkyl-amido spacer exhibited a planar and fairly rigid geometry (modelled by DFT computation). They were the best irreversible and reversible G-quadruplex binders, respectively. The above NDI-QMP was able to alkylate the telomeric G-quadruplex DNA in the nanomolar range and resulted 100-1000 times more selective on G-quadruplex versus single- and double-stranded oligonucleotides. This compound was also the most cytotoxic against a lung carcinoma cell line. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  6. Applications of Isothermal Titration Calorimetry in Biophysical Studies of G-quadruplexes

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    Concetta Giancola

    2009-07-01

    Full Text Available G-quadruplexes are higher-order nucleic acids structures formed by G-rich sequences that are stabilized by tetrads of hydrogen-bonded guanine bases. Recently, there has been growing interest in the study of G-quadruplexes because of their possible involvement in many biological processes. Isothermal titration calorimetry (ITC has been proven to be a useful tool to study the energetic aspects of G-quadruplex interactions. Particularly, ITC has been applied many times to determine the thermodynamic properties of drug-quadruplex interactions to screening among various drugs and to address drug design. In the present review, we will focus on the ITC studies of G-quadruplex structures and their interaction with proteins and drugs and the most significant results will be discussed.

  7. Evidences for Piperine inhibiting cancer by targeting human G-quadruplex DNA sequences.

    Science.gov (United States)

    Tawani, Arpita; Amanullah, Ayeman; Mishra, Amit; Kumar, Amit

    2016-12-20

    Piperine, a naturally occurring alkaloid, is well known as anti-oxidant, anti-mutagenic, anti-tumor and anti-proliferative agent. Piperine exerts such pharmacological activities by binding or interacting with various cellular targets. Recently, the first report for Piperine interaction with duplex DNA has been published last year but its interaction with G-quadruplex structures has not been studied yet. Herein, we report for the first time the interaction of Piperine with various DNA G-quadruplex structures. Comprehensive biophysical techniques were employed to determine the basis of interaction for the complex formed between Piperine and G-quadruplex DNA sequences. Piperine showed specificity for G-quadruplex DNA over double stranded DNA, with highest affinity for G-quadruplex structure formed at c-myc promoter region. Further, in-vitro studies show that Piperine causes apoptosis-mediated cell death that further emphasizes the potential of this natural product, Piperine, as a promising candidate for targeting G-quadruplex structure and thus, acts as a potent anti-cancer agent.

  8. Exploration of G-quadruplex function in c-Myb gene and its transcriptional regulation by topotecan.

    Science.gov (United States)

    Li, Fangyuan; Zhou, Jiang; Xu, Ming; Yuan, Gu

    2017-10-05

    Our bioinformatics research shows that there are four G-rich sequences (S1-S4) in the upstream region of the transcription start site of c-Myb gene, and we have proved that these sequences have the ability to form G-quadruplex structures. This work mainly focuses on G-quadruplex function, recognition and transcription regulation in c-Myb gene, revealing a novel regulatory element in c-Myb proximal promoter region, and its transcription regulation by G-quadruplex binder. The research has identified that the enhancer effect in c-Myb transcription was primarily affected by the G-quadruplex formed by S1 sequence, and the up-regulation effect may due to the removal of repressive progress of MZF-1 by stabilizing G-quadruplex. Attentions were being paid to the development of G-quadruplex binders for selective recognition, and topotecan was found to have high binding affinity in vitro and could effectively affect the c-Myb transcription activities in cells. The regulation of G-quadruplex with binders in transcriptional, translational levels by Q-RT-PCR and western blot was in expectation of providing a strategy for gene expression modulation. In conclusion, our study revealed a G-quadruplex structure in c-Myb proximal promoter region, which was of great importance in the regulation of c-Myb function. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. G Quadruplex in Plants: A Ubiquitous Regulatory Element and Its Biological Relevance

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    Vikas Yadav

    2017-07-01

    Full Text Available G quadruplexes (G4 are higher-order DNA and RNA secondary structures formed by G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential G4 quadruplex sequences have been identified in G-rich eukaryotic non-telomeric and telomeric genomic regions. Upon function, G4 formation is known to involve in chromatin remodeling, gene regulation and has been associated with genomic instability, genetic diseases and cancer progression. The natural role and biological validation of G4 structures is starting to be explored, and is of particular interest for the therapeutic interventions for human diseases. However, the existence and physiological role of G4 DNA and G4 RNA in plants species have not been much investigated yet and therefore, is of great interest for the development of improved crop varieties for sustainable agriculture. In this context, several recent studies suggests that these highly diverse G4 structures in plants can be employed to regulate expression of genes involved in several pathophysiological conditions including stress response to biotic and abiotic stresses as well as DNA damage. In the current review, we summarize the recent findings regarding the emerging functional significance of G4 structures in plants and discuss their potential value in the development of improved crop varieties.

  10. A new cationic porphyrin derivative (TMPipEOPP with large side arm substituents: a highly selective G-quadruplex optical probe.

    Directory of Open Access Journals (Sweden)

    Li-Na Zhu

    Full Text Available The discovery of uncommon DNA structures and speculation about their potential functions in genes has brought attention to specific DNA structure recognition. G-quadruplexes are four-stranded nucleic acid structures formed by G-rich DNA (or RNA sequences. G-rich sequences with a high potential to form G-quadruplexes have been found in many important genomic regions. Porphyrin derivatives with cationic side arm substituents are important G-quadruplex-binding ligands. For example, 5,10,15,20-Tetrakis(N-methylpyridinium-4-yl-21H,23H-porphyrin (TMPyP4, interacts strongly with G-quadruplexes, but has poor selectivity for G-quadruplex versus duplex DNA. To increase the G-quadruplex recognition specificity, a new cationic porphyrin derivative, 5,10,15,20-tetra-{4-[2-(1-methyl-1-piperidinylethoxy]phenyl} porphyrin (TMPipEOPP, with large side arm substituents was synthesized, and the interactions between TMPipEOPP and different DNA structures were compared. The results show that G-quadruplexes cause large changes in the UV-Vis absorption and fluorescence spectra of TMPipEOPP, but duplex and single-stranded DNAs do not, indicating that TMPipEOPP can be developed as a highly specific optical probe for discriminating G-quadruplex from duplex and single-stranded DNA. Visual discrimination is also possible. Job plot and Scatchard analysis suggest that a complicated binding interaction occurs between TMPipEOPP and G-quadruplexes. At a low [G-quadruplex]/[TMPipEOPP] ratio, one G-quadruplex binds two TMPipEOPP molecules by end-stacking and outside binding modes. At a high [G-quadruplex]/[TMPipEOPP] ratio, two G-quadruplexes bind to one TMPipEOPP molecule in a sandwich-like end-stacking mode.

  11. Molecular models for intrastrand DNA G-quadruplexes

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    Corà Davide

    2009-10-01

    Full Text Available Abstract Background Independent surveys of human gene promoter regions have demonstrated an overrepresentation of G3Xn1G3Xn2G3Xn3G3 motifs which are known to be capable of forming intrastrand quadruple helix structures. In spite of the widely recognized importance of G-quadruplex structures in gene regulation and growing interest around this unusual DNA structure, there are at present only few such structures available in the Nucleic Acid Database. In the present work we generate by molecular modeling feasible G-quadruplex structures which may be useful for interpretation of experimental data. Results We have used all quadruplex DNA structures deposited in the Nucleic Acid Database in order to select a list of fragments entailing a strand of three adjacent G's paired with another strand of three adjacent G's separated by a loop of one to four residues. These fragments were further clustered and representative fragments were finally selected. Further fragments were generated by assemblying the two strands of each fragment with loops from different fragments whenever the anchor G's were superimposable. The fragments were used to assemble G quadruplex based on a superimposability criterion. Conclusion Molecular models have been generated for a large number of G3Xn1G3Xn2G3Xn3G3 sequences. For a given sequence not all topologies are possible with the available repertoire of fragments due to steric hindrance and low superimposability. Since all molecular models are generated by fragments coming from observed quadruplex structures, molecular models are in principle reliable and may be used for interpretation of experimental data. Some examples of applications are given.

  12. Tri- and tetra-substituted naphthalene diimides as potent G-quadruplex ligands.

    Science.gov (United States)

    Cuenca, Francisco; Greciano, Olga; Gunaratnam, Mekala; Haider, Shozeb; Munnur, Deeksha; Nanjunda, Rupesh; Wilson, W David; Neidle, Stephen

    2008-03-01

    A series of tri- and tetra-substituted naphthalene diimides have been designed and synthesized. Several compounds show exceptional affinity for telomeric G-quadruplex DNA in classical and competition FRET assays and SPR studies. They inhibit telomerase in the TRAP assay, and show potent senescence-based short-term anti-proliferative effects on MCF7 and A549 cancer cell lines, and localize in the nucleus and particularly the nucleolus of MCF7 cells.

  13. Multifunctional energy landscape for a DNA G-quadruplex: An evolved molecular switch

    Science.gov (United States)

    Cragnolini, Tristan; Chakraborty, Debayan; Šponer, Jiří; Derreumaux, Philippe; Pasquali, Samuela; Wales, David J.

    2017-10-01

    We explore the energy landscape for a four-fold telomere repeat, obtaining interconversion pathways between six experimentally characterised G-quadruplex topologies. The results reveal a multi-funnel system, with a variety of intermediate configurations and misfolded states. This organisation is identified with the intrinsically multi-functional nature of the system, suggesting a new paradigm for the classification of such biomolecules and clarifying issues regarding apparently conflicting experimental results.

  14. Interaction of pyrrolobenzodiazepine (PBD ligands with parallel intermolecular G-quadruplex complex using spectroscopy and ESI-MS.

    Directory of Open Access Journals (Sweden)

    Gajjela Raju

    Full Text Available Studies on ligand interaction with quadruplex DNA, and their role in stabilizing the complex at concentration prevailing under physiological condition, has attained high interest. Electrospray ionization mass spectrometry (ESI-MS and spectroscopic studies in solution were used to evaluate the interaction of PBD and TMPyP4 ligands, stoichiometry and selectivity to G-quadruplex DNA. Two synthetic ligands from PBD family, namely pyrene-linked pyrrolo[2,1-c][1,4]benzodiazepine hybrid (PBD1, mixed imine-amide pyrrolobenzodiazepine dimer (PBD2 and 5,10,15,20-tetrakis(N-methyl-4-pyridylporphyrin (TMPyP4 were studied. G-rich single-stranded oligonucleotide d(5'GGGGTTGGGG3' designated as d(T(2G(8, from the telomeric region of Tetrahymena Glaucoma, was considered for the interaction with ligands. ESI-MS and spectroscopic methods viz., circular dichroism (CD, UV-Visible, and fluorescence were employed to investigate the G-quadruplex structures formed by d(T(2G(8 sequence and its interaction with PBD and TMPyP4 ligands. From ESI-MS spectra, it is evident that the majority of quadruplexes exist as d(T(2G(8(2 and d(T(2G(8(4 forms possessing two to ten cations in the centre, thereby stabilizing the complex. CD band of PBD1 and PBD2 showed hypo and hyperchromicity, on interaction with quadruplex DNA, indicating unfolding and stabilization of quadruplex DNA complex, respectively. UV-Visible and fluorescence experiments suggest that PBD1 bind externally where as PBD2 intercalate moderately and bind externally to G-quadruplex DNA. Further, melting experiments using SYBR Green indicate that PBD1 unfolds and PBD2 stabilizes the G-quadruplex complex. ITC experiments using d(T(2G(8 quadruplex with PBD ligands reveal that PBD1 and PBD2 prefer external/loop binding and external/intercalative binding to quadruplex DNA, respectively. From experimental results it is clear that the interaction of PBD2 and TMPyP4 impart higher stability to the quadruplex complex.

  15. In-cell optical imaging of exogenous G-quadruplex DNA by fluorogenic ligands

    Science.gov (United States)

    Tseng, Ting-Yuan; Wang, Zi-Fu; Chien, Cheng-Hao; Chang, Ta-Chau

    2013-01-01

    Guanine-rich oligonucleotides (GROs) are promising therapeutic candidate for cancer treatment and other biomedical application. We have introduced a G-quadruplex (G4) ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide, to monitor the cellular uptake of naked GROs and map their intracellular localizations in living cells by using confocal microscopy. The GROs that form parallel G4 structures, such as PU22, T40214 and AS1411, are detected mainly in the lysosome of CL1-0 lung cancer cells after incubation for 2 h. On the contrary, the GROs that form non-parallel G4 structures, such as human telomeres (HT23) and thrombin binding aptamer (TBA), are rarely detected in the lysosome, but found mainly in the mitochondria. Moreover, the fluorescence resonant energy transfer studies of fluorophore-labeled GROs show that the parallel G4 structures can be retained in CL1-0 cells, whereas the non-parallel G4 structures are likely distorted in CL1-0 cells after cellular uptake. Of interest is that the distorted G4 structure of HT23 from the non-parallel G4 structure can reform to a probable parallel G4 structure induced by a G4 ligand in CL1-0 living cells. These findings are valuable to the design and rationale behind the possible targeted drug delivery to specific cellular organelles using GROs. PMID:24030712

  16. G-Quadruplex and i-Motif Are Mutually Exclusive in ILPR Double-Stranded DNA

    Science.gov (United States)

    Dhakal, Soma; Yu, Zhongbo; Konik, Ryan; Cui, Yunxi; Koirala, Deepak; Mao, Hanbin

    2012-01-01

    G-quadruplex has demonstrated its biological functions in vivo. Although G-quadruplex in single-stranded DNA (ssDNA) has been well characterized, investigation of this species in double-stranded DNA (dsDNA) lags behind. Here we use chemical footprinting and laser-tweezers-based single-molecule approaches to demonstrate that a dsDNA fragment found in the insulin-linked polymorphic region (ILPR), 5′-(ACA GGGG TGT GGGG)2 TGT, can fold into a G-quadruplex at pH 7.4 with 100 mM K+, and an i-motif at pH 5.5 with 100 mM Li+. Surprisingly, under a condition that favors the formation of both G-quadruplex and i-motif (pH 5.5, 100 mM K+), a unique determination of change in the free energy of unfolding (ΔGunfold) by laser-tweezers experiments provides compelling evidence that only one species is present in each dsDNA. Under this condition, molecules containing G-quadruplex are more stable than those with i-motif. These two species have mechanical stabilities (rupture force ≥ 17 pN) comparable to the stall force of RNA polymerases, which, from a mechanical perspective alone, could justify a regulatory mechanism for tetraplex structures in the expression of human insulin. PMID:22713573

  17. A novel colorimetric potassium sensor based on the substitution of lead from G-quadruplex.

    Science.gov (United States)

    Sun, Huijiao; Li, Xiaohong; Li, Yunchao; Fan, Louzhen; Kraatz, Heinz-Bernhard

    2013-02-21

    Potassium ions play diverse roles in biological processes, and abnormal K(+) levels are the hallmarks of diseases. However, the potential clinical application of the developed DNA-based K(+) sensors remains a challenge due to the presence of Pb(2+) in blood samples. In this contribution, a novel colorimetric potassium sensing assay that functions in the presence of Pb(2+) is reported. This approach is based on conformational switching of a hairpin DNA structure to a G-quadruplex. Specifically, the hairpin DNA containing G-rich aptamer T30695 is exposed to successive amounts of Na(+), Pb(2+) and K(+). These cations induce formation of the corresponding metal-stabilized G-quadruplex, which acts as DNAzyme (with hemin as a cofactor) for the catalytic oxidation of ABTS by H(2)O(2). Importantly, studies presented here show that K(+) replaces Pb(2+) from the G-quadruplex to form K(+)-stabilized G-quadruplex, which differ in the catalytic behavior. With Pb(2+)-stabilized G-quadruplex as a probe, a highly sensitive and selective colorimetric detection of K(+) is achieved in the presence of Pb(2+) and excessive Na(+) (140 mM) with the detection limit of 1.9 nM. This system represents the first known DNAzyme-based colorimetric K(+) sensor, which works in the presence of Pb(2+). Finally, the sensor is successfully applied for K(+) detection in a real human serum sample containing Pb(2+).

  18. A Toolbox for Predicting G-Quadruplex Formation and Stability

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    Han Min Wong

    2010-01-01

    Full Text Available G-quadruplexes are four stranded nucleic acid structures formed around a core of guanines, arranged in squares with mutual hydrogen bonding. Many of these structures are highly thermally stable, especially in the presence of monovalent cations, such as those found under physiological conditions. Understanding of their physiological roles is expanding rapidly, and they have been implicated in regulating gene transcription and translation among other functions. We have built a community-focused website to act as a repository for the information that is now being developed. At its core, this site has a detailed database (QuadDB of predicted G-quadruplexes in the human and other genomes, together with the predictive algorithm used to identify them. We also provide a QuadPredict server, which predicts thermal stability and acts as a repository for experimental data from all researchers. There are also a number of other data sources with computational predictions. We anticipate that the wide availability of this information will be of use both to researchers already active in this exciting field and to those who wish to investigate a particular gene hypothesis.

  19. Dual Binding of an Antibody and a Small Molecule Increases the Stability of TERRA G-Quadruplex.

    Science.gov (United States)

    Yangyuoru, Philip M; Di Antonio, Marco; Ghimire, Chiran; Biffi, Giulia; Balasubramanian, Shankar; Mao, Hanbin

    2015-01-12

    In investigating the binding interactions between the human telomeric RNA (TERRA) G-quadruplex (GQ) and its ligands, it was found that the small molecule carboxypyridostatin (cPDS) and the GQ-selective antibody BG4 simultaneously bind the TERRA GQ. We previously showed that the overall binding affinity of BG4 for RNA GQs is not significantly affected in the presence of cPDS. However, single-molecule mechanical unfolding experiments revealed a population (48 %) with substantially increased mechanical and thermodynamic stability. Force-jump kinetic investigations suggested competitive binding of cPDS and BG4 to the TERRA GQ. Following this, the two bound ligands slowly rearrange, thereby leading to the minor population with increased stability. Given the relevance of G-quadruplexes in the regulation of biological processes, we anticipate that the unprecedented conformational rearrangement observed in the TERRA-GQ-ligand complex may inspire new strategies for the selective stabilization of G-quadruplexes in cells.

  20. Synthesis and biophysical properties of (l)-aTNA based G-quadruplexes

    DEFF Research Database (Denmark)

    Gothelf, Kurt Vesterager; Kumar, Vipin

    2016-01-01

    Novel G-quadruplex structures are constructed by acyclic (l)-threninol nucleic acid and their synthesis and biophysical properties are described. Pyrene excimer fluorescence and circular dichroism (CD) data revealed that four strands of aTNA are oriented in antiparallel direction.......Novel G-quadruplex structures are constructed by acyclic (l)-threninol nucleic acid and their synthesis and biophysical properties are described. Pyrene excimer fluorescence and circular dichroism (CD) data revealed that four strands of aTNA are oriented in antiparallel direction....

  1. Altered biochemical specificity of G-quadruplexes with mutated tetrads

    Czech Academy of Sciences Publication Activity Database

    Švehlová, Kateřina; Lawrence, M. S.; Bednárová, Lucie; Curtis, Edward A.

    2016-01-01

    Roč. 44, č. 22 (2016), s. 10789-10803 ISSN 0305-1048 Institutional support: RVO:61388963 Keywords : G-quadruplex * G motif GTP aptamer * peroxidase deoxyribozyme Subject RIV: CE - Biochemistry Impact factor: 10.162, year: 2016 https://academic.oup.com/nar/article/44/22/10789/2333933/Altered-biochemical-specificity-of-G-quadruplexes

  2. G-quadruplex enhanced fluorescence of DNA-silver nanoclusters and their application in bioimaging

    Science.gov (United States)

    Zhu, Jinbo; Zhang, Libing; Teng, Ye; Lou, Baohua; Jia, Xiaofang; Gu, Xiaoxiao; Wang, Erkang

    2015-07-01

    Guanine proximity based fluorescence enhanced DNA-templated silver nanoclusters (AgNCs) have been reported and applied for bioanalysis. Herein, we studied the G-quadruplex enhanced fluorescence of DNA-AgNCs and gained several significant conclusions, which will be helpful for the design of future probes. Our results demonstrate that a G-quadruplex can also effectively stimulate the fluorescence potential of AgNCs. The major contribution of the G-quadruplex is to provide guanine bases, and its special structure has no measurable impact. The DNA-templated AgNCs were further analysed by native polyacrylamide gel electrophoresis and the guanine proximity enhancement mechanism could be visually verified by this method. Moreover, the fluorescence emission of C3A (CCCA)4 stabilized AgNCs was found to be easily and effectively enhanced by G-quadruplexes, such as T30695, AS1411 and TBA, especially AS1411. Benefiting from the high brightness of AS1411 enhanced DNA-AgNCs and the specific binding affinity of AS1411 for nucleolin, the AS1411 enhanced AgNCs can stain cancer cells for bioimaging.Guanine proximity based fluorescence enhanced DNA-templated silver nanoclusters (AgNCs) have been reported and applied for bioanalysis. Herein, we studied the G-quadruplex enhanced fluorescence of DNA-AgNCs and gained several significant conclusions, which will be helpful for the design of future probes. Our results demonstrate that a G-quadruplex can also effectively stimulate the fluorescence potential of AgNCs. The major contribution of the G-quadruplex is to provide guanine bases, and its special structure has no measurable impact. The DNA-templated AgNCs were further analysed by native polyacrylamide gel electrophoresis and the guanine proximity enhancement mechanism could be visually verified by this method. Moreover, the fluorescence emission of C3A (CCCA)4 stabilized AgNCs was found to be easily and effectively enhanced by G-quadruplexes, such as T30695, AS1411 and TBA, especially

  3. Blocking the binding of WT1 to bcl-2 promoter by G-quadruplex ligand SYUIQ-FM05

    Directory of Open Access Journals (Sweden)

    Yun-Xia Xiong

    2016-03-01

    Full Text Available At present, wt1, a Wilms’ tumor suppressor gene, is recognized as a critical regulator of tumorigenesis and a potential therapeutic target. WT1 shows the ability to regulate the transcription of bcl-2 by binding to a GC-rich region in the promoter, which can then fold into a special DNA secondary structure called the G-quadruplex. This function merits the exploration of the effect of a G-quadruplex ligand on the binding and subsequent regulation of WT1 on the bcl-2 promoter. In the present study, WT1 was found to bind to the double strand containing the G-quadruplex-forming sequence of the bcl-2 promoter. However, the G-quadruplex ligand SYUIQ-FM05 effectively blocked this binding by interacting with the GC-rich sequence. Our new findings are significant in the exploration of new strategies to block WT1's transcriptional regulation for cancer-cell treatment.

  4. Discovery of a natural product-like c-myc G-quadruplex DNA groove-binder by molecular docking.

    Directory of Open Access Journals (Sweden)

    Dik-Lung Ma

    Full Text Available The natural product-like carbamide (1 has been identified as a stabilizer of the c-myc G-quadruplex through high-throughput virtual screening. NMR and molecular modeling experiments revealed a groove-binding mode for 1. The biological activity of 1 against the c-myc G-quadruplex was confirmed by its ability to inhibit Taq polymerase-mediated DNA extension and c-myc expression in vitro, demonstrating that 1 is able to control c-myc gene expression at the transcriptional level presumably through the stabilization of the c-myc promoter G-quadruplex. Furthermore, the interaction between carbamide analogues and the c-myc G-quadruplex was also investigated by in vitro experiments in order to generate a brief structure-activity relationship (SAR for the observed potency of carbamide 1.

  5. Tetrasubstituted phenanthrolines as highly potent, water-soluble, and selective g-quadruplex ligands

    DEFF Research Database (Denmark)

    Larsen, Anders Foller; Nielsen, Mads Corvinius; Ulven, Trond

    2012-01-01

    Small molecules capable of stabilizing the G-quadruplex (G4) structure are of interest for the development of improved anticancer drugs. Novel 4,7-diamino-substituted 1,10-phenanthroline-2,9-dicarboxamides that represent hybrid structures of known phenanthroline-based ligands have been designed...

  6. Xanthine and 8-oxoguanine in G-quadruplexes: formation of a G·G·X·O tetrad.

    Science.gov (United States)

    Cheong, Vee Vee; Heddi, Brahim; Lech, Christopher Jacques; Phan, Anh Tuân

    2015-12-02

    G-quadruplexes are four-stranded structures built from stacked G-tetrads (G·G·G·G), which are planar cyclical assemblies of four guanine bases interacting through Hoogsteen hydrogen bonds. A G-quadruplex containing a single guanine analog substitution, such as 8-oxoguanine (O) or xanthine (X), would suffer from a loss of a Hoogsteen hydrogen bond within a G-tetrad and/or potential steric hindrance. We show that a proper arrangement of O and X bases can reestablish the hydrogen-bond pattern within a G·G·X·O tetrad. Rational incorporation of G·G·X·O tetrads in a (3+1) G-quadruplex demonstrated a similar folding topology and thermal stability to that of the unmodified G-quadruplex. pH titration conducted on X·O-modified G-quadruplexes indicated a protonation-deprotonation equilibrium of X with a pKa ∼6.7. The solution structure of a G-quadruplex containing a G·G·X·O tetrad was determined, displaying the same folding topology in both the protonated and deprotonated states. A G-quadruplex containing a deprotonated X·O pair was shown to exhibit a more electronegative groove compared to that of the unmodified one. These differences are likely to manifest in the electronic properties of G-quadruplexes and may have important implications for drug targeting and DNA-protein interactions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Modular Assembly of Cell-targeting Devices Based on an Uncommon G-quadruplex Aptamer

    DEFF Research Database (Denmark)

    Opazo, Felipe; Eiden, Laura; Hansen, Line

    2015-01-01

    Aptamers are valuable tools that provide great potential to develop cost-effective diagnostics and therapies in the biomedical field. Here, we report a novel DNA aptamer that folds into an unconventional G-quadruplex structure able to recognize and enter specifically into human Burkitt's lymphoma...

  8. Structure-based design of flavone derivatives as c-myc oncogene down-regulators.

    Science.gov (United States)

    Yang, Hui; Zhong, Hai-Jing; Leung, Ka-Ho; Chan, Daniel Shiu-Hin; Ma, Victor Pui-Yan; Fu, Wai-Chung; Nanjunda, Rupesh; Wilson, W David; Ma, Dik-Lung; Leung, Chung-Hang

    2013-01-23

    Based on molecular docking analysis of complexes between flavone and the c-myc G-quadruplex, we designed and screened 30 flavone derivatives containing various side chains that could potentially form interactions with the G-quadruplex grooves. As a proof-of-concept, the highest-scoring flavone derivatives containing cationic pyridinium side chains were synthesized and their interactions with the c-myc G-quadruplex were examined using a PCR-stop assay. The stabilizing effects of the flavone derivatives were found to be selective towards the c-myc G-quadruplex over other biologically relevant G-quadruplex structures, such as the human telomeric sequence (HTS). The interaction between the most potent compound of the series and the c-myc G-quadruplex was examined in depth using UV-Vis titration, molecular modeling and CD spectroscopy. Our results suggest that in addition to stabilizing the c-myc G-quadruplex, the flavone derivatives were capable of inducing the formation of the G-quadruplex structure even in the absence of monovalent cations. The flavone derivatives were found to be potent inhibitors of c-myc promoters within the cellular environment and displayed promising cytotoxic behavior against human cancer cell lines. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Biophysical Characterization of G-Quadruplex Recognition in the PITX1 mRNA by the Specificity Domain of the Helicase RHAU.

    Directory of Open Access Journals (Sweden)

    Emmanuel O Ariyo

    Full Text Available Nucleic acids rich in guanine are able to fold into unique structures known as G-quadruplexes. G-quadruplexes consist of four tracts of guanylates arranged in parallel or antiparallel strands that are aligned in stacked G-quartet planes. The structure is further stabilized by Hoogsteen hydrogen bonds and monovalent cations centered between the planes. RHAU (RNA helicase associated with AU-rich element is a member of the ATP-dependent DExH/D family of RNA helicases and can bind and resolve G-quadruplexes. RHAU contains a core helicase domain with an N-terminal extension that enables recognition and full binding affinity to RNA and DNA G-quadruplexes. PITX1, a member of the bicoid class of homeobox proteins, is a transcriptional activator active during development of vertebrates, chiefly in the anterior pituitary gland and several other organs. We have previously demonstrated that RHAU regulates PITX1 levels through interaction with G-quadruplexes at the 3'-end of the PITX1 mRNA. To understand the structural basis of G-quadruplex recognition by RHAU, we characterize a purified minimal PITX1 G-quadruplex using a variety of biophysical techniques including electrophoretic mobility shift assays, UV-VIS spectroscopy, circular dichroism, dynamic light scattering, small angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our biophysical analysis provides evidence that the RNA G-quadruplex, but not its DNA counterpart, can adopt a parallel orientation, and that only the RNA can interact with N-terminal domain of RHAU via the tetrad face of the G-quadruplex. This work extends our insight into how the N-terminal region of RHAU recognizes parallel G-quadruplexes.

  10. G-quadruplex DNA biosensor for sensitive visible detection of genetically modified food.

    Science.gov (United States)

    Jiang, Xiaohua; Zhang, Huimin; Wu, Jun; Yang, Xiang; Shao, Jingwei; Lu, Yujing; Qiu, Bin; Lin, Zhenyu; Chen, Guonan

    2014-10-01

    In this paper, a novel label-free G-quadruplex DNAzyme sensor has been proposed for colorimetric identification of GMO using CaMV 35S promoter sequence as the target. The binary probes can fold into G-quadruplex structure in the presence of DNA-T (Target DNA) and then combine with hemin to form a DNAzyme resembling horseradish peroxidase. The detection system consists of two G-rich probes with 2:2 split mode by using the absorbance and color of ABTS(2-) as signal reporter. Upon the addition of a target sequence, two probes both hybridize with target and then their G-rich sequences combine to form a G-quadruplex DNAzyme, and the DNAzyme can catalyze the reaction of ABTS(2-) with H2O2. Then the linear range is from 0.05 to 0.5 μM while detection limit is 5nM. These results demonstrate that the proposed G-quadruplex DNAzyme method could be used as a simple, sensitive and cost-effective approach for assays of GMO. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. G-Quadruplex DNAzyme Molecular Beacon for Amplified Colorimetric Biosensing of Pseudostellaria heterophylla

    Directory of Open Access Journals (Sweden)

    Juan Hu

    2013-01-01

    Full Text Available With an internal transcribed spacer of 18 S, 5.8 S and 26 S nuclear ribosomal DNA (nrDNA ITS as DNA marker, we report a colorimetric approach for authentication of Pseudostellaria heterophylla (PH and its counterfeit species based on the differentiation of the nrDNA ITS sequence. The assay possesses an unlabelled G-quadruplex DNAzyme molecular beacon (MB probe, employing complementary sequence as biorecognition element and 1:1:1:1 split G-quadruplex halves as reporter. In the absence of target DNA (T-DNA, the probe can shape intermolecular G-quadruplex structures capable of binding hemin to form G-quadruplex-hemin DNAzyme and catalyze the oxidation of ABTS2− to blue-green ABTS•− by H2O2. In the presence of T-DNA, T-DNA can hybridize with the complementary sequence to form a duplex structure, hindering the formation of the G-quadruplex structure and resulting in the loss of the catalytic activity. Consequently, a UV-Vis absorption signal decrease is observed in the ABTS2−-H2O2 system. The “turn-off” assay allows the detection of T-DNA from 1.0 × 10−9 to 3.0 × 10−7 mol·L−1 (R2 = 0.9906, with a low detection limit of 3.1 × 10−10 mol·L−1. The present study provides a sensitive and selective method and may serve as a foundation of utilizing the DNAzyme MB sensor for identifying traditional Chinese medicines.

  12. The effect of pyridyl substituents on the thermodynamics of porphyrin binding to G-quadruplex DNA.

    Science.gov (United States)

    Rowland, Gerald B; Barnett, Kerry; Dupont, Jesse I; Akurathi, Gopalakrishna; Le, Vu H; Lewis, Edwin A

    2013-12-01

    Most of the G-quadruplex interactive molecules reported to date contain extended aromatic flat ring systems and are believed to bind principally by π-π stacking on the end G-tetrads of the quadruplex structure. One such molecule, TMPyP4, (5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin), exhibits high affinity and some selectivity for G-quadruplex DNA over duplex DNA. Although not a realistic drug candidate, TMPyP4 is used in many nucleic acid research laboratories as a model ligand for the study of small molecule G-quadruplex interactions. Here we report on the synthesis and G-quadruplex interactions of four new cationic porphyrin ligands having only 1, 2, or 3 (N-methyl-4-pyridyl) substituents. The four new ligands are: P(5) (5-(N-methyl-4-pyridyl)porphyrin), P(5,10) (5,10-di(N-methyl-4-pyridyl)porphyrin), P(5,15) (5,15-di(N-methyl-4-pyridyl)porphyrin), and P(5,10,15) (5,10,15-tri(N-methyl-4-pyridyl)porphyrin). Even though these compounds have been previously synthesized, we report alternative synthetic routes that are more efficient and that result in higher yields. We have used ITC, CD, and ESI-MS to explore the effects of the number of N-methyl-4-pyridyl substituents and the substituent position on the porphyrin on the G-quadruplex binding energetics. The relative affinities for binding these ligands to the WT Bcl-2 promoter sequence G-quadruplex are: K(TMPyP4)≈K(P)(5,15)>KP(5,10,15)>KP(5,10), KP(5). The saturation stoichiometry is 2:1 for both P(5,15) and P(5,10,15), while neither P(5) nor P(5,10) exhibit significant complex formation with the WT Bcl-2 promoter sequence G-quadruplex. Additionally, binding of P(5,15) appears to interact by an 'intercalation mode' while P(5,10,15) appears to interact by an 'end-stacking mode'. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Dissecting the strand folding orientation and formation of G-quadruplexes in single- and double-stranded nucleic acids by ligand-induced photocleavage footprinting.

    Science.gov (United States)

    Zheng, Ke-wei; Zhang, Dan; Zhang, Li-xia; Hao, Yu-hua; Zhou, Xiang; Tan, Zheng

    2011-02-09

    The widespread of G-quadruplex-forming sequences in genomic DNA and their role in regulating gene expression has made G-quadruplex structures attractive therapeutic targets against a variety of diseases, such as cancer. Information on the structure of G-quadruplexes is crucial for understanding their physiological roles and designing effective drugs against them. Resolving the structures of G-quadruplexes, however, remains a challenge especially for those in double-stranded DNA. In this work, we developed a photocleavage footprinting technique to determine the folding orientation of each individual G-tract in intramolecular G-quadruplex formed in both single- and double-stranded nucleic acids. Based on the differential photocleavage induced by a ligand tetrakis(2-trimethylaminoethylethanol) phthalocyaninato zinc tetraiodine (Zn-TTAPc) to the guanines between the two terminal G-quartets in a G-quadruplex, this method identifies the guanines hosted in each terminal G-quartets to reveal G-tract orientation. The method is extremely intuitive, straightforward, and requires little expertise. Besides, it also detects G-quadruplex formation in long single- and double-stranded nucleic acids.

  14. A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Hao; Suslov, Nikolai B.; Li, Nan-Sheng; Shelke, Sandip A.; Evans, Molly E.; Koldobskaya, Yelena; Rice, Phoebe A.; Piccirilli, Joseph A. [UC

    2014-08-21

    Spinach is an in vitro–selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence. Spinach is thus an RNA analog of GFP and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2-Å and 2.4-Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially preformed binding site for the fluorophore. The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers.

  15. Templated Formation of Discrete RNA and DNA:RNA Hybrid G-Quadruplexes and Their Interactions with Targeting Ligands.

    Science.gov (United States)

    Bonnat, Laureen; Dejeu, Jérôme; Bonnet, Hugues; Génnaro, Béatrice; Jarjayes, Olivier; Thomas, Fabrice; Lavergne, Thomas; Defrancq, Eric

    2016-02-24

    G-rich RNA and DNA oligonucleotides derived from the human telomeric sequence were assembled onto addressable cyclopeptide platforms through oxime ligations and copper-catalyzed azide-alkyne cycloaddition (CuAAc) reactions. The resulting conjugates were able to fold into highly stable RNA and DNA:RNA hybrid G-quadruplex (G4) architectures as demonstrated by UV, circular dichroism (CD), and NMR spectroscopic analysis. Whereas rationally designed parallel RNA and DNA:RNA hybrid G4 topologies could be obtained, we could not force the formation of an antiparallel RNA G4 structure, thus supporting the idea that this topology is strongly disfavored. The binding affinities of four representative G4 ligands toward the discrete RNA and DNA:RNA hybrid G4 topologies were compared to the one obtained with the corresponding DNA G4 structure. Surface plasmon resonance (SPR) binding analysis suggests that the accessibility to G4 recognition elements is different among the three structures and supports the idea that G4 ligands might be shaped to achieve structure selectivity in a biological context. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. A mRNA-Responsive G-Quadruplex-Based Drug Release System

    Directory of Open Access Journals (Sweden)

    Hidenobu Yaku

    2015-04-01

    Full Text Available G-quadruplex-based drug delivery carriers (GDDCs were designed to capture and release a telomerase inhibitor in response to a target mRNA. Hybridization between a loop on the GDDC structure and the mRNA should cause the G-quadruplex structure of the GDDC to unfold and release the bound inhibitor, anionic copper(II phthalocyanine (CuAPC. As a proof of concept, GDDCs were designed with a 10-30-mer loop, which can hybridize with a target sequence in epidermal growth factor receptor (EGFR mRNA. Structural analysis using circular dichroism (CD spectroscopy showed that the GDDCs form a (3 + 1 type G-quadruplex structure in 100 mM KCl and 10 mM MgCl2 in the absence of the target RNA. Visible absorbance titration experiments showed that the GDDCs bind to CuAPC with Ka values of 1.5 × 105 to 5.9 × 105 M−1 (Kd values of 6.7 to 1.7 μM at 25 °C, depending on the loop length. Fluorescence titration further showed that the G-quadruplex structure unfolds upon binding to the target RNA with Ka values above 1.0 × 108 M−1 (Kd values below 0.01 μM at 25 °C. These results suggest the carrier can sense and bind to the target RNA, which should result in release of the bound drug. Finally, visible absorbance titration experiments demonstrated that the GDDC release CuAPC in response to the target RNA.

  17. A triple stranded G-quadruplex formation in the promoter region of human myosin β(Myh7) gene.

    Science.gov (United States)

    Singh, Anju; Kukreti, Shrikant

    2017-09-19

    Regulatory regions in human genome, enriched in guanine-rich DNA sequences have the propensity to fold into G-quadruplex structures. On exploring the genome for search of G-tracts, it was interesting to find that promoter of Human Myosin Gene (MYH7) contains a conserved 23-mer G-rich sequence (HM-23). Mutations in this gene are associated with familial cardiomyopathy. Enrichment of MYH7 gene in G-rich sequences could possibly play a critical role in its regulation. We used polyacrylamide gel electrophoresis (PAGE), UV-Thermal denaturation (UV-Tm) and Circular Dichroism (CD), to demonstrate the formation of a G-quadruplex by 23-mer G-rich sequence HM23 in promoter location of MYH7 gene. We observed that the wild G-rich sequence HM23 containing consecutive G5 stretch in two stacks adopt G-quadruplexes of diverse molecularity by involvement of four-strand, three-strand and two-strands with same parallel topology. Interestingly, the mutated sequence in the absence of continuous G5 stretch obstructs the formation of three-stranded G-quadruplex. We demonstrated that continuous G5 stretch is mandatory for the formation of a unique three-stranded G-quadruplex. Presence of various transcription factors (TF) in vicinity of the sequence HM23 leave fair possibility of recognition by TF binding sites, and so modulate gene expression. These findings may add on our understanding about the effect of base change in the formation of varied structural species in similar solution condition. This study may give insight about structural polymorphism arising due to recognition of non-Watson-Crick G-quadruplex structures by cellular proteins and designing structure specific molecules.

  18. Evidences for Piperine inhibiting cancer by targeting human G-quadruplex DNA sequences

    OpenAIRE

    Arpita Tawani; Ayeman Amanullah; Amit Mishra; Amit Kumar

    2016-01-01

    Piperine, a naturally occurring alkaloid, is well known as anti-oxidant, anti-mutagenic, anti-tumor and anti-proliferative agent. Piperine exerts such pharmacological activities by binding or interacting with various cellular targets. Recently, the first report for Piperine interaction with duplex DNA has been published last year but its interaction with G-quadruplex structures has not been studied yet. Herein, we report for the first time the interaction of Piperine with various DNA G-quadru...

  19. G-rich VEGF aptamer with locked and unlocked nucleic acid modifications exhibits a unique G-quadruplex fold

    DEFF Research Database (Denmark)

    Marusic, Maja; Veedu, Rakesh N; Wengel, Jesper

    2013-01-01

    The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K(+) rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes......-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general....

  20. G-Quadruplex Identification in the Genome of Protozoan Parasites Points to Naphthalene Diimide Ligands as New Antiparasitic Agents.

    Science.gov (United States)

    Belmonte-Reche, Efres; Martínez-García, Marta; Guédin, Aurore; Zuffo, Michela; Arévalo-Ruiz, Matilde; Doria, Filippo; Campos-Salinas, Jenny; Maynadier, Marjorie; López-Rubio, José Juan; Freccero, Mauro; Mergny, Jean-Louis; Pérez-Victoria, José María; Morales, Juan Carlos

    2018-02-08

    G-quadruplexes (G4) are DNA secondary structures that take part in the regulation of gene expression. Putative G4 forming sequences (PQS) have been reported in mammals, yeast, bacteria, and viruses. Here, we present PQS searches on the genomes of T. brucei, L. major, and P. falciparum. We found telomeric sequences and new PQS motifs. Biophysical experiments showed that EBR1, a 29 nucleotide long highly repeated PQS in T. brucei, forms a stable G4 structure. G4 ligands based on carbohydrate conjugated naphthalene diimides (carb-NDIs) that bind G4's including hTel could bind EBR1 with selectivity versus dsDNA. These ligands showed important antiparasitic activity. IC 50 values were in the nanomolar range against T. brucei with high selectivity against MRC-5 human cells. Confocal microscopy confirmed these ligands localize in the nucleus and kinetoplast of T. brucei suggesting they can reach their potential G4 targets. Cytotoxicity and zebrafish toxicity studies revealed sugar conjugation reduces intrinsic toxicity of NDIs.

  1. 9-N-Substituted berberine derivatives: stabilization of G-quadruplex DNA and down-regulation of oncogene c-myc.

    Science.gov (United States)

    Ma, Yan; Ou, Tian-Miao; Hou, Jin-Qiang; Lu, Yu-Jing; Tan, Jia-Heng; Gu, Lian-Quan; Huang, Zhi-Shu

    2008-08-15

    A series of 9-N-substituted berberine derivatives (2a-j) were synthesized and evaluated as a new class of G-quadruplex binding ligands. G-quadruplex of DNA had been proven to be the transcription controller of human c-myc gene. The interaction of 9-N-substituted berberine derivatives with G-quadruplex DNA in c-myc was examined via EMSA, CD spectroscopy, FRET-melting method, PCR-stop assay, competitive dialysis, cell proliferation assay, and RT-PCR assay. The experiment results indicated that these derivatives could selectively induce and stabilize the formation of intramolecular parallel G-quadruplex in c-myc, which led to down-regulation of transcription of the c-myc in the HL60 lymphomas cell line. The related structure-activity relationships were also discussed.

  2. “Turn-off-on” fluorescent sensor for (N-methyl-4-pyridyl) porphyrin -DNA and G-quadruplex interactions based on ZnCdSe quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Dan; Fan, Yao; Gao, Fang; Yang, Tian-ming, E-mail: tmyang@mail.scuec.edu.cn

    2015-08-12

    As a new detection model, the reversible fluorescence “turn-off-on” sensor based on quantum dots (QDs) has already been successfully employed in the detections of many biochemical materials, especially in the researches on the interactions between anticancer drugs. The previous studies, however, mainly focused on simple-structured oligonucleotides and Calf thymus DNA. G-quadruplex, an important target for anti-cancer drug with special secondary structure, has been stimulating increasing research interests. In this paper, we report a new detection method based on the fluorescence “turn-off-on” model with water-soluble ZnCdSe QDs as the fluorescent probe, to analyze the interactions between anticancer drug (N-methyl-4-pyridyl) porphyrin (TMPyP) and nucleic acid, especially the G-quadruplex. The fluorescence of QDs can be quenched by TMPyP via photo-induced electron transfer and fluorescence resonance energy transfer, while on the other hand, the combination between TMPyP and G-quadruplex releases QDs from their quenchers and thus recovers the fluorescence. Most importantly, the fluorescence “turn-off-on” model has been employed, for the first time, to analyze the impacts of special factors on the interaction between TMPyP and G-quadruplex. The excellent selectivity of the system has been verified in the studies of the interactions between TMPyP and different DNAs (double-stranded DNA, single-stranded G-quadruplex, and different types of G-quadruplexes) in Na{sup +} or K{sup +}-containing buffer. - Highlights: • Reversible fluorescence sensor was firstly used on TMPyP and G-quadruplex study. • SsDNA and various G-quadruplexes were successfully recognized by fluorescence. • The new quantum dot is hypotoxicity and can be extensively applied.

  3. Phenanthroline-2,9-bistriazoles as selective G-quadruplex ligands

    DEFF Research Database (Denmark)

    Nielsen, Mads Corvinius; Larsen, Anders Foller; Abdikadir, Faisal Hussein

    2014-01-01

    (I)-catalyzed azide-alkyne cycloaddition (CuAAC) in CH2Cl2 and water in the presence of a complexing agent. Characterization of the target compounds on telomeric and c-KIT G4 sequences led to the identification of guanidinium-substituted compounds as potent G4 DNA ligands with high selectivity over duplex DNA......G-quadruplex (G4) ligands are currently receiving considerable attention as potential anticancer therapeutics. A series of phenanthroline-2,9-bistriazoles carrying tethered positive end groups has been synthesized and evaluated as G4 stabilizers. The compounds were efficiently assembled by copper...... were thus identified as potent G4 ligands with high selectivity over duplex DNA, and preliminary results indicate that the scaffold may form basis for the development of subtype-specific G4 ligands....

  4. Comparative investigation of binding interactions with three steroidal derivatives of d(GGGT)4G-quadruplex aptamer.

    Science.gov (United States)

    Huang, Shan; Liang, Yu; Cui, Jianguo; Xie, Jiangning; Liu, Yi; Hu, Baoqing; Xiao, Qi

    2018-02-16

    Steroidal derivatives have attracted tremendous attentions in biological and biomedical areas, due to their variety biological activities. The investigation of structural influences helps in understanding their biological activities. The interactions of steroidal derivatives with DNA may play important roles in biological activities, however only a few investigations were reported on this issue. Herein, the structural influences of three steroidal derivatives were investigated based on their binding interactions with d(GGGT) 4 G-quadruplex aptamer by spectroscopic approaches, nuclear magnetic resonance (NMR), electrochemical methods, and molecular modeling techniques. Three compounds were found to selectively bind with parallel G-quadruplex aptamer to form three complexes through end-stacking binding modes. Three compounds stabilized the G-quadruplex structure of the aptamer at different levels, which enhanced the biological activity of this aptamer to some extent. The space steric hindrance was responsible for differences in the binding interactions between d(GGGT) 4 G-quadruplex aptamer and three compounds. These results provide new information for the molecular understanding of binding interactions of steroidal derivatives with DNA and the strategy for research of structural influences. Copyright © 2018. Published by Elsevier Inc.

  5. G-quadruplexes regulate Epstein-Barr virus-encoded nuclear antigen 1 mRNA translation.

    Science.gov (United States)

    Murat, Pierre; Zhong, Jie; Lekieffre, Lea; Cowieson, Nathan P; Clancy, Jennifer L; Preiss, Thomas; Balasubramanian, Shankar; Khanna, Rajiv; Tellam, Judy

    2014-05-01

    Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cis-acting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals for translational control and immune evasion. Furthermore, these findings suggest alternative therapeutic strategies focused on targeting RNA structure within viral ORFs.

  6. Target guided synthesis using DNA nano-templates for selectively assembling a G-quadruplex binding c-MYC inhibitor

    Science.gov (United States)

    Panda, Deepanjan; Saha, Puja; Das, Tania; Dash, Jyotirmayee

    2017-07-01

    The development of small molecules is essential to modulate the cellular functions of biological targets in living system. Target Guided Synthesis (TGS) approaches have been used for the identification of potent small molecules for biological targets. We herein demonstrate an innovative example of TGS using DNA nano-templates that promote Huisgen cycloaddition from an array of azide and alkyne fragments. A G-quadruplex and a control duplex DNA nano-template have been prepared by assembling the DNA structures on gold-coated magnetic nanoparticles. The DNA nano-templates facilitate the regioselective formation of 1,4-substituted triazole products, which are easily isolated by magnetic decantation. The G-quadruplex nano-template can be easily recovered and reused for five reaction cycles. The major triazole product, generated by the G-quadruplex inhibits c-MYC expression by directly targeting the c-MYC promoter G-quadruplex. This work highlights that the nano-TGS approach may serve as a valuable strategy to generate target-selective ligands for drug discovery.

  7. Exploration of binding affinity and selectivity of brucine with G-quadruplex in the c-myb proto-oncogene by electrospray ionization mass spectrometry.

    Science.gov (United States)

    Li, Huihui; Hai, Jinhui; Zhou, Jiang; Yuan, Gu

    2016-02-15

    The c-myb gene is a potential therapeutic target for human tumors and leukemias. Active ingredients from natural products may be used as drugs in chemotherapy for human cancers. Here, electrospray ionization mass spectrometry (ESI-MS) was used to probe the formation and recognition of the G-quadruplex structure from the G-rich sequence that is found in the c-myb gene promoter, 5'-GGGCTGGGCTGGGCGGGG-3'. The aim of our study is to evaluate a potential binder for the c-myb gene from natural products, and thereby to modulate c-myb gene expression. ESI-MS, as an effective method, was utilized not only to characterize the formation of the G-quadruplex in the c-myb oncogene, but also as a tool to probe the binding characteristics of alkaloid molecules with the target G-quadruplex DNA. ESI-MS results with the support of circular dichroism (CD) spectra demonstrated the formation of an intramolecular parallel-stranded G-quadruplex in the c-myb oncogene promoter. A screening of six alkaloid molecules showed that brucine (P1) had a strong binding affinity to the c-myb G-quadruplex DNA. It is notable that P1 can bind selectively to the c-myb G-quadruplex with respect to duplex DNAs, as well as to G-quadruplexes in other types of gene sequences. According to ESI-MS results, in which the stability was tested by capillary heating and collision-induced dissociation, the binding of P1 could thermally stabilize the c-myb G-quadruplex DNA. In this work, brucine (P1), an alkaloid molecule, has been found to bind to the intramolecular parallel G-quadruplex in the c-myb oncogene promoter with high affinity and selectivity, and could thermally stabilize the c-myb G-quadruplex DNA, indicating that the binding of P1 has the potential to modulate c-myb gene expression. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  8. Interaction of a Cationic Porphyrin and Its Metal Derivatives with G-Quadruplex DNA.

    Science.gov (United States)

    Boschi, Eric; Davis, Supriya; Taylor, Scott; Butterworth, Andrew; Chirayath, Lilyan A; Purohit, Vaishali; Siegel, Laura K; Buenaventura, Janesha; Sheriff, Alexandra H; Jin, Rowen; Sheardy, Richard; Yatsunyk, Liliya A; Azam, Mahrukh

    2016-12-22

    G-quadruplex (GQ) structures formed from guanine-rich sequences are found throughout the genome and are overrepresented in the promoter regions of some oncogenes, at the telomeric ends of eukaryotic chromosomes, and at the 5'-untranslated regions of mRNA. Interaction of small molecule ligands with GQ DNA is an area of great research interest to develop novel anticancer therapeutics and GQ sensors. In this paper we examine the interactions of TMPyP4, its isomer TMPyP2 (containing N-methyl-2-pyridyl substituents, N-Me-2Py) as well as two metal derivatives ZnTMPyP4 and CuTMPyP4 with GQs formed by dT4G4 and dT4G4T in 100 mM K+ or Na+ conditions. The DNA sequences were chosen to elucidate the effect of the 3'-T on the stabilization effect of porphyrins, binding modes, affinities, and stoichiometries determined via circular dichroism melting studies, UV-vis titrations, continuous variation analysis, and fluorescence studies. Our findings demonstrate that the stabilizing abilities of porphyrins are stronger toward (dT4G4)4 as compared to (dT4G4T)4 (ΔTm is 4.4 vs -6.4 for TMPyP4; 12.7 vs 5.7 for TMPyP2; 16.4 vs 12.1 for ZnTMPyP4; and 1.9 vs -8.4 °C for CuTMPyP4) suggesting that the 3'G-tetrad presents at least one of the binding sites. The binding affinity was determined to be moderate (Ka ∼ 106-107 μM-1) with a typical binding stoichiometry of 1:1 or 2:1 porphyrin-to-GQ. In all studies, ZnTMPyP4 emerged as a ligand superior to TMPyP4. Overall, our work contributes to clearer understanding of interactions between porphyrins and GQ DNA.

  9. Role of loop residues and cations on the formation and stability of dimeric DNA G-quadruplexes.

    Science.gov (United States)

    Cevec, Mirko; Plavec, Janez

    2005-11-22

    Formation of guanine-quadruplexes by four DNA oligonucleotides with common sequence dG4-loop-dG4 has been studied by a combination of NMR and UV spectroscopy. The loops consisted of 1',2'-dideoxyribose, propanediol, hexaethylene glycol, and thymine residues. The comparison of data on modified and parent oligonucleotides gave insight into the role of loop residues on formation and stability of dimeric G-quadruplexes. All modified oligonucleotides fold into dimeric fold-back G-quadruplexes in the presence of sodium ions. Multiple structures form in the presence of potassium and ammonium ions, which is in contrast to the parent oligonucleotide with dT4 loop. 15N-filtered 1H NMR spectra demonstrate that all studied G-quadruplexes exhibit three 15NH4(+) ion binding sites. Topology of intermolecular G-quadruplexes was evaluated by NMR measurements and diffusion experiments. The spherical, prolate-ellipsoid and symmetric cylinder models were used to interpret experimental translational diffusion constants in terms of diameters and lengths of unfolded oligonucleotides and their respective G-quadruplexes. UV melting and annealing curves show that oligonucleotides with non-nucleosidic loop residues fold faster, exhibit no hysteresis, and are less stable than dimeric d(G4T4G4)2 which can be attributed to the absence of H-bonds, stacking between loop residues and the outer G-quartets as well as cation-pi interactions. Oligonucleotide consisting of hexaethylene glycol linkage with only two phosphate groups in the loop exhibits higher melting temperature and more negative deltaH(o) and deltaG(o) values than oligonucleotides with four 1',2'-dideoxyribose or propanediol residues.

  10. G-Quadruplexes in DNA Replication: A Problem or a Necessity?

    Science.gov (United States)

    Valton, Anne-Laure; Prioleau, Marie-Noëlle

    2016-11-01

    DNA replication is a highly regulated process that ensures the correct duplication of the genome at each cell cycle. A precise cell type-specific temporal program controls the duplication of complex vertebrate genomes in an orderly manner. This program is based on the regulation of both replication origin firing and replication fork progression. G-quadruplexes (G4s), DNA secondary structures displaying noncanonical Watson-Crick base pairing, have recently emerged as key controllers of genome duplication. Here we discuss the various means by which G4s affect this fundamental cellular process. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Influence of base stacking geometry on the nature of excited states in G-quadruplexes: a time-dependent DFT study.

    Science.gov (United States)

    Lech, Christopher J; Phan, Anh Tuân; Michel-Beyerle, Maria-Elisabeth; Voityuk, Alexander A

    2015-03-05

    G-quadruplexes are four-stranded structures of nucleic acids that are formed from the association of guanine nucleobases into cyclical arrangements known as tetrads. G-quadruplexes are involved in a host of biological processes and are of interest in nanomaterial applications. However, not much is known about their electronic properties. In this paper, we analyze electronic excited states of G-quadruplexes using a combination of time-dependent DFT calculations and molecular dynamics simulations. We systematically consider experimentally observed arrangements of stacked guanine tetrads. The effects of structural features on exciton delocalization and photoinduced charge separation are explored using a quantitative analysis of the transition electron density. It is shown that collective coherent excitations shared between two guanine nucleobases dominate in the absorption spectrum of stacked G-tetrads. These excitations may also include a significant contribution of charge transfer states. Large variation in exciton localization is also observed between different structures with a general propensity toward localization between two bases. We reveal large differences in how charge separation occurs within different nucleobase arrangements, with some geometries favoring separation within a single tetrad and others favoring separation between tetrads. We also investigate the effects of the coordinating K(+) ion located in the central cavity of G-quadruplexes on the relative excited state properties of such systems. Our results demonstrate how the nature of excited states in G-quadruplexes depends on the nucleobase stacking geometry resulting from the mutual arrangement of guanine tetrads.

  12. Spectral properties and thermal stability of AS1411 G-quadruplex.

    Science.gov (United States)

    Bagheri, Zeinab; Ranjbar, Bijan; Latifi, Hamid; Zibaii, Mohammad Ismail; Moghadam, Tahereh Tohidi; Azizi, Azade

    2015-01-01

    G-quadruplexes are supramolecular structures of G-rich nucleic acid, formed by non-canonical base pairing in the presence of specific environmental inducers. These structures have been vastly considered in diagnostic and therapeutic applications. However, detailed information on structure, optical properties and thermal stability of G-quadruplex potent oligonucleotides is scarce. Herein, optical properties and thermodynamic stability of AS1411 quadruplex is reported for various concentrations of potassium and lead ions. Circular dichroism showed that AS1411 ss-DNA folds into parallel conformation in the presence of metal ions and molecular crowding condition. UV-vis spectroscopy indicated formation of quadruplex and fluorescent spectroscopy revealed intercalation of PicoGreen in its structure, with enhancement of emission intensity upon increment of metal ion concentration. This investigation also proposes high-throughput and reliable analysis of AS1411 quadruplex's thermal stability by real-time PCR technique, which can be further applied for other quadruplex structures. Copyright © 2014. Published by Elsevier B.V.

  13. Stabilization of G-quadruplex DNA with platinum(II) Schiff base complexes: luminescent probe and down-regulation of c-myc oncogene expression.

    Science.gov (United States)

    Wu, Peng; Ma, Dik-Lung; Leung, Chung-Hang; Yan, Siu-Cheong; Zhu, Nianyong; Abagyan, R; Che, Chi-Ming

    2009-12-07

    The interactions of a series of platinum(II) Schiff base complexes with c-myc G-quadruplex DNA were studied. Complex [PtL(1a)] (1 a; H(2)L(1a)=N,N'-bis(salicylidene)-4,5-methoxy-1,2-phenylenediamine) can moderately inhibit c-myc gene promoter activity in a cell-free system through stabilizing the G-quadruplex structure and can inhibit c-myc oncogene expression in cultured cells. The interaction between 1 a and G-quadruplex DNA has been examined by (1)H NMR spectroscopy. By using computer-aided structure-based drug design for hit-to-lead optimization, an in silico G-quadruplex DNA model has been constructed for docking-based virtual screening to develop new platinum(II) Schiff base complexes with improved inhibitory activities. Complex [PtL(3)] (3; H(2)L(3)=N,N'-bis{4-[1-(2-propylpiperidine)oxy]salicylidene}-4,5-methoxy-1,2-phenylenediamine) has been identified with a top score in the virtual screening. This complex was subsequently prepared and experimentally tested in vitro for its ability to stabilize or induce the formation of the c-myc G-quadruplex. The inhibitory activity of 3 (IC(50)=4.4 muM) is tenfold more than that of 1 a. The interaction between 1 a or 3 with c-myc G-quadruplex DNA has been examined by absorption titration, emission titration, molecular modeling, and NMR titration experiments, thus revealing that both 1 a and 3 bind c-myc G-quadruplex DNA through an external end-stacking mode at the 3' terminal face of the G-quadruplex. Such binding of G-quadruplex DNA with 3 is accompanied by up to an eightfold increase in the intensity of photoluminescence at lambda(max)=652 nm. Complex 3 also effectively down-regulated the expression of c-myc in human hepatocarcinoma cells.

  14. Quinone methides tethered to naphthalene diimides as selective G-quadruplex alkylating agents.

    Science.gov (United States)

    Di Antonio, Marco; Doria, Filippo; Richter, Sara N; Bertipaglia, Carolina; Mella, Mariella; Sissi, Claudia; Palumbo, Manlio; Freccero, Mauro

    2009-09-16

    We have developed novel G-quadruplex (G-4) ligand/alkylating hybrid structures, tethering the naphthalene diimide moiety to quaternary ammonium salts of Mannich bases, as quinone-methide precursors, activatable by mild thermal digestion (40 degrees C). The bis-substituted naphthalene diimides were efficiently synthesized, and their reactivity as activatable bis-alkylating agents was investigated in the presence of thiols and amines in aqueous buffered solutions. The electrophilic intermediate, quinone-methide, involved in the alkylation process was trapped, in the presence of ethyl vinyl ether, in a hetero Diels-Alder [4 + 2] cycloaddition reaction, yielding a substituted 2-ethoxychroman. The DNA recognition and alkylation properties of these new derivatives were investigated by gel electrophoresis, circular dichroism, and enzymatic assays. The alkylation process occurred preferentially on the G-4 structure in comparison to other DNA conformations. By dissecting reversible recognition and alkylation events, we found that the reversible process is a prerequisite to DNA alkylation, which in turn reinforces the G-quadruplex structural rearrangement.

  15. Development of Fluorescent Protein Probes Specific for Parallel DNA and RNA G-Quadruplexes.

    Science.gov (United States)

    Dang, Dung Thanh; Phan, Anh Tuân

    2016-01-01

    We have developed fluorescent protein probes specific for parallel G-quadruplexes by attaching cyan fluorescent protein to the G-quadruplex-binding motif of the RNA helicase RHAU. Fluorescent probes containing RHAU peptide fragments of different lengths were constructed, and their binding to G-quadruplexes was characterized. The selective recognition and discrimination of G-quadruplex topologies by the fluorescent protein probes was easily detected by the naked eye or by conventional gel imaging. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Another step toward DNA selective targeting: Ni(II) and Cu(II) complexes of a Schiff base ligand able to bind gene promoter G-quadruplexes.

    Science.gov (United States)

    Terenzi, Alessio; Lötsch, Daniela; van Schoonhoven, Sushilla; Roller, Alexander; Kowol, Christian R; Berger, Walter; Keppler, Bernhard K; Barone, Giampaolo

    2016-05-04

    DNA G-rich sequences are able to form four-stranded structures organized in stacked guanine tetrads. These structures, called G-quadruplexes, were found to have an important role in the regulation of oncogenes expression and became, for such a reason, appealing targets for anticancer drugs. Aiming at finding selective G-quadruplex binders, we have designed, synthesized and characterized a new water soluble Salen-like Schiff base ligand and its Ni(II) and Cu(II) metal complexes. UV-Vis, circular dichroism and FRET measurements indicated that the nickel complex can stabilize oncogene promoter G-quadruplexes with high selectivity, presenting no interactions with duplex DNA at all. The same compound exhibited dose-dependent cytotoxic activity in MCF-7 breast cancer cells when combined with lipofectamine as lipophilic carrier.

  17. An intermolecular G-quadruplex as the basis for GTP recognition in the class V–GTP aptamer

    Science.gov (United States)

    Nasiri, Amir H.; Wurm, Jan Philip; Immer, Carina; Weickhmann, Anna Katharina; Wöhnert, Jens

    2016-01-01

    Many naturally occurring or artificially created RNAs are capable of binding to guanine or guanine derivatives with high affinity and selectivity. They bind their ligands using very different recognition modes involving a diverse set of hydrogen bonding and stacking interactions. Apparently, the potential structural diversity for guanine, guanosine, and guanine nucleotide binding motifs is far from being fully explored. Szostak and coworkers have derived a large set of different GTP-binding aptamer families differing widely in sequence, secondary structure, and ligand specificity. The so-called class V–GTP aptamer from this set binds GTP with very high affinity and has a complex secondary structure. Here we use solution NMR spectroscopy to demonstrate that the class V aptamer binds GTP through the formation of an intermolecular two-layered G-quadruplex structure that directly incorporates the ligand and folds only upon ligand addition. Ligand binding and G-quadruplex formation depend strongly on the identity of monovalent cations present with a clear preference for potassium ions. GTP binding through direct insertion into an intermolecular G-quadruplex is a previously unobserved structural variation for ligand-binding RNA motifs and rationalizes the previously observed specificity pattern of the class V aptamer for GTP analogs. PMID:27659052

  18. An intermolecular G-quadruplex as the basis for GTP recognition in the class V-GTP aptamer.

    Science.gov (United States)

    Nasiri, Amir H; Wurm, Jan Philip; Immer, Carina; Weickhmann, Anna Katharina; Wöhnert, Jens

    2016-11-01

    Many naturally occurring or artificially created RNAs are capable of binding to guanine or guanine derivatives with high affinity and selectivity. They bind their ligands using very different recognition modes involving a diverse set of hydrogen bonding and stacking interactions. Apparently, the potential structural diversity for guanine, guanosine, and guanine nucleotide binding motifs is far from being fully explored. Szostak and coworkers have derived a large set of different GTP-binding aptamer families differing widely in sequence, secondary structure, and ligand specificity. The so-called class V-GTP aptamer from this set binds GTP with very high affinity and has a complex secondary structure. Here we use solution NMR spectroscopy to demonstrate that the class V aptamer binds GTP through the formation of an intermolecular two-layered G-quadruplex structure that directly incorporates the ligand and folds only upon ligand addition. Ligand binding and G-quadruplex formation depend strongly on the identity of monovalent cations present with a clear preference for potassium ions. GTP binding through direct insertion into an intermolecular G-quadruplex is a previously unobserved structural variation for ligand-binding RNA motifs and rationalizes the previously observed specificity pattern of the class V aptamer for GTP analogs. © 2016 Nasiri et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  19. A Label-Free Luminescent Switch-On Assay for ATP Using a G-Quadruplex-Selective Iridium(III) Complex

    OpenAIRE

    Ka-Ho Leung; Lihua Lu; Modi Wang; Tsun-Yin Mak; Daniel Shiu-Hin Chan; Fung-Kit Tang; Chung-Hang Leung; Hiu-Yee Kwan; Zhiling Yu; Dik-Lung Ma

    2013-01-01

    We report herein the G-quadruplex-selective property of a luminescent cyclometallated iridium(III) complex for the detection of adenosine-5'-triphosphate (ATP) in aqueous solution. The ATP-binding aptamer was employed as the ATP recognition unit, while the iridium(III) complex was used to monitor the formation of the G-quadruplex structure induced by ATP. The sensitivity and fold enhancement of the assay were higher than those of the previously reported assay using the organic dye crystal vio...

  20. Ball with hair: modular functionalization of highly stable G-quadruplex DNA nano-scaffolds through N2-guanine modification.

    Science.gov (United States)

    Lech, Christopher Jacques; Phan, Anh Tuân

    2017-06-20

    Functionalized nanoparticles have seen valuable applications, particularly in the delivery of therapeutic and diagnostic agents in biological systems. However, the manufacturing of such nano-scale systems with the consistency required for biological application can be challenging, as variation in size and shape have large influences in nanoparticle behavior in vivo. We report on the development of a versatile nano-scaffold based on the modular functionalization of a DNA G-quadruplex. DNA sequences are functionalized in a modular fashion using well-established phosphoramidite chemical synthesis with nucleotides containing modification of the amino (N2) position of the guanine base. In physiological conditions, these sequences fold into well-defined G-quadruplex structures. The resulting DNA nano-scaffolds are thermally stable, consistent in size, and functionalized in a manner that allows for control over the density and relative orientation of functional chemistries on the nano-scaffold surface. Various chemistries including small modifications (N2-methyl-guanine), bulky aromatic modifications (N2-benzyl-guanine), and long chain-like modifications (N2-6-amino-hexyl-guanine) are tested and are found to be generally compatible with G-quadruplex formation. Furthermore, these modifications stabilize the G-quadruplex scaffold by 2.0-13.3 °C per modification in the melting temperature, with concurrent modifications producing extremely stable nano-scaffolds. We demonstrate the potential of this approach by functionalizing nano-scaffolds for use within the biotin-avidin conjugation approach. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Construction of a controllable Förster resonance energy transfer system based on G-quadruplex for DNA sensing.

    Science.gov (United States)

    Yue, Qiaoli; Shen, Tongfei; Wang, Changna; Wang, Lei; Li, Haibo; Xu, Shuling; Wang, Huaisheng; Liu, Jifeng

    2013-02-15

    Conjugations of oligonucleotides, chromophores, and gold nanoparticles (GNPs) can be used for energy transfer assays to detect DNA. Herein, a homogenous Förster resonance energy transfer (FRET) system employing two-step modification of oligonucleotide on GNPs was reported. The distance between the donor (fluorescein attached onto DNA) and the acceptor (GNPs) was controlled by using the G-rich DNA. In the presence of porphyrin or berberine, which can act as ligands of G-quadruplexes, the G-rich DNA spacer can result into G-quadruplex structure. Therefore, the intimate contact between the fluorophore and the GNP results in efficient energy transfer and fluorescence quenching. After hybridization with target DNA, the G-quadruplex stretched and resulted in an enhancement of fluorescence. So the present FRET system can be used for target DNA sensing with detection limit as low as 40 pM (S/N=3). In this study, a relation between the fluorescence quenching efficiency and GNP sizes was found and bigger GNPs had higher fluorescence enhancement after hybridization with target DNA. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Simple G-quadruplex-based 2-aminopurine fluorescence probe for highly sensitive and amplified detection of microRNA-21.

    Science.gov (United States)

    Li, Shiyu; Liu, Chan; Gong, Hang; Chen, Chunyan; Chen, Xiaoming; Cai, Changqun

    2018-02-01

    Based on 2-aminopurine (2-AP) probe in conjunction with a G-quadruplex structure and signal amplification technique, a simple and highly sensitive fluorescence sensor for detecting microRNA (miRNA) is developed for high signal-to-background ratio and wide linear range. The proposed sensor contains two hairpins DNA: H1 and H2. H1 is labeled by 2-AP incorporated into a G-rich sequence. Upon the addition of a target miRNA, H1 is unfolded and forms DNA/RNA complexes that contain a G-quadruplex, thereby significantly enhancing 2-AP fluorescence due to the protection provided by the G-quadruplex. Subsequently, H2 can displace the miRNA from the DNA/RNA complexes and induce signal amplification, resulting in further enhanced fluorescence intensity. Hence, the sensor is highly sensitive and its low limit of detection (L.O.D.) can reach as low as 1.48pM. Furthermore, the proposed sensor is used to detect overexpressed miRNA-21 from human breast cancer cell lysate. The result demonstrates the potential of the proposed sensor to monitor different miRNA biomarkers for the early diagnosis of various cancers. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Telomere Chromatin Condensation Assay (TCCA): a novel approach to study structural telomere integrity.

    Science.gov (United States)

    Gonzalez-Vasconcellos, Iria; Alonso-Rodríguez, Silvia; López-Baltar, Isidoro; Fernández, José Luis

    2015-01-01

    Telomeres, the DNA-protein complexes located at the end of linear eukaryotic chromosomes are essential for genome stability. Improper higher-order chromatin organization at the chromosome ends can give rise to telomeric recombination and genomic instability. We report the development of an assay to quantify differences in the condensation of telomeric chromatin, thereby offering new opportunities to study telomere biology and stability. We have combined a DNA nuclease digestion with a quantitative PCR (qPCR) assay of telomeric DNA, which we term the Telomere Chromatin Condensation Assay (TCCA). By quantifying the relative quantities of telomeric DNA that are progressively digested with the exonuclease Bal 31 the method can discriminate between different levels of telomeric chromatin condensation. The structural chromatin packaging at telomeres shielded against exonuclease digestion delivered an estimate, which we term Chromatin Protection Factor (CPF) that ranged from 1.7 to 2.3 fold greater than that present in unpacked DNA. The CPF was significantly decreased when cell cultures were incubated with the DNA hypomethylating agent 5-azacytidine, demonstrating the ability of the TCCA assay to discriminate between packaging levels of telomeric DNA. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Targeting C-myc G-Quadruplex: Dual Recognition by Aminosugar-Bisbenzimidazoles with Varying Linker Lengths

    Directory of Open Access Journals (Sweden)

    Nihar Ranjan

    2013-11-01

    Full Text Available G-quadruplexes are therapeutically important biological targets. In this report, we present biophysical studies of neomycin-Hoechst 33258 conjugates binding to a G-quadruplex derived from the C-myc promoter sequence. Our studies indicate that conjugation of neomycin to a G-quadruplex binder, Hoechst 33258, enhances its binding. The enhancement in G-quadruplex binding of these conjugates varies with the length and composition of the linkers joining the neomycin and Hoechst 33258 units.

  5. A highly selective G-quadruplex-based luminescent switch-on probe for the detection of gene deletion.

    Science.gov (United States)

    He, Hong-Zhang; Chan, Daniel Shiu-Hin; Leung, Chung-Hang; Ma, Dik-Lung

    2012-10-04

    A G-quadruplex-based switch-on luminescence assay has been developed for the detection of gene deletion using a cyclometallated iridium(III) complex as a G-quadruplex-selective probe. Upon hybridization with the target DNA, the two split G-quadruplex-forming sequences assemble into a split G-quadruplex, which greatly enhances the luminescence emission of the iridium(III) probe. The assay is simple and highly selective.

  6. Complexes of the ATP-dependent Lon protease and DNA aptamers with G-quadruplexes as a model for developing a nanosensor biomagnetic immunoassay system

    Science.gov (United States)

    Spiridonova, V. A.; Sizov, V. A.; Kuzmenko, E. O.; Melnichuk, A. V.; Oleinichenko, E. A.; Kudzhaev, A. M.; Rotanova, T. V.; Snigirev, O. V.

    2017-07-01

    The binding to Lon protease through biotinylated aptamers whose structures contain G-quadruplex fragments with magnetic nanoparticles (MNPs) functionalized by streptavidin was investigated. The conditions of binding of target aptamers to MNPs are met. The resulting complexes are proposed for detection of Lon protease in different biological sources and for constructing a novel biomagnetic nanosensor immunoassay system.

  7. Telomere Capping Proteins are Structurally Related to RPA with an additional Telomere-Specific Domain

    Energy Technology Data Exchange (ETDEWEB)

    Gelinas, A.; Paschini, M; Reyes, F; Heroux, A; Batey, R; Lundblad, V; Wuttke, D

    2009-01-01

    Telomeres must be capped to preserve chromosomal stability. The conserved Stn1 and Ten1 proteins are required for proper capping of the telomere, although the mechanistic details of how they contribute to telomere maintenance are unclear. Here, we report the crystal structures of the C-terminal domain of the Saccharomyces cerevisiae Stn1 and the Schizosaccharomyces pombe Ten1 proteins. These structures reveal striking similarities to corresponding subunits in the replication protein A complex, further supporting an evolutionary link between telomere maintenance proteins and DNA repair complexes. Our structural and in vivo data of Stn1 identify a new domain that has evolved to support a telomere-specific role in chromosome maintenance. These findings endorse a model of an evolutionarily conserved mechanism of DNA maintenance that has developed as a result of increased chromosomal structural complexity.

  8. Distinct differences in metal ion specificity of RNA and DNA G-quadruplexes.

    Science.gov (United States)

    Guiset Miserachs, Helena; Donghi, Daniela; Börner, Richard; Johannsen, Silke; Sigel, Roland K O

    2016-12-01

    RNA G-quadruplexes, as their well-studied DNA analogs, require the presence of cations to fold and remain stable. This is the first comprehensive study on the interaction of RNA quadruplexes with metal ions. We investigated the formation and stability of two highly conserved and biologically relevant RNA quadruplex-forming sequences (24nt-TERRA and 18nt-NRAS) in the presence of several monovalent and divalent metal ions, namely Li + , Na + , K + , Rb + , Cs + , NH 4 + , Mg 2+ , Ca 2+ , Sr 2+ , and Ba 2+ . Circular dichroism was used to probe the influence of these metal ions on the folded fraction of the parallel G-quadruplexes, and UV thermal melting experiments allowed to assess the relative stability of the structures in each cationic condition. Our results show that the RNA quadruplexes are more stable than their DNA counterparts under the same buffer conditions. We have observed that the addition of mainly Na + , K + , Rb + , NH 4 + , as well as Sr 2+ and Ba 2+ in water, shifts the equilibrium to the folded quadruplex form, whereby the NRAS sequence responds stronger than TERRA. However, only K + and Sr 2+ lead to a significant increase in the stability of the folded structures, which is consistent with their coordination to the O6 atoms from the G-quartet guanosines. Compared to the respective DNA motives, dNRAS and htelo, the RNA sequences are not stabilized by Na + ions. Finally, the difference in response between NRAS and TERRA, as well as to the corresponding DNA sequences with respect to different metal ions, could potentially be exploited for selective targeting purposes.

  9. G-quadruplex oligonucleotide AS1411 as a cancer-targeting agent: Uses and mechanisms.

    Science.gov (United States)

    Bates, Paula J; Reyes-Reyes, Elsa M; Malik, Mohammad T; Murphy, Emily M; O'Toole, Martin G; Trent, John O

    2017-05-01

    AS1411 is a 26-mer G-rich DNA oligonucleotide that forms a variety of G-quadruplex structures. It was identified based on its cancer-selective antiproliferative activity and subsequently determined to be an aptamer to nucleolin, a multifunctional protein that preferentially binds quadruplex nucleic acids and which is present at high levels on the surface of cancer cells. AS1411 has exceptionally efficient cellular internalization compared to non-quadruplex DNA sequences. Recent developments related to AS1411 will be examined, with a focus on its use for targeted delivery of therapeutic and imaging agents. Numerous research groups have used AS1411 as a targeting agent to deliver nanoparticles, oligonucleotides, and small molecules into cancer cells. Studies in animal models have demonstrated that AS1411-linked materials can accumulate selectively in tumors following systemic administration. The mechanism underlying the cancer-targeting ability of AS1411 is not completely understood, but recent studies suggest a model that involves: (1) initial uptake by macropinocytosis, a form of endocytosis prevalent in cancer cells; (2) stimulation of macropinocytosis by a nucleolin-dependent mechanism resulting in further uptake; and (3) disruption of nucleolin-mediated trafficking and efflux leading to cargoes becoming trapped inside cancer cells. Human trials have indicated that AS1411 is safe and can induce durable remissions in a few patients, but new strategies are needed to maximize its clinical impact. A better understanding of the mechanisms by which AS1411 targets and kills cancer cells may hasten the development of promising technologies using AS1411-linked nanoparticles or conjugates for cancer-targeted therapy and imaging. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016. Published by Elsevier B.V.

  10. DNA breaks and repair in interstitial telomere sequences: Influence of chromatin structure; Etude des cassures de l'ADN et des mecanismes de reparation dans les sequences telomeriques interstitielles: Influence de la structure chromatinienne

    Energy Technology Data Exchange (ETDEWEB)

    Revaud, D.

    2009-06-15

    Interstitial Telomeric Sequences (ITS) are over-involved in spontaneous and radiationinduced chromosome aberrations in chinese hamster cells. We have performed a study to investigate the origin of their instability, spontaneously or after low doses irradiation. Our results demonstrate that ITS have a particular chromatin structure: short nucleotide repeat length, less compaction of the 30 nm chromatin fiber, presence of G-quadruplex structures. These features would modulate breaks production and would favour the recruitment of alternative DNA repair mechanisms, which are prone to produce chromosome aberrations. These pathways could be at the origin of chromosome aberrations in ITS whereas NHEJ and HR Double Strand Break repair pathways are rather required for a correct repair in these regions. (author)

  11. Separation of the potential G-quadruplex ligands from the butanol extract of Zanthoxylum ailanthoides Sieb. & Zucc. by countercurrent chromatography and preparative high performance liquid chromatography.

    Science.gov (United States)

    Han, Tian; Cao, Xueli; Xu, Jing; Pei, Hairun; Zhang, Hong; Tang, Yalin

    2017-07-21

    G-quadruplex DNA structure is considered to be a very attractive target for antitumor drug design due to its unique role in maintaining telomerase activities. Therefore, discovering ligands with high stability of G-quadruplex structure is of great interest. In this paper, pH-zone refining counter current chromatography (CCC) and preparative high performance liquid chromatography (HPLC) were employed for the separation of potent G-quadruplex ligands from the n-butanol fraction of the crude extract of Zanthoxylum ailanthoides, which is a traditional Chinese medicine recently found to display high inhibitory activity against several human cancer cells. The 75% aqueous ethanol extract of the stem bark of Z. ailanthoides and its fractions with petroleum ether, ethyl acetate and n-butanol displayed almost the same G-quadruplex stabilization ability. Here, pH-zone refining CCC was used for the separation of the alkaloids from the n-butanol fraction by a seldom used solvent system composed of dichloromethane-methanol-water (4:1:2.5) with 10mM TEA in the organic stationary phase as retainer and 10mM HCl in the aqueous mobile phase as eluter. Compounds I, II and III were obtained, with purity greater than 95%, in the quantities of 31.2, 94.0, and 26.4mg respectively from 300mg of lipophilic fraction within 80min, which were identified as three tetrahydroprotoberberines isolated for the first time in this plant. In addition, a phenylpropanoid glycoside compound IV (Syringin), an isoquinoline (Magnoflorine, V), and two lignin isomers (+)-lyoniresiol-3α-O-β-d-glucopyranoside (VI) and (-)-lyoniresinol -3α-O-β-D -glucopyranoside (VII) were isolated by traditional CCC together with preparative HPLC. Compounds IV, V, VI and VII were obtained, with purity greater than 95%, in the quantities of 4.0, 13.2, 6.7, and 6.5mg respectively from 960mg of hydrophilic fraction. Among the seven isolated compounds, tetrahydroprotoberberine I, II and III were found to display remarkable

  12. A novel electrochemical aptasensor for highly sensitive detection of thrombin based on the autonomous assembly of hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme nanowires

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Shunbi, E-mail: xieshunbi@163.com; Chai, Yaqin, E-mail: yaqinchai@swu.edu.cn; Yuan, Yali; Bai, Lijuan; Yuan, Ruo, E-mail: yuanruo@swu.edu.cn

    2014-06-01

    Highlights: • This assay is label-free, the signal can be read out by measuring the electrochemical signal of hemin. • The hemin/G-quadruplex HRP-DNAzyme nanowires were formed via EXPAR reaction and HCR. • The prepared aptasensor exhibited low detection limit and wide linear range to TB. - Abstract: In this work, a new signal amplified strategy was constructed based on isothermal exponential amplification reaction (EXPAR) and hybridization chain reaction (HCR) generating the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-mimicking DNAzyme) nanowires as signal output component for the sensitive detection of thrombin (TB). We employed EXPAR’s ultra-high amplification efficiency to produce a large amount of two hairpin helper DNAs within a minutes. And then the resultant two hairpin helper DNAs could autonomously assemble the hemin/G-quadruplex HRP-mimicking DNAzymes nanowires as the redox-active reporter units on the electrode surface via hybridization chain reaction (HCR). The hemin/G-quadruplex structures simultaneously served as electron transfer medium and electrocatalyst to amplify the signal in the presence of H{sub 2}O{sub 2}. Specifically, only when the EXPAR reaction process has occurred, the HCR could be achieved and the hemin/G-quadruplex complexes could be formed on the surface of an electrode to give a detectable signal. The proposed strategy combines the amplification power of the EXPAR, HCR, and the inherent high sensitivity of the electrochemical detection. With such design, the proposed assay showed a good linear relationship within the range of 0.1 pM–50 nM with a detection limit of 33 fM (defined as S/N = 3) for TB.

  13. The G-quadruplex-forming aptamer AS1411 potently inhibits HIV-1 attachment to the host cell

    OpenAIRE

    Perrone, Rosalba; Butovskaya, Elena; Lago, Sara; Garzino-Demo, Alfredo; Pannecouque, Christophe; Palù, Giorgio; Richter, Sara N.

    2016-01-01

    AS1411 is a G-rich aptamer that forms a stable G-quadruplex structure and displays antineoplastic properties both in vitro and in vivo. This oligonucleotide has undergone phase 2 clinical trials. The major molecular target of AS1411 is nucleolin (NCL), a multifunctional nucleolar protein also present in the cell membrane where it selectively mediates the binding and uptake of AS1411. Cell-surface NCL has been recognised as a low-affinity co-receptor for human immunodeficiency virus type 1 (HI...

  14. Chiral ruthenium(II polypyridyl complexes: stabilization of g-quadruplex DNA, inhibition of telomerase activity and cellular uptake.

    Directory of Open Access Journals (Sweden)

    Qianqian Yu

    Full Text Available Two ruthenium(II complexes, Λ-[Ru(phen(2(p-HPIP](2+ and Δ-[Ru(phen(2(p-HPIP](2+, were synthesized and characterized via proton nuclear magnetic resonance spectroscopy, electrospray ionization-mass spectrometry, and circular dichroism spectroscopy. This study aims to clarify the anticancer effect of metal complexes as novel and potent telomerase inhibitors and cellular nucleus target drug. First, the chiral selectivity of the compounds and their ability to stabilize quadruplex DNA were studied via absorption and emission analyses, circular dichroism spectroscopy, fluorescence-resonance energy transfer melting assay, electrophoretic mobility shift assay, and polymerase chain reaction stop assay. The two chiral compounds selectively induced and stabilized the G-quadruplex of telomeric DNA with or without metal cations. These results provide new insights into the development of chiral anticancer agents for G-quadruplex DNA targeting. Telomerase repeat amplification protocol reveals the higher inhibitory activity of Λ-[Ru(phen(2(p-HPIP](2+ against telomerase, suggesting that Λ-[Ru(phen(2(p-HPIP](2+ may be a potential telomerase inhibitor for cancer chemotherapy. MTT assay results show that these chiral complexes have significant antitumor activities in HepG2 cells. More interestingly, cellular uptake and laser-scanning confocal microscopic studies reveal the efficient uptake of Λ-[Ru(phen(2(p-HPIP](2+ by HepG2 cells. This complex then enters the cytoplasm and tends to accumulate in the nucleus. This nuclear penetration of the ruthenium complexes and their subsequent accumulation are associated with the chirality of the isomers as well as with the subtle environment of the ruthenium complexes. Therefore, the nucleus can be the cellular target of chiral ruthenium complexes for anticancer therapy.

  15. G-quadruplex DNAzymes-induced highly selective and sensitive colorimetric sensing of free heme in rat brain.

    Science.gov (United States)

    Li, Ruimin; Jiang, Qin; Cheng, Hanjun; Zhang, Guoqiang; Zhen, Mingming; Chen, Daiqin; Ge, Jiechao; Mao, Lanqun; Wang, Chunru; Shu, Chunying

    2014-04-21

    Direct selective determination of free heme in the cerebral system is of great significance due to the crucial roles of free heme in physiological and pathological processes. In this work, a G-quadruplex DNAzymes-induced highly sensitive and selective colorimetric sensing of free heme in rat brain is established. Initially, the conformation of an 18-base G-rich DNA sequence, PS2.M (5'-GTGGGTAGGGCGGGTTGG-3'), in the presence of K(+), changes from a random coil to a "parallel" G-quadruplex structure, which can bind free heme in the cerebral system with high affinity through π-π stacking. The resulted heme/G-quadruplex complex exhibits high peroxidase-like activity, which can be used to catalyze the oxidation of colorless ABTS(2-) to green ABTS˙(-) by H2O2. The concentration of heme can be evaluated by the naked eye and determined by UV-vis spectroscopy. The signal output showed a linear relationship for heme within the concentration range from 1 to 120 nM with a detection limit of 0.637 nM. The assay demonstrated here was highly selective and free from the interference of physiologically important species such as dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), ascorbate acid (AA), cysteine, uric acid (UA), glucose and lactate in the cerebral system. The basal dialysate level of free heme in the microdialysate from the striatum of adult male Sprague-Dawley rats was determined to be 32.8 ± 19.5 nM (n = 3). The analytic protocol possesses many advantages, including theoretical simplicity, low-cost technical and instrumental demands, and responsible detection of heme in rat brain microdialysate.

  16. Mutational Dissection of Telomeric DNA Binding Requirements of G4 Resolvase 1 Shows that G4-Structure and Certain 3'-Tail Sequences Are Sufficient for Tight and Complete Binding.

    Science.gov (United States)

    Smaldino, Philip J; Routh, Eric D; Kim, Jung H; Giri, Banabihari; Creacy, Steven D; Hantgan, Roy R; Akman, Steven A; Vaughn, James P

    2015-01-01

    Ends of human chromosomes consist of the six nucleotide repeat d[pTTAGGG]n known as telomeric DNA, which protects chromosomes. We have previously shown that the DHX36 gene product, G4 Resolvase 1 (G4R1), binds parallel G-quadruplex (G4) DNA with an unusually tight apparent Kd. Recent work associates G4R1 with the telomerase holoenzyme, which may allow it to access telomeric G4-DNA. Here we show that G4R1 can tightly bind telomeric G4-DNA, and in the context of the telomeric sequence, we determine length, sequence, and structural requirements sufficient for tight G4R1 telomeric binding. Specifically, G4R1 binds telomeric DNA in the K+-induced "3+1" G4-topology with an apparent Kd = 10 ± 1.9 pM, a value similar as previously found for binding to unimolecular parallel G4-DNA. G4R1 binds to the Na+-induced "2+2" basket G4-structure formed by the same DNA sequence with an apparent Kd = 71 ± 2.2 pM. While the minimal G4-structure is not sufficient for G4R1 binding, a 5' G4-structure with a 3' unstructured tail containing a guanine flanked by adenine(s) is sufficient for maximal binding. Mutations directed to disrupt G4-structure similarly disrupt G4R1 binding; secondary mutations that restore G4-structure also restore G4R1 binding. We present a model showing that a replication fork disrupting a T-loop could create a 5' quadruplex with an opened 3'tail structure that is recognized by G4R1.

  17. Mutational Dissection of Telomeric DNA Binding Requirements of G4 Resolvase 1 Shows that G4-Structure and Certain 3’-Tail Sequences Are Sufficient for Tight and Complete Binding

    Science.gov (United States)

    Smaldino, Philip J.; Routh, Eric D.; Kim, Jung H.; Giri, Banabihari; Creacy, Steven D.; Hantgan, Roy R.; Akman, Steven A.; Vaughn, James P.

    2015-01-01

    Ends of human chromosomes consist of the six nucleotide repeat d[pTTAGGG]n known as telomeric DNA, which protects chromosomes. We have previously shown that the DHX36 gene product, G4 Resolvase 1 (G4R1), binds parallel G-quadruplex (G4) DNA with an unusually tight apparent Kd. Recent work associates G4R1 with the telomerase holoenzyme, which may allow it to access telomeric G4-DNA. Here we show that G4R1 can tightly bind telomeric G4-DNA, and in the context of the telomeric sequence, we determine length, sequence, and structural requirements sufficient for tight G4R1 telomeric binding. Specifically, G4R1 binds telomeric DNA in the K+-induced “3+1” G4-topology with an apparent Kd = 10 ±1.9 pM, a value similar as previously found for binding to unimolecular parallel G4-DNA. G4R1 binds to the Na+-induced “2+2” basket G4-structure formed by the same DNA sequence with an apparent Kd = 71 ± 2.2 pM. While the minimal G4-structure is not sufficient for G4R1 binding, a 5’ G4-structure with a 3’ unstructured tail containing a guanine flanked by adenine(s) is sufficient for maximal binding. Mutations directed to disrupt G4-structure similarly disrupt G4R1 binding; secondary mutations that restore G4-structure also restore G4R1 binding. We present a model showing that a replication fork disrupting a T-loop could create a 5’ quadruplex with an opened 3’tail structure that is recognized by G4R1. PMID:26172836

  18. Selective G-Quadruplex DNA Recognition by a New Class of Designed Cyanines

    Directory of Open Access Journals (Sweden)

    Markus W. Germann

    2013-11-01

    Full Text Available A variety of cyanines provide versatile and sensitive agents acting as DNA stains and sensors and have been structurally modified to bind in the DNA minor groove in a sequence dependent manner. Similarly, we are developing a new set of cyanines that have been designed to achieve highly selective binding to DNA G-quadruplexes with much weaker binding to DNA duplexes. A systematic set of structurally analogous trimethine cyanines has been synthesized and evaluated for quadruplex targeting. The results reveal that elevated quadruplex binding and specificity are highly sensitive to the polymethine chain length, heterocyclic structure and intrinsic charge of the compound. Biophysical experiments show that the compounds display significant selectivity for quadruplex binding with a higher preference for parallel stranded quadruplexes, such as cMYC. NMR studies revealed the primary binding through an end-stacking mode and SPR studies showed the strongest compounds have primary KD values below 100 nM that are nearly 100-fold weaker for duplexes. The high selectivity of these newly designed trimethine cyanines for quadruplexes as well as their ability to discriminate between different quadruplexes are extremely promising features to develop them as novel probes for targeting quadruplexes in vivo.

  19. Microfluidic-based G-quadruplex ligand displacement assay for alkaloid anticancer drug screening.

    Science.gov (United States)

    Shen, Haihui; Zhang, Bo; Xu, Huiyan; Sun, Yue; Wu, Qiwang; Shen, Hong; Liu, Yingchun

    2017-02-05

    Some natural heterocyclic alkaloids containing planar group show potential to complex with specific promoter region of protooncogene for stabilizing the G-quadruplex (G4) structure which nowadays promises to be a target in anticancer drug design. However, in view of the polymorphic characteristics and structural complexity of heterocyclic alkaloids, it is desirable to develop high-throughput and low-consumption approach for anticancer drug screening. In this paper, an intensive study on alkaloid ligand/G4 DNA interaction has been conducted, demonstrating that the end-stacking interaction is the favorable binding mode between the oncogene-related Pu22 G4 DNA and the heterocyclic alkaloid ligand. Based on structural feasibility and energy minimization, a ligand displacement assay for screening alkaloid ligand in stabilizing the oncogene target G4 has been developed, which also helps to facilitate the assessment of drug specificity. Coupled with microfluidic-based DNAzyme-catalytic chemiluminescence detection, the approach showed the advantages of high sensitivity, high throughput with low sample and reagent consumptions. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Water soluble extended naphthalene diimides as pH fluorescent sensors and G-quadruplex ligands.

    Science.gov (United States)

    Doria, Filippo; Nadai, Matteo; Sattin, Giovanna; Pasotti, Luca; Richter, Sara N; Freccero, Mauro

    2012-05-21

    Extended naphthalene diimides (NDIs) fused to 1,4-dihydropyrazine-2,3-dione, containing two solubilizing moieties, have been synthesized. Fluorescence spectra of the new NDIs were remarkably affected by pH, as the second deprotonation of the dihydropyrazinedione moiety (pK(a) 6.9) switched off the emission. Binding to a G-quadruplex folded oligonucleotide and stoichiometry were evaluated by FRET melting assay and CD analysis. G-quadruplex binding was strongly enhanced shifting from pH 7.4 to pH 6.0 as a consequence of the dihydropyrazinedione moiety protonation. Cytotoxicity studies using two human telomerase-positive cell lines (HT29 and A549) revealed that the best G-quadruplex ligand was very active against the colon cell line, with an EC(50) of 300 nM.

  1. A light-up probe targeting for Bcl-2 2345 G-quadruplex DNA with carbazole TO

    Science.gov (United States)

    Gu, Yingchun; Lin, Dayong; Tang, Yalin; Fei, Xuening; Wang, Cuihong; Zhang, Baolian; Zhou, Jianguo

    2018-02-01

    As its significant role, the selective recognition of G-quadruplex with specific structures and functions is important in biological and medicinal chemistry. Carbazole derivatives have been reported as a kind of fluorescent probe with many excellent optical properties. In the present study, the fluorescence of the dye (carbazole TO) increased almost 70 fold in the presence of bcl-2 2345 G4 compared to that alone in aqueous buffer condition with almost no fluorescence and 10-30 fold than those in the presence of other DNAs. The binding study results by activity inhibition of G4/Hemin peroxidase experiment, NMR titration and molecular docking simulation showed the high affinity and selectivity to bcl-2 2345 G4 arises from its end-stacking interaction with G-quartet. It is said that a facile approach with excellent sensitive, good selectivity and quick response for bcl-2 2345 G-quadruplex was developed and may be used for antitumor recognition or antitumor agents.

  2. Biophysical characterization of G-quadruplex forming FMR1 mRNA and of its interactions with different fragile X mental retardation protein isoforms.

    Science.gov (United States)

    Blice-Baum, Anna C; Mihailescu, Mihaela-Rita

    2014-01-01

    Fragile X syndrome, the most common form of inherited mental impairment in humans, is caused by the absence of the fragile X mental retardation protein (FMRP) due to a CGG trinucleotide repeat expansion in the 5'-untranslated region (UTR) and subsequent translational silencing of the fragile x mental retardation-1 (FMR1) gene. FMRP, which is proposed to be involved in the translational regulation of specific neuronal messenger RNA (mRNA) targets, contains an arginine-glycine-glycine (RGG) box RNA binding domain that has been shown to bind with high affinity to G-quadruplex forming mRNA structures. FMRP undergoes alternative splicing, and the binding of FMRP to a proposed G-quadruplex structure in the coding region of its mRNA (named FBS) has been proposed to affect the mRNA splicing events at exon 15. In this study, we used biophysical methods to directly demonstrate the folding of FMR1 FBS into a secondary structure that contains two specific G-quadruplexes and analyze its interactions with several FMRP isoforms. Our results show that minor splice isoforms, ISO2 and ISO3, created by the usage of the second and third acceptor sites at exon 15, bind with higher affinity to FBS than FMRP ISO1, which is created by the usage of the first acceptor site. FMRP ISO2 and ISO3 cannot undergo phosphorylation, an FMRP post-translational modification shown to modulate the protein translation regulation. Thus, their expression has to be tightly regulated, and this might be accomplished by a feedback mechanism involving the FMRP interactions with the G-quadruplex structures formed within FMR1 mRNA.

  3. RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer

    Science.gov (United States)

    Wolfe, Andrew L.; Singh, Kamini; Zhong, Yi; Drewe, Philipp; Rajasekhar, Vinagolu K.; Sanghvi, Viraj R.; Mavrakis, Konstantinos J.; Jiang, Man; Roderick, Justine E.; van der Meulen, Joni; Schatz, Jonathan H.; Rodrigo, Christina M.; Zhao, Chunying; Rondou, Pieter; de Stanchina, Elisa; Teruya-Feldstein, Julie; Kelliher, Michelle A.; Speleman, Frank; Porco, John A.; Pelletier, Jerry; Rätsch, Gunnar; Wendel, Hans-Guido

    2014-09-01

    The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5' untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

  4. A Sensitive and Label-Free Pb(II) Fluorescence Sensor Based on a DNAzyme Controlled G-Quadruplex/Thioflavin T Conformation.

    Science.gov (United States)

    Wen, Yanli; Wang, Lele; Li, Lanying; Xu, Li; Liu, Gang

    2016-12-16

    Pb(II) can cause serious damaging effects to human health, and thus, the study of Pb2+ detection methods to sensitively and selectively monitor Pb(II) pollution has significant importance. In this work, we have developed a label-free fluorescence sensing strategy based on a Pb(II) DNAzyme cleavage and the ThT/G-quadruplex complex. In the presence of Pb(II), a G-rich tail was cut and released from the substrate strand, which then would form a G-quadruplex structure by combination with ThT dye. The fluorescence signal increase was then measured for sensitive Pb(II) quantification with a limit of detection of 0.06 nM. Our sensor also demonstrated high selectivity against six different metal ions, which is very important for the analysis of complex samples.

  5. A label-free luminescent switch-on assay for ATP using a G-quadruplex-selective iridium(III) complex.

    Science.gov (United States)

    Leung, Ka-Ho; Lu, Lihua; Wang, Modi; Mak, Tsun-Yin; Chan, Daniel Shiu-Hin; Tang, Fung-Kit; Leung, Chung-Hang; Kwan, Hiu-Yee; Yu, Zhiling; Ma, Dik-Lung

    2013-01-01

    We report herein the G-quadruplex-selective property of a luminescent cyclometallated iridium(III) complex for the detection of adenosine-5'-triphosphate (ATP) in aqueous solution. The ATP-binding aptamer was employed as the ATP recognition unit, while the iridium(III) complex was used to monitor the formation of the G-quadruplex structure induced by ATP. The sensitivity and fold enhancement of the assay were higher than those of the previously reported assay using the organic dye crystal violet as a fluorescent probe. This label-free luminescent switch-on assay exhibits high sensitivity and selectivity towards ATP with a limit of detection of 2.5 µM.

  6. A label-free luminescent switch-on assay for ATP using a G-quadruplex-selective iridium(III complex.

    Directory of Open Access Journals (Sweden)

    Ka-Ho Leung

    Full Text Available We report herein the G-quadruplex-selective property of a luminescent cyclometallated iridium(III complex for the detection of adenosine-5'-triphosphate (ATP in aqueous solution. The ATP-binding aptamer was employed as the ATP recognition unit, while the iridium(III complex was used to monitor the formation of the G-quadruplex structure induced by ATP. The sensitivity and fold enhancement of the assay were higher than those of the previously reported assay using the organic dye crystal violet as a fluorescent probe. This label-free luminescent switch-on assay exhibits high sensitivity and selectivity towards ATP with a limit of detection of 2.5 µM.

  7. Structure of a Stable G-Hairpin

    Czech Academy of Sciences Publication Activity Database

    Gajarský, M.; Zivkovic, M.L.; Stadlbauer, Petr; Pagano, B.; Fiala, R.; Amato, J.; Tomáška, L´.; Šponer, Jiří; Plavec, J.; Trantírek, L.

    2017-01-01

    Roč. 139, č. 10 (2017), s. 3591-3594 ISSN 0002-7863 R&D Projects: GA ČR GA13-28310S; GA ČR(CZ) GA16-13721S Institutional support: RVO:68081707 Keywords : g-quadruplex structures * human telomeric dna * single-stranded-dna * g-triplex Subject RIV: CE - Biochemistry Impact factor: 13.858, year: 2016

  8. Label-free detection of Cu(2+ and Hg(2+ ions using reconstructed Cu(2+-specific DNAzyme and G-quadruplex DNAzyme.

    Directory of Open Access Journals (Sweden)

    Hui Li

    Full Text Available Label-free metal ion detection methods were developed. To achieve these, a reconstructed Cu(2+-specific DNA-cleaving DNAzyme (Cu(2+-specific DNAzyme with an intramolecular stem-loop structure was used. G-quadruplex-forming G-rich sequence(s, linked at the ends of double-helix stem of an intramolecular stem-loop structure, was partly caged in an intramolecular duplex or formed a split G-quadruplex. Cu(2+-triggered DNA cleavage at a specific site decreased the stability of the double-helix stem, resulting in the formation or destruction of G-quadruplex DNAzyme that can effectively catalyze the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS-H2O2 reaction. Based on these, two label-free, cost-effective and simple Cu(2+ sensors were designed. These two sensors followed different detection modes: 'turn-on' and 'turn-off'. As for the 'turn-on' sensor, the intramolecular stem-loop structure ensured a low background signal, and the co-amplification of detection signal by dual DNAzymes (Cu(2+-specific DNAzyme and G-quadruplex DNAzyme provided a high sensitivity. This sensor enabled the selective detection of aqueous Cu(2+ with a detection limit of 3.9 nM. Visual detection was possible. Although the 'turn-off' sensor gave lower detection sensitivity than the 'turn-on' one, the characteristics of cost-effectiveness and ease of operation made it an important implement to reduce the possibility of pseudo-positive or pseudo-negative results. Combining the ability of Hg(2+ ion to stabilize T-T base mismatch, above dual DNAzymes-based strategy was further used for Hg(2+ sensor design. The proposed sensor allowed the specific detection of Hg(2+ ion with a detection of 4.8 nM. Visual detection was also possible.

  9. Ion-selective formation of a guanine quadruplex on DNA origami structures.

    Science.gov (United States)

    Olejko, Lydia; Cywinski, Piotr J; Bald, Ilko

    2015-01-07

    DNA origami nanostructures are a versatile tool that can be used to arrange functionalities with high local control to study molecular processes at a single-molecule level. Here, we demonstrate that DNA origami substrates can be used to suppress the formation of specific guanine (G) quadruplex structures from telomeric DNA. The folding of telomeres into G-quadruplex structures in the presence of monovalent cations (e.g. Na(+) and K(+)) is currently used for the detection of K(+) ions, however, with insufficient selectivity towards Na(+). By means of FRET between two suitable dyes attached to the 3'- and 5'-ends of telomeric DNA we demonstrate that the formation of G-quadruplexes on DNA origami templates in the presence of sodium ions is suppressed due to steric hindrance. Hence, telomeric DNA attached to DNA origami structures represents a highly sensitive and selective detection tool for potassium ions even in the presence of high concentrations of sodium ions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. A label-free fluorescence assay for thrombin based on aptamer exonuclease protection and exonuclease III-assisted recycling amplification-responsive cascade zinc(II)-protoporphyrin IX/G-quadruplex supramolecular fluorescent labels.

    Science.gov (United States)

    Lv, Yanqin; Xue, Qingwang; Gu, Xiaohong; Zhang, Shuqiu; Liu, Jifeng

    2014-05-21

    A simple, label-free and sensitive fluorescence protein assay has been developed on the basis of aptamer exonuclease protection and exonuclease III (Exo III)-assisted recycling amplification-responsive cascade ZnPPIX/G-quadruplex supramolecular fluorescent labels. In the sensing system, a special aptamer probe containing the aptamer sequence at the 3'-terminus and the DNAzyme sequence at the 5'-terminus was applied, which has the capacity to recognize a protein target with high affinity and specificity. Exonuclease I (Exo I) can efficiently catalyze the degradation of free single stranded DNA probes in the 3' to 5' direction. In the presence of the target protein, the strong binding between the target protein and its aptamer can protect aptamer probes from degradation. Subsequently, the protected aptamer probes act as catalysators to trigger hybridization with the hairpin DNA probe that contains a partially "caged" G-quadruplex sequence. Upon interaction with the protected aptamer probes, the hairpin opens to yield the active G-quadruplex structure. In the presence of exonuclease III (Exo III), Exo III-assisted recycling amplification occurs generating numerous G-quadruplex supramolecular structures. The zinc(ii)-protoporphyrin IX (ZnPPIX) fluorophore binds to the G-quadruplexes and this results in the enhanced fluorescence of the fluorophore. The resulting fluorescence of the ZnPPIX/G-quadruplex provides the readout signal for the sensing event. Thrombin is used as the model analyte in the current proof-of-concept. The developed method was demonstrated to have very high sensitivity for the detection of proteins with a limit of detection of 0.2 pM without using washes or separations. In addition, this new method for protein detection is simple and inherits all the advantages of aptamers. The mechanism, moreover, may be generalized and used for other forms of protein analysis.

  11. Novel molecular targets for kRAS downregulation: promoter G-quadruplexes

    Science.gov (United States)

    2016-11-01

    consistency, and the proteins with the greatest enrichment will be further studied. REFERENCES 1 Le, H. T. et al. Not all G-quadruplexes are created... lithium cacodylate buffer (pH 7.2) supplemented with 10 mM potassium chloride, heated to 95 °C for 10 min, and allowed to anneal to room temperature slowly

  12. Conjugation of hemin to G-quadruplex forming oligonucleotide using click chemistry.

    Science.gov (United States)

    Kosman, J; Stanislawska, A; Gluszynska, A; Juskowiak, B

    2017-08-01

    Peroxidase-mimicking DNAzyme is one of the systems that recently gained a great interest. It has been successfully applied for designing numerous bioassays. The success of this system is connected to its advantages over a protein enzyme, horseradish peroxidase. Promising strategy for further improvement of performance of DNAzyme with peroxidase-like activity was proposed recently. It was based on the covalent attachment of hemin moiety to the G-quadruplex scaffold. We report here the first attempt of conjugating hemin to the G-quadruplex DNA using click chemistry approach. We modified hemin molecule through attachment of an azide-terminated linker to the porphyrin carboxylic group. Two click chemistry approaches were examined to conjugate the azide-modified hemin to a G-quadruplex oligonucleotide: copper-catalyzed and Cu-free cycloaddition reactions. Using Cu-free click reaction, we successfully synthesized G-quadruplex-hemin conjugate that exhibited promising peroxidase activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV.

    Science.gov (United States)

    Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P; Robertson, Erle S; Schildkraut, Carl L; Verma, Subhash C

    2016-05-05

    Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Worldwide genetic structure in 37 genes important in telomere biology

    Science.gov (United States)

    Mirabello, L; Yeager, M; Chowdhury, S; Qi, L; Deng, X; Wang, Z; Hutchinson, A; Savage, S A

    2012-01-01

    Telomeres form the ends of eukaryotic chromosomes and are vital in maintaining genetic integrity. Telomere dysfunction is associated with cancer and several chronic diseases. Patterns of genetic variation across individuals can provide keys to further understanding the evolutionary history of genes. We investigated patterns of differentiation and population structure of 37 telomere maintenance genes among 53 worldwide populations. Data from 898 unrelated individuals were obtained from the genome-wide scan of the Human Genome Diversity Panel (HGDP) and from 270 unrelated individuals from the International HapMap Project at 716 single-nucleotide polymorphism (SNP) loci. We additionally compared this gene set to HGDP data at 1396 SNPs in 174 innate immunity genes. The majority of the telomere biology genes had low to moderate haplotype diversity (45–85%), high ancestral allele frequencies (>60%) and low differentiation (FST HapMap 3. TERT had higher than expected levels of haplotype diversity, likely attributable to a lack of linkage disequilibrium, and a potential cancer-associated SNP in this gene, rs2736100, varied substantially in genotype frequency across major continental regions. It is possible that the genes under selection could influence telomere biology diseases. As a group, there appears to be less diversity and differentiation in telomere biology genes than in genes with different functions, possibly due to their critical role in telomere maintenance and chromosomal stability. PMID:21731055

  15. Conformations of Human Telomeric G-Quadruplex Studied Using a Nucleotide-Independent Nitroxide Label

    Czech Academy of Sciences Publication Activity Database

    Zhang, X.J.; Xu, C.X.; Di Felice, R.; Šponer, Jiří; Islam, B.; Stadlbauer, Petr; Ding, Y.; Mao, L.; Mao, Z.W.; Qin, P.Z.

    2016-01-01

    Roč. 55, č. 2 (2016), s. 360-372 ISSN 0006-2960 R&D Projects: GA ČR(CZ) GAP208/11/1822; GA MŠk(CZ) ED1.1.00/02.0068 Institutional support: RVO:68081707 Keywords : PARAMAGNETIC-RESONANCE SPECTROSCOPY * AMBER FORCE-FIELD * NUCLEIC- ACIDS Subject RIV: BO - Biophysics Impact factor: 2.938, year: 2016

  16. Photocrosslinking of human telomeric G-quadruplex loops by anti cyclobutane thymine dimer formation

    National Research Council Canada - National Science Library

    Dian G. T. Su; Huafeng Fang; Michael L. Gross; John-Stephen A. Taylor

    2009-01-01

    .... UVB irradiation of d[AGGG(TTAGGG) 3 ] in the presence of Na + results in a cis,syn thymine dimer between two adjacent Ts in a TTA loop and a mixture of nonadjacent anti thymine dimers between various loops...

  17. Photocrosslinking of Human Telomeric G-Quadruplex Loops by "Anti" Cyclobutane Thymine Dimer Formation

    National Research Council Canada - National Science Library

    Dian G. T. Su; Huafeng Fang; Michael L. Gross; John-Stephen A. Taylor; Philip C. Hanawalt

    2009-01-01

    .... UVB irradiation of d[AGGG(TTAGGG)₃] in the presence of Na⁺ results in a cis, syn thymine dimer between two adjacent Ts in a TTA loop and a mixture of nonadjacent anti thymine dimers between various loops...

  18. G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae.

    Science.gov (United States)

    Capra, John A; Paeschke, Katrin; Singh, Mona; Zakian, Virginia A

    2010-07-22

    G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs) across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs). Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.

  19. G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    John A Capra

    2010-07-01

    Full Text Available G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs. Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.

  20. Insight into G-DNA Structural Polymorphism and Folding from Sequence and Loop Connectivity through Free Energy Analysis

    Science.gov (United States)

    2011-01-01

    The lengths of G-tracts and their connecting loop sequences determine G-quadruplex folding and stability. Complete understanding of the sequence–structure relationships remains elusive. Here, single-loop G-quadruplexes were investigated using explicit solvent molecular dynamics (MD) simulations to characterize the effect of loop length, loop sequence, and G-tract length on the folding topologies and stability of G-quadruplexes. Eight loop types, including different variants of lateral, diagonal, and propeller loops, and six different loop sequences [d0 (i.e., no intervening residues in the loop), dT, dT2, dT3, dTTA, and dT4] were considered through MD simulation and free energy analysis. In most cases the free energetic estimates agree well with the experimental observations. The work also provides new insight into G-quadruplex folding and stability. This includes reporting the observed instability of the left propeller loop, which extends the rules for G-quadruplex folding. We also suggest a plausible explanation why human telomere sequences predominantly form hybrid-I and hybrid-II type structures in K+ solution. Overall, our calculation results indicate that short loops generally are less stable than longer loops, and we hypothesize that the extreme stability of sequences with very short loops could possibly derive from the formation of parallel multimers. The results suggest that free energy differences, estimated from MD and free energy analysis with current force fields and simulation protocols, are able to complement experiment and to help dissect and explain loop sequence, loop length, and G-tract length and orientation influences on G-quadruplex structure. PMID:21761922

  1. Telomeric D-loops containing 8-oxo-2'-deoxyguanosine are preferred substrates for Werner and Bloom syndrome helicases and are bound by POT1.

    Science.gov (United States)

    Ghosh, Avik; Rossi, Marie L; Aulds, Jason; Croteau, Deborah; Bohr, Vilhelm A

    2009-11-06

    8-Oxo-2'-deoxyguanosine (8-oxodG) is one of the most important oxidative DNA lesions, and G-rich telomeric DNA is especially susceptible to oxidative DNA damage. RecQ helicases WRN and BLM and telomere-binding protein POT1 are thought to play roles in telomere maintenance. This study examines the ability of WRN, BLM, and RecQ5 to unwind and POT1 to bind telomeric D-loops containing 8-oxodG. The results demonstrate that WRN and BLM preferentially unwind telomeric D-loops containing 8-oxodG and that POT1 binds with higher affinity to telomeric D-loops with 8-oxodG but shows no preference for telomeric single-stranded DNA with 8-oxodG. We speculate that telomeric D-loops with 8-oxodG may have a greater tendency to form G-quadruplex DNA structures than telomeric DNA lacking 8-oxodG.

  2. Real time monitoring of Pb2+-induced formation of G-quadruplex DNA with LPFG sensor

    Science.gov (United States)

    Bagheri, Z.; Latifi, H.; Ranjbar, B.; Zibaii, M. I.; Behroodi, E.

    2015-09-01

    In this paper the potential of real time monitoring of AS1411 Guanine-quadruplex folding by using long period fiber grating (LPFG) biosensor was described. AS1411 ss-DNA folds into parallel bimolecular quadruplex in solution containing metal ions. While immobilized AS1411 folds, molecular crowding of sensing area will increase and cause change the refractive index (RI) around the sensor. In addition RI change is absolutely dependent on G-quadruplex factor concentration.

  3. G-quadruplex aptamer selection using capillary electrophoresis-LED-induced fluorescence and Illumina sequencing.

    Science.gov (United States)

    Ric, Audrey; Ecochard, Vincent; Iacovoni, Jason S; Boutonnet, Audrey; Ginot, Frédéric; Ong-Meang, Varravaddheay; Poinsot, Véréna; Paquereau, Laurent; Couderc, François

    2018-03-01

    One of the major difficulties that arises when selecting aptamers containing a G-quadruplex is the correct amplification of the ssDNA sequence. Can aptamers containing a G-quadruplex be selected from a degenerate library using non-equilibrium capillary electrophoresis (CE) of equilibrium mixtures (NECEEM) along with high-throughput Illumina sequencing? In this article, we present some mismatches of the G-quadruplex T29 aptamer specific to thrombin, which was PCR amplified and sequenced by Illumina sequencing. Then, we show the proportionality between the number of sequenced molecules of T29 added to the library and the number of sequences obtained in Illumina sequencing, and we find that T29 sequences from this aptamer can be detected in a random library of ssDNA after the sample is fractionated by NECEEM, amplified by PCR, and sequenced. Treatment of the data by the counting of double-stranded DNA T29 sequences containing a maximum of two mismatches reveals a good correlation with the enrichment factor (f E ). This factor is the ratio of the number of aptamer sequences found in the collected complex sample divided by the total number of sequencing reads (aptamer and non-aptamer) plus the quantity of T29 molecules (spiked into a DNA library) injected into CE.

  4. Dynamics of protein binding to telomeres in living cells: implications for telomere structure and function.

    NARCIS (Netherlands)

    K.A. Mattern (Karin); S.J. Swiggers (Susan); A.L. Nigg (Alex); B. Löwenberg (Bob); A.B. Houtsmuller (Adriaan); J.M. Zijlmans (Mark)

    2004-01-01

    textabstractTelomeric proteins have an essential role in the regulation of the length of the telomeric DNA tract and in protection against end-to-end chromosome fusion. Telomere organization and how individual proteins are involved in different telomere functions in living cells is

  5. Interaction of pyrrolobenzodiazepine (PBD) ligands with parallel intermolecular G-quadruplex complex using spectroscopy and ESI-MS

    National Research Council Canada - National Science Library

    Raju, Gajjela; Srinivas, Ragampeta; Reddy, Vangala Santhosh; Idris, Mohammed M; Kamal, Ahmed; Nagesh, Narayana

    2012-01-01

    .... Electrospray ionization mass spectrometry (ESI-MS) and spectroscopic studies in solution were used to evaluate the interaction of PBD and TMPyP4 ligands, stoichiometry and selectivity to G-quadruplex DNA...

  6. Behavior of the guanine base in G-quadruplexes probed by the fluorescent guanine analog, 6-methyl isozanthopterin

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ji Hoon; Chitrapriya, Nataraj; Lee, Hyun Suk; Lee, Young Ae; Kim, Seog K. [Dept. of Chemistry, Yeungnam University, Gyeongsan (Korea, Republic of); Jung, Maeng Joon [Dept. of Chemistry, Kyungpook National University, Daegu (Korea, Republic of)

    2017-02-15

    In this study, circular dichroism (CD) spectrum and fluorescence techniques were used to examine the dynamic properties and microenvironment of the guanine base (G) at the central loop and at the middle of the G-stem of the G-quadruplex formed from the G{sub 3}T{sub 2}G{sub 3}TGTG{sub 3}T{sub 2}G{sub 3} sequence (G-quadruplex 1), in which the G base at the 10th and 13th position were replaced with a fluorescent G analog, 6-methyl isoxanthopterin (6MI) (G-quadruplex 2 and 3, respectively). For all G-quadruplexes, the CD spectrum revealed a positive band at 263 nm and a shoulder at 298 nm, and the thermal melting profiles were the sum of at least two sigmoidal curves. These observations indicated the presence of two conformers in the G-quadruplex. The fluorescence intensity of G-quadruplex 2 was greater than 3, as expected from the extent of stacking interaction, which is larger in the G(6MI)G sequence than the T(6MI)T sequence. The efficiency of fluorescence quenching by the polar acrylamide quencher and negatively charged I− quencher were larger for G-quadruplex 3, suggesting that 6MI in the G(6MI)G stem is exposed more to the aqueous environment compared to that in the T(6MI)T central loop. In the latter case, 6MI may direct to the center of the top G-quartet layer. The possibility of hydrogen bond formation between the carbonyl group of 6MI and the acrylamide of the G-quadruplex 3 was proposed.

  7. G-Quadruplex in the NRF2 mRNA 5' Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress.

    Science.gov (United States)

    Lee, Sang C; Zhang, Jack; Strom, Josh; Yang, Danzhou; Dinh, Thai Nho; Kappeler, Kyle; Chen, Qin M

    2017-01-01

    Inhibition of protein synthesis serves as a general measure of cellular consequences of chemical stress. A few proteins are translated selectively and influence cell fate. How these proteins can bypass the general control of translation remains unknown. We found that low to mild doses of oxidants induce de novo translation of the NRF2 protein. Here we demonstrate the presence of a G-quadruplex structure in the 5' untranslated region (UTR) of NRF2 mRNA, as measured by circular dichroism, nuclear magnetic resonance, and dimethylsulfate footprinting analyses. Such a structure is important for 5'-UTR activity, since its removal by sequence mutation eliminated H2O2-induced activation of the NRF2 5' UTR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed elongation factor 1 alpha (EF1a) as a protein binding to the G-quadruplex sequence. Cells responded to H2O2 treatment by increasing the EF1a protein association with NRF2 mRNA, as measured by RNA-protein interaction assays. The EF1a interaction with small and large subunits of ribosomes did not appear to change due to H2O2 treatment, nor did posttranslational modifications, as measured by two-dimensional (2-D) Western blot analysis. Since NRF2 encodes a transcription factor essential for protection against tissue injury, our data have revealed a novel mechanism of cellular defense involving de novo NRF2 protein translation governed by the EF1a interaction with the G-quadruplex in the NRF2 5' UTR during oxidative stress. Copyright © 2016 American Society for Microbiology.

  8. Conformational Dynamics of DNA G-Quadruplex in Solution Studied by Kinetic Capillary Electrophoresis Coupled On-line with Mass Spectrometry

    Science.gov (United States)

    Berezovski, Maxim V

    2014-01-01

    Invited for this months cover is the group of Prof. Maxim V. Berezovski. The cover picture shows the two-dimensional separation of unfolded (green) and folded (red) forms of G-quadruplex (GQ) DNA. The first dimension is kinetic capillary electrophoresis (KCE) separation of unfolded and folded DNA with different K+ concentrations in solution; the second dimension is ion mobility mass spectrometry separation of DNA conformers in the gas phase. DNA folding into a compact GQ structure is mediated by K+ ions. For more details, see the Full Paper on p. 58 ff. PMID:24808989

  9. High-throughput identification of telomere-binding ligands based on the fluorescence regulation of DNA-copper nanoparticles.

    Science.gov (United States)

    Yang, Luzhu; Wang, Yanjun; Li, Baoxin; Jin, Yan

    2017-01-15

    Formation of the G-quadruplex in the human telomeric DNA is an effective way to inhibit telomerase activity. Therefore, screening ligands of G-quadruplex has potential applications in the treatment of cancer by inhibit telomerase activity. Although several techniques have been explored for screening of telomeric G-quadruplexes ligands, high-throughput screening method for fast screening telomere-binding ligands from the large compound library is still urgently needed. Herein, a label-free fluorescence strategy has been proposed for high-throughput screening telomere-binding ligands by using DNA-copper nanoparticles (DNA-CuNPs) as a signal probe. In the absence of ligands, human telomeric DNA (GDNA) hybridized with its complementary DNA (cDNA) to form double stranded DNA (dsDNA) which can act as an efficient template for the formation of DNA-CuNPs, leading to the high fluorescence of DNA-CuNPs. In the presence of ligands, GDNA folded into G-quadruplex. Single-strdanded cDNA does not support the formation of DNA-CuNP, resulting in low fluorescence of DNA-CuNPs. Therefore, telomere-binding ligands can be high-throughput screened by monitoring the change in the fluorescence of DNA-CuNPs. Thirteen traditional chinese medicines were screened. Circular dichroism (CD) measurements demonstrated that the selected ligands could induce single-stranded telomeric DNA to form G-quadruplex. The telomere repeat amplification protocol (TRAP) assay demonstrated that the selected ligands can effectively inhibit telomerase activity. Therefore, it offers a cost-effective, label-free and reliable high-throughput way to identify G-quadruplex ligands, which holds great potential in discovering telomerase-targeted anticancer drugs. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. A selective and label-free strategy for rapid screening of telomere-binding Ligands via fluorescence regulation of DNA/silver nanocluster

    Science.gov (United States)

    Cheng, Rui; Xu, Jing; Zhang, Xiafei; Shi, Zhilu; Zhang, Qi; Jin, Yan

    2017-03-01

    Herein, the conformational switch of G-rich oligonucleotide (GDNA) demonstrated the obvious functional switch of GDNA which was found to significantly affect the fluorescence of the in-situ synthesized DNA/silver nanocluster (DNA-AgNC) in homogeneous solution. We envisioned that the allosteric interaction between GDNA and DNA-AgNC would be possible to be used for screening telomere-binding ligands. A unimolecular probe (12C5TG) is ingeniously designed consisting of three contiguous DNA elements: G-rich telomeric DNA (GDNA) as molecular recognition sequence, T-rich DNA as linker and C-rich DNA as template of DNA-AgNC. The quantum yield and stability of 12C5TG-AgNC is greatly improved because the nearby deoxyguanosines tended to protect DNA/AgNC against oxidation. However, in the presence of ligands, the formation of G-quadruplex obviously quenched the fluorescence of DNA-AgNC. By taking full advantage of intramolecular allosteric effect, telomere-binding ligands were selectively and label-free screened by using deoxyguanines and G-quadruplex as natural fluorescence enhancer and quencher of DNA-AgNC respectively. Therefore, the functional switching of G-rich structure offers a cost-effective, facile and reliable way to screen drugs, which holds a great potential in bioanalysis as well.

  11. Analysis of the Fragile X mental retardation protein isoforms 1, 2 and 3 interactions with the G-quadruplex forming semaphorin 3F mRNA.

    Science.gov (United States)

    Evans, Timothy L; Blice-Baum, Anna C; Mihailescu, Mihaela-Rita

    2012-02-01

    Fragile X syndrome, the most prevalent inheritable mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP) expression. FMRP is an RNA-binding protein with nucleo-cytoplasmic shuttle activity, proposed to act as a translation regulator of specific mRNAs in the brain. It has been shown that FMRP uses its arginine-glycine-glycine (RGG) box domain to bind a subset of mRNA targets that form a G-quadruplex structure. FMRP has also been shown to undergo the post-translational modifications of arginine methylation and phosphorylation, as well as alternative splicing, resulting in multiple isoforms. The alternative splice isoforms investigated in this study, isoform 1 (ISO1), isoform 2 (ISO2), and isoform 3 (ISO3), are created by the alternative splicing acceptor site at exon 15. FMRP ISO2 and ISO3 are truncated by 12 and 13 residues, respectively, relative to the longest FMRP isoform ISO1. These truncations, which are in the close proximity of the RGG box domain, preserve the integrity of the RGG box in all three isoforms, but eliminate the in vivo phosphorylation sites, present only on FMRP ISO1. We have expressed and purified recombinant FMRP ISO1, ISO2 and ISO3 in Escherichia coli, free of post-translational modifications, and by using fluorescence spectroscopy, we show that each FMRP isoform binds G-quadruplex RNA, albeit with different binding affinities, suggesting that naturally occurring sequence modifications in the proximity of the RGG box modulate its G-quadruplex RNA binding ability.

  12. A pseudo triple-enzyme electrochemical aptasensor based on the amplification of Pt–Pd nanowires and hemin/G-quadruplex

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yingning; Chai, Yaqin, E-mail: yqchai@swu.edu.cn; Yuan, Yali; Yuan, Ruo, E-mail: yuanruo@swu.edu.cn

    2014-06-27

    Highlights: • Pt–Pd nanowires were combined with pseudo triple-enzyme electrochemical aptasensor. • Pt–Pd nanowires served as signal enhancer. • Labeling process, deactivation and spatial distribution of enzymes were solved. - Abstract: Our present work aimed at developing a pseudo triple-enzyme cascade electrocatalytic electrochemical aptasensor for determination of thrombin with the amplification of alcohol dehydrogenase (ADH)-Pt–Pd nanowires bionanocomposite and hemin/G-quadruplex structure that simultaneously acted as NADH oxidase and HRP-mimicking DNAzyme. With the addition of ethanol to the electrolyte, the ADH immobilized on the Pt–Pd nanowires catalyzed ethanol to acetaldehyde accompanied by NAD{sup +} being converted to NADH. Then the hemin/G-quadruplex firstly served as NADH oxidase, converting the produced NADH to NAD{sup +} with the concomitant local formation of high concentration of H{sub 2}O{sub 2}. Subsequently, the hemin/G-quadruplex acted as HRP-mimicking DNAzyme, bioelectrocatalyzing the produced H{sub 2}O{sub 2}. At the same time, the Pt–Pd nanowires employed in our strategy not only provided a large surface area for immobilizing thrombin binding aptamer (TBA) and ADH, but also served as HRP-mimicking DNAzyme which rapidly bioelectrocatalyzed the reduction of the produced H{sub 2}O{sub 2}. Thus, such a pseudo triple-enzyme cascade electrochemical aptasensor could greatly promote the electron transfer of hemin and resulted in the dramatic enhancement of electrochemical signal. As a result, a wide dynamic concentration linear range from 0.2 pM to 20 nM with a low detection limit of 0.067 pM for thrombin (TB) determination was obtained. The excellent performance indicated that our strategy was a promising way for ultrasensitive assays in electrochemical aptasensors.

  13. The SARS-unique domain (SUD of SARS coronavirus contains two macrodomains that bind G-quadruplexes.

    Directory of Open Access Journals (Sweden)

    Jinzhi Tan

    2009-05-01

    Full Text Available Since the outbreak of severe acute respiratory syndrome (SARS in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV, the non-structural proteins (Nsps, have been determined. However, within the large Nsp3 (1922 amino-acid residues, the structure and function of the so-called SARS-unique domain (SUD have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389-652 ("SUD(core" of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 A resolution, respectively revealed that SUD(core forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUD(core as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5-6 nucleotides, but more extended G-stretches are found in the 3'-nontranslated regions of mRNAs coding for certain host-cell proteins

  14. The essential Schizosaccharomyces pombe Pfh1 DNA helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage.

    Science.gov (United States)

    Sabouri, Nasim; Capra, John A; Zakian, Virginia A

    2014-12-04

    G-quadruplexes (G4s) are stable non-canonical DNA secondary structures consisting of stacked arrays of four guanines, each held together by Hoogsteen hydrogen bonds. Sequences with the ability to form these structures in vitro, G4 motifs, are found throughout bacterial and eukaryotic genomes. The budding yeast Pif1 DNA helicase, as well as several bacterial Pif1 family helicases, unwind G4 structures robustly in vitro and suppress G4-induced DNA damage in S. cerevisiae in vivo. We determined the genomic distribution and evolutionary conservation of G4 motifs in four fission yeast species and investigated the relationship between G4 motifs and Pfh1, the sole S. pombe Pif1 family helicase. Using chromatin immunoprecipitation combined with deep sequencing, we found that many G4 motifs in the S. pombe genome were associated with Pfh1. Cells depleted of Pfh1 had increased fork pausing and DNA damage near G4 motifs, as indicated by high DNA polymerase occupancy and phosphorylated histone H2A, respectively. In general, G4 motifs were underrepresented in genes. However, Pfh1-associated G4 motifs were located on the transcribed strand of highly transcribed genes significantly more often than expected, suggesting that Pfh1 has a function in replication or transcription at these sites. In the absence of functional Pfh1, unresolved G4 structures cause fork pausing and DNA damage of the sort associated with human tumors.

  15. Sequencing and G-quadruplex folding of the canine proto-oncogene KIT promoter region: might dog be used as a model for human disease?

    Directory of Open Access Journals (Sweden)

    Silvia Da Ros

    Full Text Available Downregulation of gene expression by induction of non-canonical DNA structures at promotorial level is a novel attractive anticancer strategy. In human, two guanine-rich sequences (h_kit1 and h_kit2 were identified in the promotorial region of oncogene KIT. Their stabilization into G-quadruplex structures can find applications in the treatment of leukemias, mastocytosis, gastrointestinal stromal tumor, and lung carcinomas which are often associated to c-kit mis-regulation. Also the most common skin cancer in domestic dog, mast cell tumor, is linked to a mutation and/or to an over-expression of c-kit, thus supporting dog as an excellent animal model. In order to assess if the G-quadruplex mediated mechanism of regulation of c-kit expression is conserved among the two species, herein we cloned and sequenced the canine KIT promoter region and we compared it with the human one in terms of sequence and conformational equilibria in physiologically relevant conditions. Our results evidenced a general conserved promotorial sequence between the two species. As experimentally confirmed, this grants that the conformational features of the canine kit1 sequence are substantially shared with the human one. Conversely, two isoforms of the kit2 sequences were identified in the analyzed dog population. In comparison with the human counterpart, both of them showed an altered distribution among several folded conformations.

  16. G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA

    Science.gov (United States)

    Stoltenburg, Regina; Krafčiková, Petra; Víglaský, Viktor; Strehlitz, Beate

    2016-01-01

    Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5′-end also contributes essentially to the aptameric function. PMID:27650576

  17. Nucleic acid clamp-mediated recognition and stabilization of the physiologically relevant MYC promoter G-quadruplex

    Science.gov (United States)

    Hao, Taisen; Gaerig, Vanessa C.; Brooks, Tracy A.

    2016-01-01

    The MYC proto-oncogene is upregulated, often at the transcriptional level, in ∼80% of all cancers. MYC's promoter is governed by a higher order G-quadruplex (G4) structure in the NHE III1 region. Under a variety of conditions, multiple isoforms have been described to form from the first four continuous guanine runs (G41–4) predominating under the physiologically relevant supercoiled conditions. In the current study, short oligonucleotides complementing the 5′- and 3′-regions flanking the G4 have been connected by an abasic linker to form G4 clamps, varying both linker length and G4 isoform being targeted. Clamp A with an 18 Å linker was found to have marked affinity for its target isomer (G41–4) over the other major structures (G42–5 and G41–5, recognized by clamps B and C, respectively), and to be able to shift equilibrating DNA to foster greater G4 formation. In addition, clamp A, but not B or C, is able to modulate MYC promoter activity with a significant and dose-dependent effect on transcription driven by the Del4 plasmid. This linked clamp-mediated approach to G4 recognition represents a novel therapeutic mechanism with specificity for an individual promoter structure, amenable to a large array of promoters. PMID:27789698

  18. Nucleotide Pool Depletion Induces G-Quadruplex-Dependent Perturbation of Gene Expression

    Directory of Open Access Journals (Sweden)

    Charikleia Papadopoulou

    2015-12-01

    Full Text Available Nucleotide pool imbalance has been proposed to drive genetic instability in cancer. Here, we show that slowing replication forks by depleting nucleotide pools with hydroxyurea (HU can also give rise to both transient and permanent epigenetic instability of a reporter locus, BU-1, in DT40 cells. HU induces stochastic formation of Bu-1low variants in dividing cells, which have lost the H3K4me3 present in untreated cells. This instability is potentiated by an intragenic G quadruplex, which also promotes local H2Ax phosphorylation and transient heterochromatinization. Genome-wide, gene expression changes induced by HU significantly overlap with those resulting from loss of the G4-helicases FANCJ, WRN, and BLM. Thus, the effects of global replication stress induced by nucleotide pool depletion can be focused by local replication impediments caused by G quadruplex formation to induce epigenetic instability and changes in gene expression, a mechanism that may contribute to selectable transcriptional changes in cancer.

  19. Development of a luminescent G-quadruplex-selective iridium(III) complex for the label-free detection of adenosine

    Science.gov (United States)

    Lu, Lihua; Zhong, Hai-Jing; He, Bingyong; Leung, Chung-Hang; Ma, Dik-Lung

    2016-01-01

    A panel of six luminescent iridium(III) complexes were synthesized and evaluated for their ability to act as G-quadruplex-selective probes. The novel iridium(III) complex 1 was found to be highly selective for G-quadruplex DNA, and was employed for the construction of a label-free G-quadruplex-based adenosine detection assay in aqueous solution. Two different detection strategies were investigated for adenosine detection, and the results showed that initial addition of adenosine to the adenosine aptamer gave superior results. The assay exhibited a linear response for adenosine in the concentration range of 5 to 120 μM (R2 = 0.992), and the limit of detection for adenosine was 5 μM. Moreover, this assay was highly selective for adenosine over other nucleosides, and exhibited potential use for biological sample analysis.

  20. A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex.

    Science.gov (United States)

    Kusov, Yuri; Tan, Jinzhi; Alvarez, Enrique; Enjuanes, Luis; Hilgenfeld, Rolf

    2015-10-01

    The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the "SARS-unique domain". The X domain was proposed to be an ADP-ribose-1"-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. A colorimetric and smartphone readable method for uracil-DNA glycosylase detection based on the target-triggered formation of G-quadruplex.

    Science.gov (United States)

    Nie, Huaijun; Wang, Wei; Li, Wang; Nie, Zhou; Yao, Shouzhuo

    2015-04-21

    A simple, visible and smartphone readable strategy for the sensitive detection of uracil-DNA glycosylase (UDG) activity has been developed. This is based on the UDG-catalyzed removal of uracil bases, which induces the formation of a G-quadruplex-cofactor complex. The guanine-rich DNA probe can form a special G-quadruplex structure with hemin to display peroxidase activity and catalyze the H2O2-mediated oxidation of ABTS(2-) to the colored ABTS˙(-), providing a visible signal for UDG detection. The proposed sensing platform exhibits a good linear response to UDG concentrations ranging from 0.008 to 0.2 U mL(-1), with a low detection limit of 0.008 U mL(-1). Based on the theory of chromatics, we can directly read out the color value using a smartphone app to reflect the content of UDG with high resolution, providing a new vision for the portable assay strategy. Furthermore, the utility of this method for screening potential UDG inhibitors has also been demonstrated.

  2. Circular dichroism spectroscopic investigation of double-decker phthalocyanine with G-Quadruplex as promising telomerase inhibitor

    Science.gov (United States)

    Baǧda, Efkan; Baǧda, Esra; Yabaş, Ebru

    2017-01-01

    In the present study, interaction of a double-decker phthalocyanine with two G-quadruplex DNA, Tel 21 and cMYC, was investigated. To the best of our knowledge, this is the first study about G-quadruplex-double decker phthalocyanine interaction. The spectrophotometric titration method was used for binding constant calculations. From the binding constants, it can be said that double-decker phthalocyanine more likely to bind Tel 21 rather than cMYC. The conformational changes upon binding were monitored via circular dichroism spectroscopy. The ethidium bromide replacement assay was investigated spectrofluorometrically.

  3. A G-rich element forms a G-quadruplex and regulates BACE1 mRNA alternative splicing.

    Science.gov (United States)

    Fisette, Jean-François; Montagna, Daniel R; Mihailescu, Mihaela-Rita; Wolfe, Michael S

    2012-06-01

    β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the transmembrane aspartyl protease that catalyzes the first cleavage step in the proteolysis of the APP to the amyloid β-protein (Aβ), a process involved in the pathogenesis of Alzheimer disease. BACE1 pre-mRNA undergoes complex alternative splicing, the regulation of which is not well understood. We identified a G-rich sequence within exon 3 of BACE1 involved in controlling splice site selection. Mutation of the G-rich sequence decreased use of the normal 5' splice site of exon 3, which leads to full-length and proteolytically active BACE1, and increased use of an alternative splice site, which leads to a shorter, essentially inactive isoform. Nuclease protection assays, nuclear magnetic resonance, and circular dichroism spectroscopy revealed that this sequence folds into a G-quadruplex structure. Several proteins were identified as capable of binding to the G-rich sequence, and one of these, heterogeneous nuclear ribonucleoprotein H, was found to regulate BACE1 exon 3 alternative splicing and in a manner dependent on the G-rich sequence. Knockdown of heterogeneous nuclear ribonucleoprotein H led to a decrease in the full-length BACE1 mRNA isoform as well as a decrease in Aβ production from APP, suggesting new possibilities for therapeutic approaches to Alzheimer's disease. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

  4. Fuzzy logic sensing of G-quadruplex DNA and its cleavage reagents based on reduced graphene oxide.

    Science.gov (United States)

    Huang, Wei Tao; Zhang, Jian Rong; Xie, Wan Yi; Shi, Yan; Luo, Hong Qun; Li, Nian Bing

    2014-07-15

    Herein, by combining the merits of nanotechnology and fuzzy logic theory, we develop a simple, label-free, and general strategy based on an organic dye-graphene hybrid system for fluorescence intelligent sensing of G-quadruplexes (G4) formation, hydroxyl radical (HO∙), and Fe(2+) in vitro. By exploiting acridine orange (AO) dyes-graphene as a nanofilter and nanoswitch and the ability of graphene to interact with DNA with different structures, our approach can efficiently distinguish, quantitatively detect target analytes. In vitro assays with G4DNA demonstrated increases in fluorescence intensity of the AO-rGO system with a linear range of 16-338 nM and a detection limit as low as 2.0 nM. The requenched fluorescence of the G4TBA-AO-rGO system has a non-linear response to Fenton reagent. But this requenching reduces the fluorescence intensity in a manner proportional to the logarithm to the base 10 of the concentration of Fenton reagent in the range of 0.1-100 μM and 100-2000 μM, respectively. Furthermore, we develop a novel and intelligent sensing method based on fuzzy logic which mimics human reasoning, solves complex and non-linear problems, and transforms the numerical output into the language description output for potential application in biochemical systems, environmental monitoring systems, and molecular-level fuzzy logic computing system. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. The G-quadruplex-forming aptamer AS1411 potently inhibits HIV-1 attachment to the host cell.

    Science.gov (United States)

    Perrone, Rosalba; Butovskaya, Elena; Lago, Sara; Garzino-Demo, Alfredo; Pannecouque, Christophe; Palù, Giorgio; Richter, Sara N

    2016-04-01

    AS1411 is a G-rich aptamer that forms a stable G-quadruplex structure and displays antineoplastic properties both in vitro and in vivo. This oligonucleotide has undergone phase 2 clinical trials. The major molecular target of AS1411 is nucleolin (NCL), a multifunctional nucleolar protein also present in the cell membrane where it selectively mediates the binding and uptake of AS1411. Cell-surface NCL has been recognised as a low-affinity co-receptor for human immunodeficiency virus type 1 (HIV-1) anchorage on target cells. Here we assessed the anti-HIV-1 properties and underlying mechanism of action of AS1411. The antiviral activity of AS1411 was determined towards different HIV-1 strains, host cells and at various times post-infection. Acutely, persistently and latently infected cells were tested, including HIV-1-infected peripheral blood mononuclear cells from a healthy donor. Mechanistic studies to exclude modes of action other than virus binding via NCL were performed. AS1411 efficiently inhibited HIV-1 attachment/entry into the host cell. The aptamer displayed antiviral activity in the absence of cytotoxicity at the tested doses, therefore displaying a wide therapeutic window and favourable selectivity indexes. These findings, besides validating cell-surface-expressed NCL as an antiviral target, open the way for the possible use of AS1411 as a new potent and promisingly safe anti-HIV-1 agent. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Crystal structure of four-stranded Oxytricha telomeric DNA

    Science.gov (United States)

    Kang, C.; Zhang, X.; Ratliff, R.; Moyzis, R.; Rich, A.

    1992-01-01

    The sequence d(GGGGTTTTGGGG) from the 3' overhang of the Oxytricha telomere has been crystallized and its three-dimensional structure solved to 2.5 A resolution. The oligonucleotide forms hairpins, two of which join to make a four-stranded helical structure with the loops containing four thymine residues at either end. The guanine residues are held together by cyclic hydrogen bonding and an ion is located in the centre. The four guanine residues in each segment have a glycosyl conformation that alternates between anti and syn. There are two four-stranded molecules in the asymmetric unit showing that the structure has some intrinsic flexibility.

  7. Translational repression of cyclin D3 by a stable G-quadruplex in its 5′ UTR: implications for cell cycle regulation

    National Research Council Canada - National Science Library

    Weng, Heng-You; Huang, Hui-Lin; Zhao, Pan-Pan; Zhou, Hui; Qu, Liang-Hu

    2012-01-01

    .... In this study, we report that a conserved RNA G-quadruplex-forming sequence (hereafter CRQ), located in the 5′ UTR of mammalian CCND3 mRNA, is able to fold into an extremely stable, intramolecular, parallel G-quadruplex in vitro...

  8. G-quadruplex DNA/protoporphyrin IX-based synergistic platform for targeted photodynamic cancer therapy.

    Science.gov (United States)

    Zhou, Zhixue; Li, Dan; Zhang, Libing; Wang, Erkang; Dong, Shaojun

    2015-03-01

    Photodynamic therapy (PDT) is an emerging technique to induce cancer cell death. However, the tumor specificity, cellular uptake and biodistribution of many photosensitizers urgently need to be improved. In this regard, we show here that the integrated nanoassemblies based on G-quadruplex DNAs (GQDs)/protoporphyrin IX (PPIX) can serve as a synergistic platform for targeted high-performance PDT. In the nanoassemblies, GQDs function as carriers of sensitiser PPIX and confers the system cancer cell targeting ability. After nucleolin-mediated efficient binding and cellular uptake of GQDs/PPIX assemblies, the strong red fluorescence of GQDs/PPIX complex provides a powerful tool for biological imaging. Moreover, the reactive oxygen species (ROS) generated by GQDs/PPIX under light illumination can effectively kill cancer cells. The present approach is simply composed by DNA and photosensitizers, thereby avoiding any complicated and time-consuming covalent modification or chemical labeling procedure. Copyright © 2014. Published by Elsevier B.V.

  9. G-quadruplex fluorescence sensing by core-extended naphthalene diimides.

    Science.gov (United States)

    Zuffo, Michela; Doria, Filippo; Botti, Silvia; Bergamaschi, Greta; Freccero, Mauro

    2017-05-01

    Fluorescent sensing of G-quadruplex nucleic acids (G4s) is an effective strategy to elucidate their role in vitro and in vivo. Small molecule ligands have often been exploited, producing an emission light up upon binding. Naphthalene diimides (NDIs), although potent G4 binders exhibiting red-NIR fluorophores, have only been marginally exploited, as they are usually quenched upon binding. Contrary, aggregating core-extended naphthalene diimides (c ex -NDIs) proved to be effective probes. We prepared a library of eighteen c ex -NDIs by organic synthesis, characterising their aggregation-dependent absorption and emission properties. Absorption and emission titrations, fluorescent intercalator displacement assay (FID) and circular dichroism (CD) analysis were performed to elucidate their behavior as G4 fluorescent sensors, selectivity and binding mode. c ex -NDIs aggregate under aqueous solvents and as a result, their fluorescence is mostly quenched under physiological conditions. Upon G4 binding, they disaggregate into binding monomers, producing a fluorescent light-up with anti-parallel and hybrid G4s. Contrary, with parallel G4s a light-off was recorded. For the formers a groove-like interaction was inferred by ICD signals, while for the latter an end-stacking interaction mode was hypothesized by G4-FID data. c ex -NDIs G4 sensing mechanism works via a induced disaggregation. The emission response depends on the G4 topology, which dictates the prevailing -groove or end-stacking- binding mode. This study highlights the potential of c ex -NDIs as G4 fluorescent probes. Besides being readily synthesized and conveniently emitting above 600nm, they light-up upon binding to anti-parallel and hybrid G4, complementing a number of other probes' selectivity for the parallel topology. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Intermolecular G-quadruplex induces Hyaluronic Acid-DNA superpolymers causing cancer cell swelling, blebbing and death.

    Science.gov (United States)

    Beals, Nathan; Model, Michael; Worden, Matt; Hegmann, Torsten; Basu, Soumitra

    2018-02-05

    Over the past decade, nanomedicine has gained considerable traction through its relevance, for example in "smart" delivery, thus creating platforms for novel treatments. Here we report a natural polymer-DNA conjugate that undergoes self-assembly in a K+ dependent fashion to form a G-quadruplex (GQ) and generate superpolymeric structures. We derivatized the naturally occurring glycosaminoglycan polymer hyaluronic acid (HA) with short G-rich DNA (HASH-DNA) that can form intermolecular non-canonical GQ structure. Gel mobility shift assay and Circular Dichroism measurements confirmed HASH conjugation to DNA and K+ dependent GQ formation respectively. TEM and SEM results indicated that the addition of K+ to the HASH-DNA conjugate led to the formation of micron range structures, while control samples remained unordered and as nebulous globular form. Confocal microscopy of a fluorescently labeled form of the superpolymer verified increased cellular uptake. The HASH-DNA conjugates showed toxicity in HeLa cells while a scrambled DNA (Mut) conjugate HASH-Mut showed no cytotoxicity, presumably due to non-formation of the superpolymeric structure. To understand the mechanism of cell death and if the superpolymeric structure is responsible for it, we monitored the cell size and observed an average of 23% increase in size compared to 4.5% in control cells at 4.5 hours. We believe that cellular stress is generated presumably by the intracellular assembly of this large superpolymeric nanostructures causing cell blebbing with no exit option. This approach provides a new strategy of cellular delivery of a targeted naturally occurring polymer and a novel way to induce superpolymeric structure formation that acts as a therapeutic.

  11. Development of an Efficient G-Quadruplex-Stabilised Thrombin-Binding Aptamer Containing a Three-Carbon Spacer Molecule

    DEFF Research Database (Denmark)

    Aaldering, Lukas J.; Poongavanam, Vasanthanathan; Langkjær, Niels

    2017-01-01

    The thrombin-binding aptamer (TBA), which shows anticoagulant properties, is one of the most studied G-quadruplex-forming aptamers. In this study, we investigated the impact of different chemical modifications such as a three-carbon spacer (spacer-C3), unlocked nucleic acid (UNA) and 3′-amino...

  12. Design, synthesis and evaluation of 4,7-diamino-1,10-phenanthroline G-quadruplex ligands

    DEFF Research Database (Denmark)

    Nielsen, Mads Corvinius; Borch, Jonas; Ulven, Trond

    2009-01-01

    a weak but significant G-quadruplex stabilizing effect, compared to no stabilization by 1,10-phenanthroline. We hypothesize that this effect is due to increased basicity of the phenanthroline nitrogens and protonation or ion chelation to form a central positive charge which stack on the G-tetrad above...

  13. “One Ring to Bind Them All”—Part I: The Efficiency of the Macrocyclic Scaffold for G-Quadruplex DNA Recognition

    Directory of Open Access Journals (Sweden)

    David Monchaud

    2010-01-01

    Full Text Available Macrocyclic scaffolds are particularly attractive for designing selective G-quadruplex ligands essentially because, on one hand, they show a poor affinity for the “standard” B-DNA conformation and, on the other hand, they fit nicely with the external G-quartets of quadruplexes. Stimulated by the pioneering studies on the cationic porphyrin TMPyP4 and the natural product telomestatin, follow-up studies have developed, rapidly leading to a large diversity of macrocyclic structures with remarkable-quadruplex binding properties and biological activities. In this review we summarize the current state of the art in detailing the three main categories of quadruplex-binding macrocycles described so far (telomestatin-like polyheteroarenes, porphyrins and derivatives, polyammonium cyclophanes, and in addressing both synthetic issues and biological aspects.

  14. A label-free G-quadruplex-based mercury detection assay employing the exonuclease III-mediated cleavage of T?Hg2+?T mismatched DNA

    OpenAIRE

    Wang, Wanhe; Kang, Tian-Shu; Chan, Philip Wai Hong; Lu, Jin-Jian; Chen, Xiu-Ping; Leung, Chung-Hang; Ma, Dik-Lung

    2015-01-01

    We report herein the use of an exonuclease III and G-quadruplex probe to construct a G-quadruplex-based luminescence detection platform for Hg2+. Unlike common DNA-based Hg2+ detection methods, when using the dsDNA probe to monitor the hairpin formation, the intercalation of the dsDNA probe may be influenced by the distortion of dsDNA. This ?mix-and-detect? methodology utilized the G-quadruplex probe as the signal transducer and is simple, rapid, convenient to use and can detect down to 20 nM...

  15. Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Ji-Hye [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Won Kyung [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Heeyoun [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Eunhee; Cheong, Chaejoon [Magnetic Resonance Team, Korea Basic Science Institute (KBSI), Ochang, Chungbuk 363-883 (Korea, Republic of); Cho, Myeon Haeng [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Weontae, E-mail: wlee@spin.yonsei.ac.kr [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2014-09-26

    Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminal Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2{sub 1–64}) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2{sub 1–64} and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences.

  16. Synthesis, G-quadruplexes DNA binding, and photocytotoxicity of novel cationic expanded porphyrins.

    Science.gov (United States)

    Jin, Shu-fang; Zhao, Ping; Xu, Lian-cai; Zheng, Min; Lu, Jia-zheng; Zhao, Peng-liang; Su, Qiu-lan; Chen, Hui-xian; Tang, Ding-tong; Chen, Jiong; Lin, Jia-qi

    2015-06-01

    Intensive reports allowed the conclusion that molecules with extended aromatic surfaces always do good jobs in the DNA interactions. Inspired by the previous successful researches, herein, we designed a series of cationic porphyrins with expanded planar substituents, and evaluated their binding behaviors to G-quadruplex DNA using the combination of surface-enhanced raman, circular dichroism, absorption spectroscopy and fluorescence resonance energy transfer melting assays. Asymmetrical tetracationic porphyrin with one phenyl-4-N-methyl-4-pyridyl group and three N-methyl-4-pyridyl groups exhibit the best G4-DNA binding affinities among all the designed compounds, suggesting that the bulk of the substituents should be matched to the width of the grooves they putatively lie in. Theoretical calculations applying the density functional theory have been carried out and explain the binding properties of these porphyrins reasonably. Meanwhile, these porphyrins were proved to be potential photochemotherapeutic agents since they have photocytotoxic activities against both myeloma cell (Ag8.653) and gliomas cell (U251) lines. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Getting in (and out of the loop: regulating higher order telomere structures

    Directory of Open Access Journals (Sweden)

    Sarah eLuke-Glaser

    2012-11-01

    Full Text Available The DNA at the ends of linear chromosomes (the telomere folds back onto itself and forms an intramolecular lariat-like structure. Although the telomere loop has been implicated in the protection of chromosome ends from nuclease-mediated resection and unscheduled DNA repair activities, it potentially poses an obstacle to the DNA replication machinery during S phase. Therefore, the coordinated regulation of telomere loop formation, maintenance and resolution is required in order to establish a balance between protecting the chromosome ends and promoting their duplication prior to cell division. Until recently, the only factor know to influence telomere looping in human cells was TRF2, a component of the shelterin complex. Recent work in yeast and mouse cells has uncovered additional regulatory factors that affect the loop structure at telomeres. In the following perspective we will outline what is known about telomere looping and highlight the latest results regarding the regulation of this chromosome end structure. We will speculate about how the manipulation of the telomere loop may have therapeutic implications in terms of diseases associated with telomere dysfunction and uncontrolled proliferation.

  18. ESI Mass Spectrometric Exploration of Selective Recognition of G-Quadruplex in c- myb Oncogene Promoter Using a Novel Flexible Cyclic Polyamide

    Science.gov (United States)

    Cui, Xiaojie; Zhang, Qiang; Chen, Han; Zhou, Jiang; Yuan, Gu

    2014-04-01

    In this research, electrospray ionization mass spectrometry (ESI-MS) was used to probe the binding selectivity of a flexible cyclic polyamide (cβ) to G-quadruplexes from the long G-rich sequences in the c- myb oncogene promoter. The results show that three G-rich sequences, including d[(GGA)3GGTCAC(GGA)4], d[(GGA)4GAA(GGA)4], and d[(GGA)3GGTCAC(GGA)4GAA(GGA)4] species in the c- myb promoter can form parallel G-quadruplexes, and cβ selectively binds towards these G-quadruplexes over both several other G-quadruplexes and the duplex DNA. These properties of cβ have profound implications on future studies of the regulation of c- myb oncogene expression.

  19. Single molecule conformational analysis of the biologically relevant DNA G-quadruplex in the promoter of the proto-oncogene c-MYC†

    Science.gov (United States)

    Shirude, Pravin S.; Ying, Liming; Balasubramanian, Shankar

    2008-01-01

    Single molecule fluorescence spectroscopy has been employed to resolve the conformational heterogeneity, hybridization kinetics and study mutational effects on the c-MYC promoter G-quadruplex. PMID:18536803

  20. Self-assembled Pt2L2 boxes strongly bind G-quadruplex DNA and influence gene expression in cancer cells.

    Science.gov (United States)

    Domarco, O; Lötsch, D; Schreiber, J; Dinhof, C; Van Schoonhoven, S; García, M D; Peinador, C; Keppler, B K; Berger, W; Terenzi, A

    2017-01-03

    Supramolecular Pt(ii) quadrangular boxes bind native and G-quadruplex DNA motifs in a size-dependent fashion. Three Pt molecular squares of distinct size show biological activity against cancer cells and heavily influence the expression of genes known to form G-quadruplexes in their promoter regions. The smallest Pt-box displays less activity but more selectivity for a quadruplex formed in the c-Kit gene.

  1. Structure and function of the telomeric CST complex

    Directory of Open Access Journals (Sweden)

    Cory Rice

    2016-01-01

    Full Text Available Telomeres comprise the ends of eukaryotic chromosomes and are essential for cell proliferation and genome maintenance. Telomeres are replicated by telomerase, a ribonucleoprotein (RNP reverse transcriptase, and are maintained primarily by nucleoprotein complexes such as shelterin (TRF1, TRF2, TIN2, RAP1, POT1, TPP1 and CST (Cdc13/Ctc1, Stn1, Ten1. The focus of this review is on the CST complex and its role in telomere maintenance. Although initially thought to be unique to yeast, it is now evident that the CST complex is present in a diverse range of organisms where it contributes to genome maintenance. The CST accomplishes these tasks via telomere capping and by regulating telomerase and DNA polymerase alpha-primase (polα-primase access to telomeres, a process closely coordinated with the shelterin complex in most organisms. The goal of this review is to provide a brief but comprehensive account of the diverse, and in some cases organism-dependent, functions of the CST complex and how it contributes to telomere maintenance and cell proliferation.

  2. Enhanced anti-HIV-1 activity of G-quadruplexes comprising locked nucleic acids and intercalating nucleic acids

    DEFF Research Database (Denmark)

    Pedersen, Erik Bjerregaard; Nielsen, Jakob Toudahl; Nielsen, Claus

    2011-01-01

    Two G-quadruplex forming sequences, 50-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4......-(1-pyrenylethynyl)phenylmethyl]glycerol (twisted intercalating nucleic acid, TINA). Incorporation of LNA or INA/TINA monomers provide as much as 8-fold improvement of anti-HIV-1 activity. We demonstrate for the first time a detailed analysis of the effect the incorporation of INA/TINA monomers in quadruplex forming...

  3. K(+)-Responsive off-to-on switching of hammerhead ribozyme through dual G-quadruplex formation requiring no heating and cooling treatment.

    Science.gov (United States)

    Yamaoki, Yudai; Nagata, Takashi; Mashima, Tsukasa; Katahira, Masato

    Functional RNAs that switch their activities in response to K(+) may sense the intracellular (100 mM) and extracellular (5 mM) K(+) concentrations and regulate their functions accordingly. Previously, we developed a quadruplex hammerhead ribozyme (QHR) whose conformational change, from a duplex to a G-quadruplex, triggered by K(+) results in expression of the activity. However, this QHR required heating and cooling treatment (annealing) to induce the K(+)-responsive conformational change and activity. Here, we developed a new quadruplex hammerhead ribozyme (QHR) system that does not require annealing to induce the K(+)-responsive conformational change and activity. This system is composed of QHR and a G-quadruplex-forming complementary DNA strand (QCS). In the absence of K(+), QCS formed a duplex with QHR, which suppressed the residual activity. Upon elevation of the K(+) concentration, QCS dissociated from QHR was trapped in a G-quadruplex, and then QHR could form a G-quadruplex and exerted the activity. The 11.6-fold higher activity was induced by K(+) with an EC50 value of 23 mM, but not by Na(+), which is desirable when the activity switching between the intra-/extracellular environment is aimed at. This is the first report of the activation of functional RNA through a 'dual G-quadruplex formation system'. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Telomerer og telomerase

    DEFF Research Database (Denmark)

    Bendix, Laila; Kølvraa, Steen

    2010-01-01

    In 2009 the Nobel Prize in Medicine was awarded to EH Blackburn, CW Greider and JW Szostak for their work on "How chromosomes are protected by telomeres and the enzyme telomerase". Telomeres are specialized DNA structures localized at the end of linear chromosomes. Telomeres are known as the biol......In 2009 the Nobel Prize in Medicine was awarded to EH Blackburn, CW Greider and JW Szostak for their work on "How chromosomes are protected by telomeres and the enzyme telomerase". Telomeres are specialized DNA structures localized at the end of linear chromosomes. Telomeres are known...... as the biological clock of the cell, since they shorten with each cell division. Telomerase can elongate telomeres. Telomeres protect chromosome ends against being recognized as double stranded DNA breaks, and are thought to be a guard against cancer. It has furthermore been suggested that telomeres may play a role...

  5. Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures.

    Science.gov (United States)

    de Lara, J; Wydner, K L; Hyland, K M; Ward, W S

    1993-11-01

    The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.

  6. Regulation of the equilibrium between G-quadruplex and duplex DNA in promoter of human c-myc oncogene by a pyrene derivative.

    Science.gov (United States)

    Zhang, Zhenjiang; He, Xiangwei; Yuan, Gu

    2011-12-01

    It has been established that the equilibrium between duplex and G-quadruplex of the nuclease hypersensitivity element III1 (NHE III1) in human c-myc promoter is linked with this gene's transcription. Using NMR and ESI-MS, we have found a pyrene derivative, DMAPP, is able to modulate this equilibrium and, thus, might have the potential to regulate this oncogene's transcription. DMAPP has shown as a G-quadruplex binding agent and could induce c-myc G-quadruplex formation out of duplex. These results provide new clue for rational drug design to target transcription control of c-myc. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Quinolino-benzo-[5, 6]-dihydroisoquindolium compounds derived from berberine: a new class of highly selective ligands for G-quadruplex DNA in c-myc oncogene.

    Science.gov (United States)

    Ma, Yan; Ou, Tian-Miao; Tan, Jia-Heng; Hou, Jin-Qiang; Huang, Shi-Liang; Gu, Lian-Quan; Huang, Zhi-Shu

    2011-05-01

    A series of quinolino-benzo-[5, 6]-dihydroisoquindolium compounds (3a, 3f, 3g, and 3j) derived from alkaloid berberine were designed and synthesized as novel G-quadruplex ligands. Subsequent biophysical and biochemical evaluation demonstrated that the addition of pyridine ring and amino group into berberine improved the binding ability and selectivity towards G-quadruplex DNA in comparison with the previously reported 9-N-substituted berberine derivatives. Furthermore, qRT-PCR assay showed compound 3j led the down-regulation of c-myc gene transcription in leukemia cell line HL60, while little effect on normal cell line ECV-304, which was consistent with the behavior of an effective G-quadruplex ligand targeting c-myc oncogene. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  8. A cyclometallated iridium(III) complex as a c-myc G-quadruplex stabilizer and down-regulator of c-myc oncogene expression.

    Science.gov (United States)

    Yang, H; Ma, V P-Y; Chan, D S-H; He, H-Z; Leung, C-H; Ma, D-L

    2013-01-01

    A new cyclometallated iridium(III) complex with the 2,2'-biquinoline N-donor ligand has been synthesized and characterized. The interaction and affinity of the complex towards c-myc G-quadruplex and duplex DNA have been investigated using UV/Vis spectroscopy and gel mobility shift assay. These studies revealed that complex 1 binds to c-myc G-quadruplexes (Pu22 and Pu27) with high affinity but does not interact with duplex DNA either by intercalation or groove binding. The ability of 1 to stabilize c-myc G-quadruplex DNA in vitro has also been examined through a PCR stop assay and a cell-based luciferase reporter assay. Complex 1 displays promising cytotoxic activity against the HeLa cell line with sub-micromolar potency.

  9. Inducement of G-quadruplex DNA forming and down-regulation of oncogene c-myc by bile acid-amino acid conjugate-BAA.

    Science.gov (United States)

    Tian, Mingyue; Zhang, Xiufeng; Li, Yan; Ju, Yong; Xiang, Junfeng; Zhao, Changqi; Tang, Yalin

    2010-03-01

    Human c-myc gene is a central regulator of cellular proliferation and cell growth, and G-quadruplexes have been proven to be the transcriptional controller of this gene. In this study, the interaction of bile acid-amino acid conjugate (BAA) with G-quadruplexes in c-myc was investigated by circular dichroism spectroscopy, nuclear magnetic resonance (NMR) measurement, and quantitative real-time polymerase chain reaction (PCR) assay. The experimental results indicated that BAA has the ability to selectively induce the formation of parallel G-quadruplexes in c-myc, which leads to down-regulation of c-myc transcription in the human breast cancer cell MCF-7.

  10. Thioflavin T as an Efficient G-Quadruplex Inducer for the Highly Sensitive Detection of Thrombin Using a New Föster Resonance Energy Transfer System.

    Science.gov (United States)

    Liu, Xingfen; Hua, Xiaoxiao; Fan, Quli; Chao, Jie; Su, Shao; Huang, Yan-Qin; Wang, Lianhui; Huang, Wei

    2015-08-05

    We report a new Föster resonance energy transfer (FRET) system that uses a special dye, thioflavin T (ThT), as an energy acceptor and a water-soluble conjugated polymer (CP) with high fluorescence as an energy donor. A simple, label-free, and sensitive strategy for the detection of thrombin in buffer and in diluted serum was designed based on this new system using ThT as an efficient inducer of the G-quadruplex. The difference between the blank and the positive samples was amplified due to distinctive FRET signals because thrombin has little effect on the intercalation of ThT into the G-quadruplex. In the absence of the target, ThT induces the aptamer to form a G-quadruplex and intercalates into it with strong fluorescence. The electrostatic attractions between the negatively charged G-quadruplex and positively charged CP allow a short donor-acceptor distance, resulting in a high FRET signal. However, in the presence of the target, the aptamer forms a G-quadruplex-thrombin complex first, followed by the intercalation of ThT into the G-quadruplex. A long distance exists between the donor and acceptor due to the strong steric hindrance from the large-sized thrombin, which leads to a low FRET signal. Compared with previously reported strategies based on the FRET between the CP and dye, our strategy is label-free, and the sensitivity was improved by an order of magnitude. Our strategy also shows the advantages of being simple, rapid (about 50 min), sensitive, label-free, and low-cost in comparison to strategies based on the FRET between quantum dots and dyes.

  11. Electrochemical immunosensor for detecting the spore wall protein of Nosema bombycis based on the amplification of hemin/G-quadruplex DNAzyme concatamers functionalized Pt@Pd nanowires.

    Science.gov (United States)

    Wang, Qin; Song, Yue; Chai, Yaqin; Pan, Guoqing; Li, Tian; Yuan, Yali; Yuan, Ruo

    2014-10-15

    In this work, an ultrasensitive electrochemical immunosensor for detecting the Pebrine disease related spore wall protein of Nosema bombycis (SWP N.b) was fabricated based on the amplification of hemin/G-quadruplex functionalized Pt@Pd nanowires (Pt@PdNWs). The synthesized Pt@PdNWs possessed large surface area, which could effectively improve the immobilization amount of hemin/G-quadruplex DNAzyme concatamers produced via hybridization chain reaction (HCR). In the presence of SWP N.b, the hemin/G-quadruplex labeled Pt@PdNWs bioconjugations was captured on electrode surface and thus obtained electrochemical signal. After the addition of NADH into the electrolytic cell, hemin/G-quadruplex firstly acted as an NADH oxidase to locally produce H2O2 in the presence of dissolved O2. Then, the generated H2O2 would be quickly reduced via hemin/G-quadruplex as a horseradish peroxidase mimicking (HRP-mimicking) DNAzyme, which finally promoted the self-redox reaction of hemin/G-quadruplex and a greatly enhanced electrochemical signal was obtained. Furthermore, Pt@PdNWs with excellent electrocatalytic performance could also amplify electrochemical signal. With these amplification factors, the electrochemical immunosensor exhibited a wide linear range from 0.001 ng mL(-1) to 100 ng mL(-1) with a detection limit (LOD) of 0.24 pg mL(-1), providing a new promise for the diagnosis of Pebrine disease. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Human DNA repair genes possess potential G-quadruplex sequences in their promoters and 5`-untranslated regions.

    Science.gov (United States)

    Fleming, Aaron M; Zhu, Judy; Ding, Yun; Visser, Joshua A; Zhu, Julia; Burrows, Cynthia J

    2018-01-10

    The cellular response to oxidative stress includes transcriptional changes, particularly for genes involved in DNA repair. Recently, our laboratory demonstrated that oxidation of 2`-deoxyguanosine (G) to 8-oxo-7,8-dihydro-2`-deoxyguanosine (OG) in G-rich potential G-quadruplex sequences (PQSs) in gene promoters impacts the level of gene expression up or down depending on the position of the PQS in the promoter. In the present report, bioinformatic analysis found that the 390 human DNA repair genes in the genome ontology initiative harbor 2,936 PQSs in their promoters and 5`-untranslated regions (5`-UTRs). The average density of PQSs in human DNA repair genes was found to be nearly twofold greater than the average density of PQSs in all coding and non-coding human genes (7.5 vs. 4.3 per gene). The distribution of the PQSs in the DNA repair genes on the non-transcribed (coding) vs. transcribed strands reflects that of PQSs in all human genes. Next, literature data were interrogated to select 30 PQSs to catalog their ability to adopt G-quadruplex (G4) folds in vitro using five different experimental tests. The G4 characterization experiments concluded that 26 of the 30 sequences could adopt G4 topologies in solution. Last, four PQSs were synthesized into the promoter of a luciferase plasmid and co-transfected with the G4-specific ligands pyridostatin, Phen-DC3, or BRACO-19 in human cells to determine whether the PQSs could adopt G4 folds. The cell studies identified changes in luciferase expression when the G4 ligands were present, and the magnitude of the expression changes dependent on the PQS and the coding vs. template strand on which the sequence resided. Our studies demonstrate PQSs exist at a high density in human DNA repair gene promoters and a subset of the identified sequences fold in vitro and in vivo.

  13. The Myb/SANT domain of the telomere-binding protein TRF2 alters chromatin structure.

    Science.gov (United States)

    Baker, Asmaa M; Fu, Qiang; Hayward, William; Lindsay, Stuart M; Fletcher, Terace M

    2009-08-01

    Eukaryotic DNA is packaged into chromatin, which regulates genome activities such as telomere maintenance. This study focuses on the interactions of a myb/SANT DNA-binding domain from the telomere-binding protein, TRF2, with reconstituted telomeric nucleosomal array fibers. Biophysical characteristics of the factor-bound nucleosomal arrays were determined by analytical agarose gel electrophoresis (AAGE) and single molecules were visualized by atomic force microscopy (AFM). The TRF2 DNA-binding domain (TRF2 DBD) neutralized more negative charge on the surface of nucleosomal arrays than histone-free DNA. Binding of TRF2 DBD at lower concentrations increased the radius and conformational flexibility, suggesting a distortion of the fiber structure. Additional loading of TRF2 DBD onto the nucleosomal arrays reduced the flexibility and strongly blocked access of micrococcal nuclease as contour lengths shortened, consistent with formation of a unique, more compact higher-order structure. Mirroring the structural results, TRF2 DBD stimulated a strand invasion-like reaction, associated with telomeric t-loops, at lower concentrations while inhibiting the reaction at higher concentrations. Full-length TRF2 was even more effective at stimulating this reaction. The TRF2 DBD had less effect on histone-free DNA structure and did not stimulate the t-loop reaction with this substrate, highlighting the influence of chromatin structure on the activities of DNA-binding proteins.

  14. Yeast Sub1 and human PC4 are G-quadruplex binding proteins that suppress genome instability at co-transcriptionally formed G4 DNA.

    Science.gov (United States)

    Lopez, Christopher R; Singh, Shivani; Hambarde, Shashank; Griffin, Wezley C; Gao, Jun; Chib, Shubeena; Yu, Yang; Ira, Grzegorz; Raney, Kevin D; Kim, Nayun

    2017-06-02

    G-quadruplex or G4 DNA is a non-B secondary DNA structure consisting of a stacked array of guanine-quartets that can disrupt critical cellular functions such as replication and transcription. When sequences that can adopt Non-B structures including G4 DNA are located within actively transcribed genes, the reshaping of DNA topology necessary for transcription process stimulates secondary structure-formation thereby amplifying the potential for genome instability. Using a reporter assay designed to study G4-induced recombination in the context of an actively transcribed locus in Saccharomyces cerevisiae, we tested whether co-transcriptional activator Sub1, recently identified as a G4-binding factor, contributes to genome maintenance at G4-forming sequences. Our data indicate that, upon Sub1-disruption, genome instability linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly augmented and that its highly conserved DNA binding domain or the human homolog PC4 is sufficient to suppress G4-associated genome instability. We also show that Sub1 interacts specifically with co-transcriptionally formed G4 DNA in vivo and that yeast cells become highly sensitivity to G4-stabilizing chemical ligands by the loss of Sub1. Finally, we demonstrate the physical and genetic interaction of Sub1 with the G4-resolving helicase Pif1, suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Microwave-assisted synthesis of ruthenium(II) complexes with alkynes as potential inhibitor by selectively recognizing c-myc G-quadruplex DNA.

    Science.gov (United States)

    Zhang, Shuangyan; Wu, Qiong; Zhang, Hao; Wang, Qi; Wang, Xicheng; Mei, Wenjie; Wu, Xiaohui; Zheng, Wenjie

    2017-11-01

    Herein, two polypyridyl ruthenium(II) complexes with alkynes, [Ru(bpy)2L](ClO4)2 (L=p-TEPIP (1) and p-BEPIP (2); bpy=2,2'-bipyridine; p-TEPIP=2-(4-trimethylsilylpropargyl)-1H-imidazo[4,5f][1,10]phenanthroline; p-BEPIP=2-(4-phenyacetylenephenyl)-1H-imidazo[4,5f][1,10]phenanthroline) have been successfully achieved in yields of 32%-89% by a Sonogashira coupling reaction under microwave irradiation. We studied these complexes as potential stabilizers of c-myc G-quadruplex DNA. Observations revealed that both complexes could selectively bind to and stabilize c-myc G-quadruplex DNA with a constant of approximately 1.61±0.78 and 9.47±4.20×10(3)M(-1), respectively, as determined from ITC (isothermal ttitration calorimetry) experiments, FRET (fluorescence resonance energy ttransfer) assay and competitive FRET assay. Moreover, the melting point (Tm) of the c-myc G-quadruplex DNA increased in the presence of 1 and 2 ([Ru]=0.2μM) by approximately 9 and 19.9°C, respectively. It is noteworthy that the conformation of the c-myc G-quadruplex DNA appeared to change when titrated with 1 and 2, which was accompanied by a negative-induced CD (circular dichroism) signal that appeared at a wavelength of 295nm. Furthermore, the conformational change in c-myc G-quadruplex DNA induced by 1 and 2have also been confirmed by TEM (transmission electron microscopy) and AFM (atomic force microscopy). Consequently, the replication of c-myc DNA was blocked by 1 and 2, and especially by 2, as verified by PCR (polymerase chain reaction) -stop assay and Western-blot assay. Thus, these ruthenium(II) complexes can be developed as potential inhibitors in chemotherapy through their binding and stabilization of c-myc G-quadruplex DNA. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Synergic Role of Nucleophosmin Three-helix Bundle and a Flanking Unstructured Tail in the Interaction with G-quadruplex DNA*

    Science.gov (United States)

    Arcovito, Alessandro; Chiarella, Sara; Della Longa, Stefano; Di Matteo, Adele; Lo Sterzo, Carlo; Scaglione, Giovanni Luca; Federici, Luca

    2014-01-01

    Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a number of functions in ribosome biogenesis and export, cell cycle control, and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia; mutations map to the C-terminal domain of the protein and cause its denaturation and aberrant cytoplasmic translocation. NPM1 C-terminal domain binds G-quadruplex regions at ribosomal DNA and at gene promoters, including the well characterized sequence from the nuclease-hypersensitive element III region of the c-MYC promoter. These activities are lost by the leukemic variant. Here we analyze the NPM1/G-quadruplex interaction, focusing on residues belonging to both the NPM1 terminal three-helix bundle and a lysine-rich unstructured tail, which has been shown to be necessary for high affinity recognition. We performed extended site-directed mutagenesis and measured binding rate constants through surface plasmon resonance analysis. These data, supported by molecular dynamics simulations, suggest that the unstructured tail plays a double role in the reaction mechanism. On the one hand, it facilitates the formation of an encounter complex through long range electrostatic interactions; on the other hand, it directly contacts the G-quadruplex scaffold through multiple and transient electrostatic interactions, significantly enlarging the contact surface. PMID:24952945

  17. Synergic role of nucleophosmin three-helix bundle and a flanking unstructured tail in the interaction with G-quadruplex DNA.

    Science.gov (United States)

    Arcovito, Alessandro; Chiarella, Sara; Della Longa, Stefano; Di Matteo, Adele; Lo Sterzo, Carlo; Scaglione, Giovanni Luca; Federici, Luca

    2014-08-01

    Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a number of functions in ribosome biogenesis and export, cell cycle control, and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia; mutations map to the C-terminal domain of the protein and cause its denaturation and aberrant cytoplasmic translocation. NPM1 C-terminal domain binds G-quadruplex regions at ribosomal DNA and at gene promoters, including the well characterized sequence from the nuclease-hypersensitive element III region of the c-MYC promoter. These activities are lost by the leukemic variant. Here we analyze the NPM1/G-quadruplex interaction, focusing on residues belonging to both the NPM1 terminal three-helix bundle and a lysine-rich unstructured tail, which has been shown to be necessary for high affinity recognition. We performed extended site-directed mutagenesis and measured binding rate constants through surface plasmon resonance analysis. These data, supported by molecular dynamics simulations, suggest that the unstructured tail plays a double role in the reaction mechanism. On the one hand, it facilitates the formation of an encounter complex through long range electrostatic interactions; on the other hand, it directly contacts the G-quadruplex scaffold through multiple and transient electrostatic interactions, significantly enlarging the contact surface. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Telomeres: Hallmarks of radiosensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Ayouaz, A.; Raynaud, C.; Heride, C.; Revaud, D.; Sabatier, L. [CEA, DSV, IRCM/SRO, F-92265 Fontenay Aux Roses (France)

    2008-07-01

    Telomeres are the very ends of the chromosomes. They can be seen as natural double-strand breaks (DSB), specialized structures which prevent DSB repair and activation of DNA damage checkpoints. In somatic cells, attrition of telomeres occurs after each cell division until replicative senescence. In the absence of telomerase, telomeres shorten due to incomplete replication of the lagging strand at the very end of chromosome termini. Moreover, oxidative stress and accumulating reactive oxygen species (ROS) lead to an increased telomere shortening due to a less efficient repair of SSB in telomeres. The specialized structures at telomeres include proteins involved in both telomere maintenance and DNA repair. However when a telomere is damaged and has to be repaired, those proteins might fail to perform an accurate repair of the damage.This is the starting point of this article in which we first summarize the well-established relationships between DNA repair processes and maintenance of functional telomeres. We then examine how damaged telomeres would be processed, and show that irradiation alters telomere maintenance leading to possibly dramatic consequences. Our point is to suggest that those consequences are not restricted to the short term effects such as increased radiation-induced cell death. On the contrary, we postulate that the major impact of the loss of telomere integrity might occur in the long term, during multistep carcinogenesis. Its major role would be to act as an amplifying event unmasking in one single step recessive radiation-induced mutations among thousands of genes and providing cellular proliferative advantage. Moreover, the chromosomal instability generated by damaged telomeres will favour each step of the transformation from normal to fully transformed cells. (authors)

  19. A G-quadruplex-binding compound showing anti-tumour activity in an in vivo model for pancreatic cancer

    Science.gov (United States)

    Ohnmacht, Stephan A; Marchetti, Chiara; Gunaratnam, Mekala; Besser, Rachael J; Haider, Shozeb M; Di Vita, Gloria; Lowe, Helen L; Mellinas-Gomez, Maria; Diocou, Seckou; Robson, Mathew; Šponer, Jiri; Islam, Barira; Barbara Pedley, R; Hartley, John A; Neidle, Stephen

    2015-01-01

    We report here that a tetra-substituted naphthalene-diimide derivative (MM41) has significant in vivo anti-tumour activity against the MIA PaCa-2 pancreatic cancer xenograft model. IV administration with a twice-weekly 15 mg/kg dose produces ca 80% tumour growth decrease in a group of tumour-bearing animals. Two animals survived tumour-free after 279 days. High levels of MM41 are rapidly transported into cell nuclei and were found to accumulate in the tumour. MM41 is a quadruplex-interactive compound which binds strongly to the quadruplexes encoded in the promoter sequences of the BCL-2 and k-RAS genes, both of which are dis-regulated in many human pancreatic cancers. Levels of BCL-2 were reduced by ca 40% in tumours from MM41-treated animals relative to controls, consistent with BCL-2 being a target for MM41. Molecular modelling suggests that MM41 binds to a BCL-2 quadruplex in a manner resembling that previously observed in co-crystal structures with human telomeric quadruplexes. This supports the concept that MM41 (and by implication other quadruplex-targeting small molecules) can bind to quadruplex-forming promoter regions in a number of genes and down-regulate their transcription. We suggest that quadruplexes within those master genes that are up-regulated drivers for particular cancers, may be selective targets for compounds such as MM41. PMID:26077929

  20. Stephen Neidle on cancer therapy and G-quadruplex inhibitors. Interview by Joanna De Souza.

    Science.gov (United States)

    Neidle, Stephen

    2004-09-15

    Stephen Neidle was educated at Imperial College, London, where he graduated in chemistry and then proceeded to do a PhD in crystallography. After a period as an ICI Fellow, he joined the Biophysics Department at King's College, which ignited his interest in nucleic acid structural studies. He was appointed as one of the first Cancer Research Campaign Career Development Awardees, becoming a Life Fellow on moving to the Institute of Cancer Research. He was appointed to the Chair of Biophysics at the Institute of Cancer Research in 1990, and moved to the new Chair of Chemical Biology at the School of Pharmacy in the University of London in 2002, where he also directs the Cancer Research UK Biomolecular Structure Group. He is currently Chairman of the Chemical Biology Forum of the Royal Society of Chemistry, which is involved in developing the interface between chemistry and the life sciences. He will shortly assume the Directorship of the newly-established Centre for Cancer Medicines at the School. Stephen Neidle has received several awards for his work on drug-nucleic acid recognition and drug design, including the 2000 prize of the Biological and Medicinal Chemistry Sector of the Royal Society of Chemistry, and its 2002 Interdisciplinary Award. He was the 2004 Paul Ehrlich Lecturer of the French Societé de Chimie Therapeutique, and was recently awarded the 2004 Aventis Prize in Medicinal Chemistry.

  1. G-quadruplex recognition activities of E. Coli MutS

    Directory of Open Access Journals (Sweden)

    Ehrat Edward A

    2012-07-01

    Full Text Available Abstract Background Guanine quadruplex (G4 DNA is a four-stranded structure that contributes to genome instability and site-specific recombination. G4 DNA folds from sequences containing tandemly repetitive guanines, sequence motifs that are found throughout prokaryote and eukaryote genomes. While some cellular activities have been identified with binding or processing G4 DNA, the factors and pathways governing G4 DNA metabolism are largely undefined. Highly conserved mismatch repair factors have emerged as potential G4-responding complexes because, in addition to initiating heteroduplex correction, the human homologs bind non-B form DNA with high affinity. Moreover, the MutS homologs across species have the capacity to recognize a diverse range of DNA pairing variations and damage, suggesting a conserved ability to bind non-B form DNA. Results Here, we asked if E. coli MutS and a heteroduplex recognition mutant, MutS F36A, were capable of recognizing and responding to G4 DNA structures. We find by mobility shift assay that E. coli MutS binds to G4 DNA with high affinity better than binding to G-T heteroduplexes. In the same assay, MutS F36A failed to recognize G-T mismatched oligonucleotides, as expected, but retained an ability to bind to G4 DNA. Association with G4 DNA by MutS is not likely to activate the mismatch repair pathway because nucleotide binding did not promote release of MutS or MutS F36A from G4 DNA as it does for heteroduplexes. G4 recognition activities occur under physiological conditions, and we find that M13 phage harboring G4-capable DNA poorly infected a MutS deficient strain of E. coli compared to M13mp18, suggesting functional roles for mismatch repair factors in the cellular response to unstable genomic elements. Conclusions Taken together, our findings demonstrate that E. coli MutS has a binding activity specific for non-B form G4 DNA, but such binding appears independent of canonical heteroduplex repair activation.

  2. Identifying the impact of G-quadruplexes on Affymetrix 3' arrays using cloud computing.

    Science.gov (United States)

    Memon, Farhat N; Owen, Anne M; Sanchez-Graillet, Olivia; Upton, Graham J G; Harrison, Andrew P

    2010-01-15

    A tetramer quadruplex structure is formed by four parallel strands of DNA/ RNA containing runs of guanine. These quadruplexes are able to form because guanine can Hoogsteen hydrogen bond to other guanines, and a tetrad of guanines can form a stable arrangement. Recently we have discovered that probes on Affymetrix GeneChips that contain runs of guanine do not measure gene expression reliably. We associate this finding with the likelihood that quadruplexes are forming on the surface of GeneChips. In order to cope with the rapidly expanding size of GeneChip array datasets in the public domain, we are exploring the use of cloud computing to replicate our experiments on 3' arrays to look at the effect of the location of G-spots (runs of guanines). Cloud computing is a recently introduced high-performance solution that takes advantage of the computational infrastructure of large organisations such as Amazon and Google. We expect that cloud computing will become widely adopted because it enables bioinformaticians to avoid capital expenditure on expensive computing resources and to only pay a cloud computing provider for what is used. Moreover, as well as financial efficiency, cloud computing is an ecologically-friendly technology, it enables efficient data-sharing and we expect it to be faster for development purposes. Here we propose the advantageous use of cloud computing to perform a large data-mining analysis of public domain 3' arrays.

  3. G-Tetraplex-Induced FRET within Telomeric Repeat Sequences Using (Py) A-(Per) A as Energy Donor-Acceptor Pair.

    Science.gov (United States)

    Kundu, Rajen

    2016-01-01

    G-tetraplex induced fluorescence resonance energy transfer (FRET) within telomeric repeat sequences has been studied using a nucleoside-tethered FRET pair embedded in the human telomeric G-quadruplex forming sequence (5'-A GGG TT(Py) A GGG TT(Per) A GGG TTA GGG-3', Py=pyrene, Per=perylene). Conformational change from a single strand to an anti-parallel G-quadruplex leads to FRET from energy donor ((Py) A) to acceptor ((Per) A). The distance between the FRET donor/acceptor partners was controlled by changing the number of G-quartet spacer units. The FRET efficiency decreases with increase in G-quartet units. Overall findings indicate that this could be further used for the development of FRET-based sensing and measurement techniques. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Telomerers rolle ved aldersbetingede sygdomme

    DEFF Research Database (Denmark)

    Bendix, Laila; Kølvraa, Steen

    2010-01-01

    Telomeres are specialized DNA structures, protecting the ends of linear chromosomes. The association between telomeres and cellular aging is well-established, and it has been shown that there is a negative correlation between telomere length and chronological age for many types of human tissue. O...

  5. A sensitive fluorescence anisotropy method for detection of lead (II) ion by a G-quadruplex-inducible DNA aptamer

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Dapeng [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085 (China); Yin, Lei; Meng, Zihui [School of Chemical Engineering and Environment, Beijing Institute of Technology, Beijing, 100081 (China); Yu, Anchi [Department of Chemistry, Renmin University of China, Beijing, 100872 (China); Guo, Lianghong [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085 (China); Wang, Hailin, E-mail: hlwang@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085 (China)

    2014-02-17

    Graphical abstract: -- Highlights: •A fluorescence anisotropy approach for detection of Pb{sup 2+} was developed. •The strategy was based on binding-induced allosteric conformational change of aptamer probe. •The sensing mechanism was established by testing the photoinduced electron transfer interaction. -- Abstract: Sensitive and selective detection of Pb{sup 2+} is of great importance to both human health and environmental protection. Here we propose a novel fluorescence anisotropy (FA) approach for sensing Pb{sup 2+} in homogeneous solution by a G-rich thrombin binding aptamer (TBA). The TBA labeled with 6-carboxytetramethylrhodamine (TMR) at the seventh thymine nucleotide was used as a fluorescent probe for signaling Pb{sup 2+}. It was found that the aptamer probe had a high FA in the absence of Pb{sup 2+}. This is because the rotation of TMR is restricted by intramolecular interaction with the adjacent guanine bases, which results in photoinduced electron transfer (PET). When the aptamer probe binds to Pb{sup 2+} to form G-quadruplex, the intramolecular interaction should be eliminated, resulting in faster rotation of the fluorophore TMR in solution. Therefore, FA of aptamer probe is expected to decrease significantly upon binding to Pb{sup 2+}. Indeed, we observed a decrease in FA of aptamer probe upon Pb{sup 2+} binding. Circular dichroism, fluorescence spectra, and fluorescence lifetime measurement were used to verify the reliability and reasonability of the sensing mechanism. By monitoring the FA change of the aptamer probe, we were able to real-time detect binding between the TBA probe and Pb{sup 2+}. Moreover, the aptamer probe was exploited as a recognition element for quantification of Pb{sup 2+} in homogeneous solution. The change in FA showed a linear response to Pb{sup 2+} from 10 nM to 2.0 μM, with 1.0 nM limit of detection. In addition, this sensing system exhibited good selectivity for Pb{sup 2+} over other metal ions. The method is simple

  6. Epigenetic suppression of human telomerase (hTERT) is mediated by the metastasis suppressor NME2 in a G-quadruplex-dependent fashion.

    Science.gov (United States)

    Saha, Dhurjhoti; Singh, Ankita; Hussain, Tabish; Srivastava, Vivek; Sengupta, Suman; Kar, Anirban; Dhapola, Parashar; Dhople, Vishnu; Ummanni, Ramesh; Chowdhury, Shantanu

    2017-09-15

    Transcriptional activation of the human telomerase reverse transcriptase (hTERT) gene, which remains repressed in adult somatic cells, is critical during tumorigenesis. Several transcription factors and the epigenetic state of the hTERT promoter are known to be important for tight control of hTERT in normal tissues, but the molecular mechanisms leading to hTERT reactivation in cancer are not well-understood. Surprisingly, here we found occupancy of the metastasis suppressor non-metastatic 2 (NME2) within the hTERT core promoter in HT1080 fibrosarcoma cells and HCT116 colon cancer cells and NME2-mediated transcriptional repression of hTERT in these cells. We also report that loss of NME2 results in up-regulated hTERT expression. Mechanistically, additional results indicated that the RE1-silencing transcription factor (REST)-lysine-specific histone demethylase 1 (LSD1) co-repressor complex associates with the hTERT promoter in an NME2-dependent way and that this assembly is required for maintaining repressive chromatin at the hTERT promoter. Interestingly, a G-quadruplex motif at the hTERT promoter was essential for occupancy of NME2 and the REST repressor complex on the hTERT promoter. In light of this mechanistic insight, we studied the effects of G-quadruplex-binding ligands on hTERT expression and observed that several of these ligands repressed hTERT expression. Together, our results support a mechanism of hTERT epigenetic control involving a G-quadruplex promoter motif, which potentially can be targeted by tailored small molecules. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Switch-on fluorescence scheme for antibiotics based on a magnetic composite probe with aptamer and hemin/G-quadruplex coimmobilized nano-Pt-luminol as signal tracer.

    Science.gov (United States)

    Miao, Yang-Bao; Gan, Ning; Ren, Hong-Xia; Li, Tianhua; Cao, Yuting; Hu, Futao; Chen, Yinji

    2016-01-15

    A selective and facile fluorescence "switch-on" scheme is developed to detect antibiotics residues in food, using chloramphenicol (CAP) as model, based on a novel magnetic aptamer probe (aptamer-Pt-luminol nanocomposite labeled with hemin/G-quadruplex). Firstly, the composite probe is prepared through the immuno-reactions between the capture beads (anti-dsDNA antibody labeled on magnetic Dynabeads) and the nanotracer (nano-Pt-luminol labeled with double-strand aptamer, as ds-Apt, and hemin/G-quadruplex). When the composite probe is mixed with CAP, the aptamer preferentially reacted with CAP to decompose the double-strand aptamer to ssDNA, which cannot be recognized by the anti-dsDNA antibody on the capture probes. Thus, after magnetic separation, the nanotracer can be released into the supernatant. Because the hemin/G-quadruplex and PtNPs in nanotracer can catalyze luminol-H2O2 system to emit fluorescence. Thus a dual-amplified "switch-on" signal appeared, of which intensity is proportional to the concentration of CAP between 0.001 and 100ng mL(-1) with detection limit of 0.0005ng mL(-1) (S/N=3). Besides, our method has good selectivity and was employed for CAP detection in real milk samples. The results agree well with those from conventional gas chromatograph-mass spectrometer (GC-MS). The switch-on signal is produced by one-step substitution reaction between aptamer in nanotracer and target. When the analyte is changed, the probe can be refabricated only by changing the corresponding aptamer. Thus, all features above prove our strategy to be a facile, feasible and selective method in antibiotics screening for food safety. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Thioflavin T binds dimeric parallel-stranded GA-containing non-G-quadruplex DNAs: a general approach to lighting up double-stranded scaffolds.

    Science.gov (United States)

    Liu, Shuangna; Peng, Pai; Wang, Huihui; Shi, Lili; Li, Tao

    2017-12-01

    A molecular rotor thioflavin T (ThT) is usually used as a fluorescent ligand specific for G-quadruplexes. Here, we demonstrate that ThT can tightly bind non-G-quadruplex DNAs with several GA motifs and dimerize them in a parallel double-stranded mode, accompanied by over 100-fold enhancement in the fluorescence emission of ThT. The introduction of reverse Watson-Crick T-A base pairs into these dimeric parallel-stranded DNA systems remarkably favors the binding of ThT into the pocket between G•G and A•A base pairs, where ThT is encapsulated thereby restricting its two rotary aromatic rings in the excited state. A similar mechanism is also demonstrated in antiparallel DNA duplexes where several motifs of two consecutive G•G wobble base pairs are incorporated and serve as the active pockets for ThT binding. The insight into the interactions of ThT with non-G-quadruplex DNAs allows us to introduce a new concept for constructing DNA-based sensors and devices. As proof-of-concept experiments, we design a DNA triplex containing GA motifs in its Hoogsteen hydrogen-bonded two parallel strands as a pH-driven nanoswitch and two GA-containing parallel duplexes as novel metal sensing platforms where C-C and T-T mismatches are included. This work may find further applications in biological systems (e.g. disease gene detection) where parallel duplex or triplex stretches are involved. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Coarse-Grained Simulations Complemented by Atomistic Molecular Dynamics Provide New Insights into Folding and Unfolding of Human Telomeric G-Quadruplexes

    Czech Academy of Sciences Publication Activity Database

    Stadlbauer, Petr; Mazzanti, L.; Cragnolini, T.; Wales, D.J.; Derreumaux, P.; Pasquali, C.; Sponer, Jiri

    2016-01-01

    Roč. 12, č. 12 (2016), s. 6077-6097 ISSN 1549-9618 R&D Projects: GA ČR(CZ) GA16-13721S Institutional support: RVO:68081707 Keywords : particle mesh ewald * amber force-field * free-energy profiles * binding dna aptamer Subject RIV: BO - Biophysics Impact factor: 5.245, year: 2016

  10. Shedding lights on the flexible-armed porphyrins: Human telomeric G4 DNA interaction and cell photocytotoxicity research.

    Science.gov (United States)

    Sun, Xiang-Yu; Zhao, Ping; Jin, Shu-Fang; Liu, Min-Chao; Wang, Xia-Hong; Huang, Yu-Min; Cheng, Zhen-Feng; Yan, Si-Qi; Li, Yan-Yu; Chen, Ya-Qing; Zhong, Yan-Mei

    2017-08-01

    DNA polymorphism exerts a fascination on a large scientific community. Without crystallographic structural data, clarification of the binding modes between G-quadruplex (G4) and ligand (complex) is a challenging job. In the present work, three porphyrin compounds with different flexible carbon chains (arms) were designed, synthesized and characterized. Their binding, folding and stabilizing abilities to human telomeric G4 DNA structures were comparatively researched. Positive charges at the end of the flexible carbon chains seem to be favorable for the DNA-porphyrin interactions, which were evidenced by the spectral results and further confirmed by the molecular docking calculations. Biological function analysis demonstrated that these porphyrins show no substantial inhibition to Hela, A549 and BEL 7402 cancer cell lines under dark while exhibit broad inhibition under visible light. This significantly enhanced photocytotoxicity relative to the dark control is an essential property of photochemotherapeutic agents. The feature of the flexible arms emerges as critical influencing factors in the cell photocytotoxicity. Moreover, an ROS-mediated mitochondrial dysfunction pathway was suggested for the cell apoptosis induced by these flexible-armed porphyrins. It is found that the porphyrins with positive charges located at the end of the flexible arms represent an exciting opportunity for photochemotherapeutic anti-cancer drug design. Copyright © 2017. Published by Elsevier B.V.

  11. What Makes Telomeres Unique?

    Science.gov (United States)

    Sieradzan, Adam K; Krupa, Paweł; Wales, David J

    2017-03-16

    Telomeres are repetitive nucleotide sequences, which are essential for protecting the termini of chromosomes. Thousands of such repetitions are necessary to maintain the stability of the whole chromosome. Several similar repeated telomeric sequences have been found in different species, but why has nature chosen them? What features do telomeres have in common? In this article, we study the physical properties of human-like (TTAGGG), plant (TTTAGG), insect (TTAGG), and Candida guilermondi (GGTGTAC) telomeres in comparison with seven control, nontelomeric sequences. We used steered molecular dynamics with the nucleic acid united residue (NARES) coarse-grained force field, which we compared with the all-atom AMBER14 force field and experimental data. Our results reveal important features in all of the telomeric sequences, including their exceptionally high mechanical resistance and stability to untangling and stretching, compared to those of nontelomeric sequences. We find that the additional stability of the telomeres comes from their ability to form triplex structures and wrap around loose chains of linear DNA by regrabbing the chain. We find that, with slower pulling speed, regrabbing and triplex formation is more frequent. We also found that some of the sequences can form triplexes experimentally, such as TTTTTCCCC, and can mimic telomeric properties.

  12. Microwave-Assisted Synthesis of Arene Ru(II Complexes Induce Tumor Cell Apoptosis Through Selectively Binding and Stabilizing bcl-2 G-Quadruplex DNA

    Directory of Open Access Journals (Sweden)

    Yanhua Chen

    2016-05-01

    Full Text Available A series of arene Ru(II complexes coordinated with phenanthroimidazole derivatives, [(η6-C6H6Ru(lCl]Cl(1b L = p-ClPIP = 2-(4-Chlorophenylimidazole[4,5f] 1,10-phenanthroline; 2b L = m-ClPIP = 2-(3-Chlorophenylimidazole[4,5f] 1,10-phenanthroline; 3b L = p-NPIP = 2-(4-Nitrophenylimidazole[4,5f] 1,10-phenanthroline; 4b L = m-NPIP = 2-(3-Nitrophenyl imidazole [4,5f] 1,10-phenanthroline were synthesized in yields of 89.9%–92.7% under conditions of microwave irradiation heating for 30 min to liberate four arene Ru(II complexes (1b, 2b, 3b, 4b. The anti-tumor activity of 1b against various tumor cells was evaluated by MTT assay. The results indicated that this complex blocked the growth of human lung adenocarcinoma A549 cells with an IC50 of 16.59 μM. Flow cytometric analysis showed that apoptosis of A549 cells was observed following treatment with 1b. Furthermore, the in vitro DNA-binding behaviors that were confirmed by spectroscopy indicated that 1b could selectively bind and stabilize bcl-2 G-quadruplex DNA to induce apoptosis of A549 cells. Therefore, the synthesized 1b has impressive bcl-2 G-quadruplex DNA-binding and stabilizing activities with potential applications in cancer chemotherapy.

  13. Telomeres: Implications for Cancer Development

    Directory of Open Access Journals (Sweden)

    Aina Bernal

    2018-01-01

    Full Text Available Telomeres facilitate the protection of natural ends of chromosomes from constitutive exposure to the DNA damage response (DDR. This is most likely achieved by a lariat structure that hides the linear telomeric DNA through protein-protein and protein-DNA interactions. The telomere shortening associated with DNA replication in the absence of a compensatory mechanism culminates in unmasked telomeres. Then, the subsequent activation of the DDR will define the fate of cells according to the functionality of cell cycle checkpoints. Dysfunctional telomeres can suppress cancer development by engaging replicative senescence or apoptotic pathways, but they can also promote tumour initiation. Studies in telomere dynamics and karyotype analysis underpin telomere crisis as a key event driving genomic instability. Significant attainment of telomerase or alternative lengthening of telomeres (ALT-pathway to maintain telomere length may be permissive and required for clonal evolution of genomically-unstable cells during progression to malignancy. We summarise current knowledge of the role of telomeres in the maintenance of chromosomal stability and carcinogenesis.

  14. Role of TERRA in the regulation of telomere length.

    Science.gov (United States)

    Wang, Caiqin; Zhao, Li; Lu, Shiming

    2015-01-01

    Telomere dysfunction is closely associated with human diseases such as cancer and ageing. Inappropriate changes in telomere length and/or structure result in telomere dysfunction. Telomeres have been considered to be transcriptionally silent, but it was recently demonstrated that mammalian telomeres are transcribed into telomeric repeat-containing RNA (TERRA). TERRA, a long non-coding RNA, participates in the regulation of telomere length, telomerase activity and heterochromatinization. The correct regulation of telomere length may be crucial to telomeric homeostasis and functions. Here, we summarize recent advances in our understanding of the crucial role of TERRA in the maintenance of telomere length, with focus on the variety of mechanisms by which TERRA is involved in the regulation of telomere length. This review aims to enable further understanding of how TERRA-targeted drugs can target telomere-related diseases.

  15. New Insights into Transcription Fidelity: Thermal Stability of Non-Canonical Structures in Template DNA Regulates Transcriptional Arrest, Pause, and Slippage: e90580

    National Research Council Canada - National Science Library

    Hisae Tateishi-Karimata; Noburu Isono; Naoki Sugimoto

    2014-01-01

      The thermal stability and topology of non-canonical structures of G-quadruplexes and hairpins in template DNA were investigated, and the effect of non-canonical structures on transcription fidelity...

  16. New insights into transcription fidelity: thermal stability of non-canonical structures in template DNA regulates transcriptional arrest, pause, and slippage

    National Research Council Canada - National Science Library

    Tateishi-Karimata, Hisae; Isono, Noburu; Sugimoto, Naoki

    2014-01-01

    The thermal stability and topology of non-canonical structures of G-quadruplexes and hairpins in template DNA were investigated, and the effect of non-canonical structures on transcription fidelity...

  17. Telomere homeostasis in IUGR placentas - A review.

    Science.gov (United States)

    Biron-Shental, Tal; Sadeh-Mestechkin, Dana; Amiel, Aliza

    2016-03-01

    Telomeres are nucleoprotein structures located at the termini of chromosomes. They are essential for chromosome stability. Telomeres become shorter due to mitotic cycles and environmental factors. When telomeres are shortened and therefore dysfunctional, cellular senescence occurs and organ dysfunction might develop. During pregnancy, fetal growth restriction secondary to placental insufficiency has been linked to impaired telomere homeostasis in which telomeres are shorter, telomerase is decreased, and compensatory mechanisms of telomere capture are enhanced. These characteristics, along with increased signs of senescence, indicate telomere dysfunction in trophoblasts from placentas affected by intrauterine growth restriction (IUGR). This review summarizes the information currently available regarding telomere homeostasis in trophoblasts from human pregnancies affected by IUGR. Improved understanding of placental physiology might help in the development of treatment options for fetuses with IUGR. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. The JIL-1 kinase affects telomere expression in the different telomere domains of Drosophila.

    Directory of Open Access Journals (Sweden)

    Rute Silva-Sousa

    Full Text Available In Drosophila, the non-LTR retrotransposons HeT-A, TART and TAHRE build a head-to-tail array of repetitions that constitute the telomere domain by targeted transposition at the end of the chromosome whenever needed. As a consequence, Drosophila telomeres have the peculiarity to harbor the genes in charge of telomere elongation. Understanding telomere expression is important in Drosophila since telomere homeostasis depends in part on the expression of this genomic compartment. We have recently shown that the essential kinase JIL-1 is the first positive regulator of the telomere retrotransposons. JIL-1 mediates chromatin changes at the promoter of the HeT-A retrotransposon that are necessary to obtain wild type levels of expression of these telomere transposons. With the present study, we show how JIL-1 is also needed for the expression of a reporter gene embedded in the telomere domain. Our analysis, using different reporter lines from the telomere and subtelomere domains of different chromosomes, indicates that JIL-1 likely acts protecting the telomere domain from the spreading of repressive chromatin from the adjacent subtelomere domain. Moreover, the analysis of the 4R telomere suggests a slightly different chromatin structure at this telomere. In summary, our results strongly suggest that the action of JIL-1 depends on which telomere domain, which chromosome and which promoter is embedded in the telomere chromatin.

  19. Cytogenetic study on antlions (Neuroptera, Myrmeleontidae: first data on telomere structure and rDNA location

    Directory of Open Access Journals (Sweden)

    Valentina G. Kuznetsova

    2016-11-01

    Full Text Available Myrmeleontidae, commonly known as “antlions”, are the most diverse family of the insect order Neuroptera, with over 1700 described species (in 191 genera of which 37 species (in 21 genera have so far been studied in respect to standard karyotypes. In the present paper we provide first data on the occurrence of the “insect-type” telomeric repeat (TTAGGn and location of 18S rDNA clusters in the antlion karyotypes studied using fluorescence in situ hybridization (FISH. We show that males of Palpares libelluloides (Linnaeus, 1764 (Palparinae, Acanthaclisis occitanica (Villers, 1789 (Acanthaclisinae and Distoleon tetragrammicus (Fabricius, 1798 (Nemoleontinae have rDNA clusters on a large bivalent, two last species having an additional rDNA cluster on one of the sex chromosomes, most probably the X. (TTAGGn - containing telomeres are clearly characteristic of P. libelluloides and A. occitanica; the presence of this telomeric motif in D. tetragrammicus is questionable. In addition, we detected the presence of the (TTAGGn telomeric repeat in Libelloides macaronius (Scopoli, 1763 from the family Ascalaphidae (owlflies, a sister group to the Myrmeleontidae. We presume that the “insect” motif (TTAGGn was present in a common ancestor of the families Ascalaphidae and Myrmeleontidae within the neuropteran suborder Myrmeleontiformia.

  20. Cytogenetic study on antlions (Neuroptera, Myrmeleontidae): first data on telomere structure and rDNA location.

    Science.gov (United States)

    Kuznetsova, Valentina G; Khabiev, Gadzhimurad N; Anokhin, Boris A

    2016-01-01

    Myrmeleontidae, commonly known as "antlions", are the most diverse family of the insect order Neuroptera, with over 1700 described species (in 191 genera) of which 37 species (in 21 genera) have so far been studied in respect to standard karyotypes. In the present paper we provide first data on the occurrence of the "insect-type" telomeric repeat (TTAGG) n and location of 18S rDNA clusters in the antlion karyotypes studied using fluorescence in situ hybridization (FISH). We show that males of Palpares libelluloides (Linnaeus, 1764) (Palparinae), Acanthaclisis occitanica (Villers, 1789) (Acanthaclisinae) and Distoleon tetragrammicus (Fabricius, 1798) (Nemoleontinae) have rDNA clusters on a large bivalent, two last species having an additional rDNA cluster on one of the sex chromosomes, most probably the X. (TTAGG) n - containing telomeres are clearly characteristic of Palpares libelluloides and Acanthaclisis occitanica ; the presence of this telomeric motif in Distoleon tetragrammicus is questionable. In addition, we detected the presence of the (TTAGG) n telomeric repeat in Libelloides macaronius (Scopoli, 1763) from the family Ascalaphidae (owlflies), a sister group to the Myrmeleontidae. We presume that the "insect" motif (TTAGG) n was present in a common ancestor of the families Ascalaphidae and Myrmeleontidae within the neuropteran suborder Myrmeleontiformia.

  1. Flower bud transcriptome analysis of Sapium sebiferum (Linn. Roxb. and primary investigation of drought induced flowering: pathway construction and G-quadruplex prediction based on transcriptome.

    Directory of Open Access Journals (Sweden)

    Minglei Yang

    Full Text Available Sapium sebiferum (Linn. Roxb. (Chinese Tallow Tree is a perennial woody tree and its seeds are rich in oil which hold great potential for biodiesel production. Despite a traditional woody oil plant, our understanding on S. sebiferum genetics and molecular biology remains scant. In this study, the first comprehensive transcriptome of S. sebiferum flower has been generated by sequencing and de novo assembly. A total of 149,342 unigenes were generated from raw reads, of which 24,289 unigenes were successfully matched to public database. A total of 61 MADS box genes and putative pathways involved in S. sebiferum flower development have been identified. Abiotic stress response network was also constructed in this work, where 2,686 unigenes are involved in the pathway. As for lipid biosynthesis, 161 unigenes have been identified in fatty acid (FA and triacylglycerol (TAG biosynthesis. Besides, the G-Quadruplexes in RNA of S. sebiferum also have been predicted. An interesting finding is that the stress-induced flowering was observed in S. sebiferum for the first time. According to the results of semi-quantitative PCR, expression tendencies of flowering-related genes, GA1, AP2 and CRY2, accorded with stress-related genes, such as GRX50435 and PRXⅡ39562. This transcriptome provides functional genomic information for further research of S. sebiferum, especially for the genetic engineering to shorten the juvenile period and improve yield by regulating flower development. It also offers a useful database for the research of other Euphorbiaceae family plants.

  2. Exploiting Polydopamine Nanospheres to DNA Computing: A Simple, Enzyme-Free and G-Quadruplex-Free DNA Parity Generator/Checker for Error Detection during Data Transmission.

    Science.gov (United States)

    Fan, Daoqing; Wang, Erkang; Dong, Shaojun

    2017-01-18

    Molecular logic devices with various functions play an indispensable role in molecular data transmission/processing. However, during any kinds of data transmission, a constant and unavoidable circumstance is the appearance of bit errors, which have serious effects on the regular logic computation. Fortunately, these errors can be detected via plugging a parity generator (pG) at the transmitting terminal and a parity checker (pC) at the receiving terminal. Herein, taking advantage of the efficient adsorption/quenching ability of polydopamine nanospheres toward fluorophore-labeled single-stranded DNA, we explored this biocompatible nanomaterial to DNA logic computation and constructed the first simple, enzyme-free, and G-quadruplex-free DNA pG/pC for error detection through data transmission. Besides, graphene oxide (GO) was innovatively introduced as the "corrective element" to perform the output-correction function of pC. All the erroneous outputs were corrected to normal conditions completely, ensuring the regular operation of later logic computing. The total operation of this non-G4 pG/pC system (error checking/output-correction) could be completed within 1 h (about 1/3 of previous G4 platform) in a simpler and more efficient way. Notably, the odd pG/pC with analogous functions was also achieved through negative logic conversion to the fabricated even one. Furthermore, the same system could also perform three-input concatenated logic computation (XOR-INHIBIT), enriching the complexity of PDs-based logic computation.

  3. The differential processing of telomeres in response to increased telomeric transcription and RNA-DNA hybrid accumulation.

    Science.gov (United States)

    Balk, Bettina; Dees, Martina; Bender, Katharina; Luke, Brian

    2014-01-01

    Telomeres are protective nucleoprotein structures at the ends of eukaryotic chromosomes. Despite the heterochromatic state of telomeres they are transcribed, generating non-coding telomeric repeat-containing RNA (TERRA). Strongly induced TERRA transcription has been shown to cause telomere shortening and accelerated senescence in the absence of both telomerase and homology-directed repair (HDR). Moreover, it has recently been demonstrated that TERRA forms RNA-DNA hybrids at chromosome ends. The accumulation of RNA-DNA hybrids at telomeres also leads to rapid senescence and telomere loss in the absence of telomerase and HDR. Conversely, in the presence of HDR, telomeric RNA-DNA hybrid accumulation and increased telomere transcription promote telomere recombination, and hence, delayed senescence. Here, we demonstrate that despite these similar phenotypic outcomes, telomeres that are highly transcribed are not processed in the same manner as those that accumulate RNA-DNA hybrids.

  4. Telomestatin-induced telomere uncapping is modulated by POT1 through G-overhang extension in HT1080 human tumor cells.

    Science.gov (United States)

    Gomez, Dennis; Wenner, Thomas; Brassart, Bertrand; Douarre, Céline; O'Donohue, Marie-Françoise; El Khoury, Victoria; Shin-Ya, Kazuo; Morjani, Hamid; Trentesaux, Chantal; Riou, Jean-François

    2006-12-15

    Telomestatin is a potent G-quadruplex ligand that interacts with the 3' telomeric overhang, leading to its degradation, and induces a delayed senescence and apoptosis of cancer cells. POT1 and TRF2 were recently identified as specific telomere-binding proteins involved in telomere capping and t-loop maintenance and whose interaction with telomeres is modulated by telomestatin. We show here that the treatment of HT1080 human tumor cells by telomestatin induces a rapid decrease of the telomeric G-overhang and of the double-stranded telomeric repeats. Telomestatin treatment also provokes a strong decrease of POT1 and TRF2 from their telomere sites, suggesting that the ligand triggers the uncapping of the telomere ends. The effect of the ligand is associated with an increase of the gamma-H2AX foci, one part of them colocalizing at telomeres, thus indicating the occurrence of a DNA damage response at the telomere, but also the presence of additional DNA targets for telomestatin. Interestingly, the expression of GFP-POT1 in HT1080 cells increases both telomere and G-overhang length. As compared with HT1080 cells, HT1080GFP-POT1 cells presented a resistance to telomestatin treatment characterized by a protection to the telomestatin-induced growth inhibition and the G-overhang shortening. This protection is related to the initial G-overhang length rather than to its degradation rate and is overcome by increased telomestatin concentration. Altogether these results suggest that telomestatin induced a telomere dysfunction in which G-overhang length and POT1 level are important factors but also suggest the presence of additional DNA sites of action for the ligand.

  5. Telomeric repeat-containing RNA TERRA: a noncoding RNA connecting telomere biology to genome integrity.

    Science.gov (United States)

    Cusanelli, Emilio; Chartrand, Pascal

    2015-01-01

    Telomeres are dynamic nucleoprotein structures that protect the ends of chromosomes from degradation and activation of DNA damage response. For this reason, telomeres are essential to genome integrity. Chromosome ends are enriched in heterochromatic marks and proper organization of telomeric chromatin is important to telomere stability. Despite their heterochromatic state, telomeres are transcribed giving rise to long noncoding RNAs (lncRNA) called TERRA (telomeric repeat-containing RNA). TERRA molecules play critical roles in telomere biology, including regulation of telomerase activity and heterochromatin formation at chromosome ends. Emerging evidence indicate that TERRA transcripts form DNA-RNA hybrids at chromosome ends which can promote homologous recombination among telomeres, delaying cellular senescence and sustaining genome instability. Intriguingly, TERRA RNA-telomeric DNA hybrids are involved in telomere length homeostasis of telomerase-negative cancer cells. Furthermore, TERRA transcripts play a role in the DNA damage response (DDR) triggered by dysfunctional telomeres. We discuss here recent developments on TERRA's role in telomere biology and genome integrity, and its implication in cancer.

  6. Telomeres and Telomerase in Cardiovascular Diseases

    Directory of Open Access Journals (Sweden)

    Jih-Kai Yeh

    2016-09-01

    Full Text Available Telomeres are tandem repeat DNA sequences present at the ends of each eukaryotic chromosome to stabilize the genome structure integrity. Telomere lengths progressively shorten with each cell division. Inflammation and oxidative stress, which are implicated as major mechanisms underlying cardiovascular diseases, increase the rate of telomere shortening and lead to cellular senescence. In clinical studies, cardiovascular risk factors such as smoking, obesity, sedentary lifestyle, and hypertension have been associated with short leukocyte telomere length. In addition, low telomerase activity and short leukocyte telomere length have been observed in atherosclerotic plaque and associated with plaque instability, thus stroke or acute myocardial infarction. The aging myocardium with telomere shortening and accumulation of senescent cells limits the tissue regenerative capacity, contributing to systolic or diastolic heart failure. In addition, patients with ion-channel defects might have genetic imbalance caused by oxidative stress-related accelerated telomere shortening, which may subsequently cause sudden cardiac death. Telomere length can serve as a marker for the biological status of previous cell divisions and DNA damage with inflammation and oxidative stress. It can be integrated into current risk prediction and stratification models for cardiovascular diseases and can be used in precise personalized treatments. In this review, we summarize the current understanding of telomeres and telomerase in the aging process and their association with cardiovascular diseases. In addition, we discuss therapeutic interventions targeting the telomere system in cardiovascular disease treatments.

  7. A new signal-on method for the detection of protein based on binding-induced strategy and photoinduced electron transfer between Ag nanoclusters and split G-quadruplex-hemin complexes

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Kai, E-mail: zhangkai@jsinm.org; Wang, Ke; Zhu, Xue; Xie, Minhao

    2015-08-05

    Proteins play important roles in biological and cellular processes. The levels of proteins can be useful biomarkers for cellular events or disease diagnosis, thus the method for sensitive and selective detection of proteins is imperative to proteins express, study, and clinical diagnosis. Herein, we report a “signal-on” platform for the assay of protein based on binding-induced strategy and photoinduced electron transfer between Ag nanoclusters and split G-quadruplex-hemin complexes. By using biotin as the affinity ligand, this simple protocol could sensitively detect streptavidin with a detection limit down to 10 pM. With the use of an antibody as the affinity ligand, a method for homogeneous fluorescence detection of Prostate Specific Antigen (PSA) was also proposed with a detection limit of 10 pM. The one-step and wash-free assay showed good selectivity. Its high sensitivity, acceptable accuracy, and satisfactory versatility of analytes led to various applications in bioanalysis. - Highlights: • AgNCs have great potential for application in biomedicine. • Binding of two affinity ligands can result in binding-induced DNA assemblies. • PET can be happened between DNA/AgNCs and G-quadruplex/hemin complexes. • A platform for the detection of proteins was proposed by using PET and binding-induced strategy.

  8. Single-Molecule Studies of Telomeres and Telomerase.

    Science.gov (United States)

    Parks, Joseph W; Stone, Michael D

    2017-05-22

    Telomeres are specialized chromatin structures that protect chromosome ends from dangerous processing events. In most tissues, telomeres shorten with each round of cell division, placing a finite limit on cell growth. In rapidly dividing cells, including the majority of human cancers, cells bypass this growth limit through telomerase-catalyzed maintenance of telomere length. The dynamic properties of telomeres and telomerase render them difficult to study using ensemble biochemical and structural techniques. This review describes single-molecule approaches to studying how individual components of telomeres and telomerase contribute to function. Single-molecule methods provide a window into the complex nature of telomeres and telomerase by permitting researchers to directly visualize and manipulate the individual protein, DNA, and RNA molecules required for telomere function. The work reviewed in this article highlights how single-molecule techniques have been utilized to investigate the function of telomeres and telomerase.

  9. Telomeres, telomerase and premature ovarian failure

    Directory of Open Access Journals (Sweden)

    Renata Košir Pogačnik

    2011-11-01

    Full Text Available Telomeres are specialized structures at the ends of chromosomes, consisting of six repeated nucleotides in TTAGGG sequence. Genome stability is partly maintained by the architecture of telomeres and is gradually lost as telomeres progressively shorten with each cell replication. Critically shortened telomeres are recognized by DNA repair mechanisms as DNA damage and the cell replication cycle stops. The cell eventually dies or undergoes cell apoptosis. Telomere represents a cellular marker of biological age and are therefore also called cell mitotic clock. The enzyme that counteracts telomere shortening by adding nucleotides to the 3’ end of DNA strand is called telomerase. It is composed of the RNA subunit (TR, which is special type of messenger RNA (mRNA, the catalytic protein subunit (TERT, which works as a reverse transcriptase and numerous additional proteins. Telomerase is active in some germline, epithelial and haemopoietic cells, but in most somatic cells the activity is undetectable. In literature, the length of telomeres is closely connected with premature ovarian failure (POF. POF is generally defined as the onset of menopause before the age of 40. The causes of disease are genetical, autoimmune, iatrogenic or if we cannot establish the cause – idiopathic. A lot of studies examined correlation between idiopathic POF, length of telomeres and telomerase activity. The studies mostly show that women with POF have shortened telomeres and decreased activity of telomerase as compared to healthy women.

  10. HOT1 is a mammalian direct telomere repeat-binding protein contributing to telomerase recruitment

    NARCIS (Netherlands)

    Kappei, D.; Butter, F.; Benda, C.; Scheibe, M.; Draskovic, Irena; Stevense, M.; Novo, C.L.; Basquin, C.; Araki, M.; Araki, K.; Krastev, D.B.; Kittler, R.; Jessberger, R.; Londono-Vallejo, J.A.; Mann, M.; Buchholz, F.

    2013-01-01

    Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the

  11. Comparative biology of telomeres: where plants stand.

    Science.gov (United States)

    Watson, J Matthew; Riha, Karel

    2010-09-10

    Telomeres are essential structures at the ends of eukaryotic chromosomes. Work on their structure and function began almost 70 years ago in plants and flies, continued through the Nobel Prize winning work on yeast and ciliates, and goes on today in many model and non-model organisms. The basic molecular mechanisms of telomeres are highly conserved throughout evolution, and our current understanding of how telomeres function is a conglomeration of insights gained from many different species. This review will compare the current knowledge of telomeres in plants with other organisms, with special focus on the functional length of telomeric DNA, the search for TRF homologs, the family of POT1 proteins, and the recent discovery of members of the CST complex. Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. Telomerers rolle i cancer

    DEFF Research Database (Denmark)

    Bendix, Laila; Kølvraa, Steen

    2010-01-01

    Telomeres are a double-edged sword when it comes to cancer. On one hand, telomeres limit the cells' ability to divide and thereby restrict the uninhibited growth seen in cancer. On the other hand, short telomeres can initiate the chromosome instability that characterizes cancer. Diseases...... with the combination of short telomeres and high cancer risk are seen, but until now the use of telomeres as predictors of cancer has, in general, been unsuccessful. Telomeres and telomerase play an important role in further cancer development. Researchers are trying to exploit this in the development of new cancer...

  13. Telomeric repeat-containing RNA (TERRA) and telomerase are components of telomeres during mammalian gametogenesis.

    Science.gov (United States)

    Reig-Viader, Rita; Vila-Cejudo, Marta; Vitelli, Valerio; Buscà, Rafael; Sabaté, Montserrat; Giulotto, Elena; Caldés, Montserrat Garcia; Ruiz-Herrera, Aurora

    2014-05-01

    Telomeres are ribonucleoprotein structures at the end of chromosomes composed of telomeric DNA, specific-binding proteins, and noncoding RNA (TERRA). Despite their importance in preventing chromosome instability, little is known about the cross talk between these three elements during the formation of the germ line. Here, we provide evidence that both TERRA and the telomerase enzymatic subunit (TERT) are components of telomeres in mammalian germ cells. We found that TERRA colocalizes with telomeres during mammalian meiosis and that its expression progressively increases during spermatogenesis until the beginning of spermiogenesis. While both TERRA levels and distribution would be regulated in a gender-specific manner, telomere-TERT colocalization appears to be regulated based on species-specific characteristics of the telomeric structure. Moreover, we found that TERT localization at telomeres is maintained throughout spermatogenesis as a structural component without affecting telomere elongation. Our results represent the first evidence of colocalization between telomerase and telomeres during mammalian gametogenesis. © 2014 by the Society for the Study of Reproduction, Inc.

  14. Solution structure of the Arabidopsis thaliana telomeric repeat-binding protein DNA binding domain: a new fold with an additional C-terminal helix.

    Science.gov (United States)

    Sue, Shih-Che; Hsiao, Hsin-Hao; Chung, Ben C-P; Cheng, Ying-Hsien; Hsueh, Kuang-Lung; Chen, Chung Mong; Ho, Chia Hsing; Huang, Tai-Huang

    2006-02-10

    The double-stranded telomeric repeat-binding protein (TRP) AtTRP1 is isolated from Arabidopsis thaliana. Using gel retardation assays, we defined the C-terminal 97 amino acid residues, Gln464 to Val560 (AtTRP1(464-560)), as the minimal structured telomeric repeat-binding domain. This region contains a typical Myb DNA-binding motif and a C-terminal extension of 40 amino acid residues. The monomeric AtTRP1(464-560) binds to a 13-mer DNA duplex containing a single repeat of an A.thaliana telomeric DNA sequence (GGTTTAG) in a 1:1 complex, with a K(D) approximately 10(-6)-10(-7) M. Nuclear magnetic resonance (NMR) examination revealed that the solution structure of AtTRP1(464-560) is a novel four-helix tetrahedron rather than the three-helix bundle structure found in typical Myb motifs and other TRPs. Binding of the 13-mer DNA duplex to AtTRP1(464-560) induced significant chemical shift perturbations of protein amide resonances, which suggests that helix 3 (H3) and the flexible loop connecting H3 and H4 are essential for telomeric DNA sequence recognition. Furthermore, similar to that in hTRF1, the N-terminal arm likely contributes to or stabilizes DNA binding. Sequence comparisons suggested that the four-helix structure and the involvement of the loop residues in DNA binding may be features unique to plant TRPs.

  15. DNA Replication Origins and Fork Progression at Mammalian Telomeres

    Science.gov (United States)

    Higa, Mitsunori; Fujita, Masatoshi; Yoshida, Kazumasa

    2017-01-01

    Telomeres are essential chromosomal regions that prevent critical shortening of linear chromosomes and genomic instability in eukaryotic cells. The bulk of telomeric DNA is replicated by semi-conservative DNA replication in the same way as the rest of the genome. However, recent findings revealed that replication of telomeric repeats is a potential cause of chromosomal instability, because DNA replication through telomeres is challenged by the repetitive telomeric sequences and specific structures that hamper the replication fork. In this review, we summarize current understanding of the mechanisms by which telomeres are faithfully and safely replicated in mammalian cells. Various telomere-associated proteins ensure efficient telomere replication at different steps, such as licensing of replication origins, passage of replication forks, proper fork restart after replication stress, and dissolution of post-replicative structures. In particular, shelterin proteins have central roles in the control of telomere replication. Through physical interactions, accessory proteins are recruited to maintain telomere integrity during DNA replication. Dormant replication origins and/or homology-directed repair may rescue inappropriate fork stalling or collapse that can cause defects in telomere structure and functions. PMID:28350373

  16. Telomeres and human health

    DEFF Research Database (Denmark)

    Bojesen, S E

    2013-01-01

    Telomeres are the tips of chromosomes and consist of proteins and hexanucleotide tandem repeats of DNA. The DNA repeats are shortened at each mitotic division of normal cells, and the telomere length chronicles how many divisions the cell has undergone. Thus, telomere length is a marker of fundam...

  17. Telomeres and human reproduction.

    Science.gov (United States)

    Kalmbach, Keri Horan; Fontes Antunes, Danielle Mota; Dracxler, Roberta Caetano; Knier, Taylor Warner; Seth-Smith, Michelle Louise; Wang, Fang; Liu, Lin; Keefe, David Lawrence

    2013-01-01

    Telomeres mediate biologic aging in organisms as diverse as plants, yeast, and mammals. We propose a telomere theory of reproductive aging that posits telomere shortening in the female germ line as the primary driver of reproductive aging in women. Experimental shortening of telomeres in mice, which normally do not exhibit appreciable oocyte aging, and which have exceptionally long telomeres, recapitulates the aging phenotype of human oocytes. Telomere shortening in mice reduces synapsis and chiasmata, increases embryo fragmentation, cell cycle arrest, apoptosis, spindle dysmorphologies, and chromosome abnormalities. Telomeres are shorter in the oocytes from women undergoing in vitro fertilization, who then produce fragmented, aneuploid embryos that fail to implant. In contrast, the testes are replete with spermatogonia that can rejuvenate telomere reserves throughout the life of the man by expressing telomerase. Differences in telomere dynamics across the life span of men and women may have evolved because of the difference in the inherent risks of aging on reproduction between men and women. Additionally, growing evidence links altered telomere biology to endometriosis and gynecologic cancers, thus future studies should examine the role of telomeres in pathologies of the reproductive tract. Copyright © 2013. Published by Elsevier Inc.

  18. Telomeres and disease: enter TERRA.

    Science.gov (United States)

    Maicher, André; Kastner, Lisa; Luke, Brian

    2012-06-01

    Telomere function is tightly regulated in order to maintain chromosomal stability. When telomeres become dysfunctional, the replicative capacity of cells diminishes and cellular senescence ensues. This can lead to impaired tissue replenishment and eventually degenerative disorders, referred to as telomere syndromes. Cancer can also develop as a result of the genomic instability associated with telomere dysfunction. TERRA (TElomeric Repeat containing RNA) is a long non-coding transcript that stems from sub-telomeric regions and continues into the telomeric tract and is therefore a hybrid of both sub-telomeric and telomeric sequence. In general, increased TERRA transcription is associated with telomere shortening and compromised telomere function. Here we will briefly outline the general principles behind telomere dysfunction-associated diseases. Furthermore, we will discuss the few known links that exist between telomere transcription (TERRA) and disease. Finally, we will speculate on how the understanding, and eventual manipulation, of TERRA transcription could potentially be used in terms of therapeutic strategies.

  19. Telomeres, histone code, and DNA damage response.

    Science.gov (United States)

    Misri, S; Pandita, S; Kumar, R; Pandita, T K

    2008-01-01

    Genomic stability is maintained by telomeres, the end terminal structures that protect chromosomes from fusion or degradation. Shortening or loss of telomeric repeats or altered telomere chromatin structure is correlated with telomere dysfunction such as chromosome end-to-end associations that could lead to genomic instability and gene amplification. The structure at the end of telomeres is such that its DNA differs from DNA double strand breaks (DSBs) to avoid nonhomologous end-joining (NHEJ), which is accomplished by forming a unique higher order nucleoprotein structure. Telomeres are attached to the nuclear matrix and have a unique chromatin structure. Whether this special structure is maintained by specific chromatin changes is yet to be thoroughly investigated. Chromatin modifications implicated in transcriptional regulation are thought to be the result of a code on the histone proteins (histone code). This code, involving phosphorylation, acetylation, methylation, ubiquitylation, and sumoylation of histones, is believed to regulate chromatin accessibility either by disrupting chromatin contacts or by recruiting non-histone proteins to chromatin. The histone code in which distinct histone tail-protein interactions promote engagement may be the deciding factor for choosing specific DSB repair pathways. Recent evidence suggests that such mechanisms are involved in DNA damage detection and repair. Altered telomere chromatin structure has been linked to defective DNA damage response (DDR), and eukaryotic cells have evolved DDR mechanisms utilizing proficient DNA repair and cell cycle checkpoints in order to maintain genomic stability. Recent studies suggest that chromatin modifying factors play a critical role in the maintenance of genomic stability. This review will summarize the role of DNA damage repair proteins specifically ataxia-telangiectasia mutated (ATM) and its effectors and the telomere complex in maintaining genome stability. Copyright 2008 S. Karger

  20. Functional characterization of the TERRA transcriptome at damaged telomeres.

    Science.gov (United States)

    Porro, Antonio; Feuerhahn, Sascha; Delafontaine, Julien; Riethman, Harold; Rougemont, Jacques; Lingner, Joachim

    2014-10-31

    Telomere deprotection occurs during tumorigenesis and aging upon telomere shortening or loss of the telomeric shelterin component TRF2. Deprotected telomeres undergo changes in chromatin structure and elicit a DNA damage response (DDR) that leads to cellular senescence. The telomeric long noncoding RNA TERRA has been implicated in modulating the structure and processing of deprotected telomeres. Here, we characterize the human TERRA transcriptome at normal and TRF2-depleted telomeres and demonstrate that TERRA upregulation is occurring upon depletion of TRF2 at all transcribed telomeres. TRF2 represses TERRA transcription through its homodimerization domain, which was previously shown to induce chromatin compaction and to prevent the early steps of DDR activation. We show that TERRA associates with SUV39H1 H3K9 histone methyltransferase, which promotes accumulation of H3K9me3 at damaged telomeres and end-to-end fusions. Altogether our data elucidate the TERRA landscape and defines critical roles for this RNA in the telomeric DNA damage response.

  1. Telomere Length and Mortality

    DEFF Research Database (Denmark)

    Kimura, Masayuki; Hjelmborg, Jacob V B; Gardner, Jeffrey P

    2008-01-01

    Leukocyte telomere length, representing the mean length of all telomeres in leukocytes, is ostensibly a bioindicator of human aging. The authors hypothesized that shorter telomeres might forecast imminent mortality in elderly people better than leukocyte telomere length. They performed mortality...... telomeres predicted the death of the first co-twin better than the mTRFL did (mTRFL: 0.56, 95% confidence interval (CI): 0.49, 0.63; mTRFL(50): 0.59, 95% CI: 0.52, 0.66; mTRFL(25): 0.59, 95% CI: 0.52, 0.66; MTRFL: 0.60, 95% CI: 0.53, 0.67). The telomere-mortality association was stronger in years 3-4 than...

  2. Improved Inhibition of Telomerase by Short Twisted Intercalating Nucleic Acids under Molecular Crowding Conditions

    DEFF Research Database (Denmark)

    Agarwal, Tani; Pradhan, Devranjan; Géci, Imrich

    2012-01-01

    Human telomeric DNA has the ability to fold into a 4-stranded G-quadruplex structure. Several G-quadruplex ligands are known to stabilize the structure and thereby inhibit telomerase activity. Such ligands have demonstrated efficient telomerase inhibition in dilute conditions, but under molecular......-based telomerase repeat amplification assay (TRAP) assay as well as nondenaturing polyacrylamide gel electrophoresis-based TRAP, we demonstrate remarkable enhancement in their anti-telomerase activity even under molecular crowding conditions. This is the first time in which a G-quadruplex stabilizing agent has...

  3. SLX4 Assembles a Telomere Maintenance Toolkit by Bridging Multiple Endonucleases with Telomeres

    Directory of Open Access Journals (Sweden)

    Bingbing Wan

    2013-09-01

    Full Text Available SLX4 interacts with several endonucleases to resolve structural barriers in DNA metabolism. SLX4 also interacts with telomeric protein TRF2 in human cells. The molecular mechanism of these interactions at telomeres remains unknown. Here, we report the crystal structure of the TRF2-binding motif of SLX4 (SLX4TBM in complex with the TRFH domain of TRF2 (TRF2TRFH and map the interactions of SLX4 with endonucleases SLX1, XPF, and MUS81. TRF2 recognizes a unique HxLxP motif on SLX4 via the peptide-binding site in its TRFH domain. Telomeric localization of SLX4 and associated nucleases depend on the SLX4-endonuclease and SLX4-TRF2 interactions and the protein levels of SLX4 and TRF2. SLX4 assembles an endonuclease toolkit that negatively regulates telomere length via SLX1-catalyzed nucleolytic resolution of telomere DNA structures. We propose that the SLX4-TRF2 complex serves as a double-layer scaffold bridging multiple endonucleases with telomeres for recombination-based telomere maintenance.

  4. Telomere length modulation in human astroglial brain tumors.

    Directory of Open Access Journals (Sweden)

    Domenico La Torre

    Full Text Available BACKGROUND: Telomeres alteration during carcinogenesis and tumor progression has been described in several cancer types. Telomeres length is stabilized by telomerase (h-TERT and controlled by several proteins that protect telomere integrity, such as the Telomere Repeat-binding Factor (TRF 1 and 2 and the tankyrase-poli-ADP-ribose polymerase (TANKs-PARP complex. OBJECTIVE: To investigate telomere dysfunction in astroglial brain tumors we analyzed telomeres length, telomerase activity and the expression of a panel of genes controlling the length and structure of telomeres in tissue samples obtained in vivo from astroglial brain tumors with different grade of malignancy. MATERIALS AND METHODS: Eight Low Grade Astrocytomas (LGA, 11 Anaplastic Astrocytomas (AA and 11 Glioblastoma Multiforme (GBM samples were analyzed. Three samples of normal brain tissue (NBT were used as controls. Telomeres length was assessed through Southern Blotting. Telomerase activity was evaluated by a telomere repeat amplification protocol (TRAP assay. The expression levels of TRF1, TRF2, h-TERT and TANKs-PARP complex were determined through Immunoblotting and RT-PCR. RESULTS: LGA were featured by an up-regulation of TRF1 and 2 and by shorter telomeres. Conversely, AA and GBM were featured by a down-regulation of TRF1 and 2 and an up-regulation of both telomerase and TANKs-PARP complex. CONCLUSIONS: In human astroglial brain tumours, up-regulation of TRF1 and TRF2 occurs in the early stages of carcinogenesis determining telomeres shortening and genomic instability. In a later stage, up-regulation of PARP-TANKs and telomerase activation may occur together with an ADP-ribosylation of TRF1, causing a reduced ability to bind telomeric DNA, telomeres elongation and tumor malignant progression.

  5. Development of an Optimized Protocol for NMR Metabolomics Studies of Human Colon Cancer Cell Lines and First Insight from Testing of the Protocol Using DNA G-Quadruplex Ligands as Novel Anti-Cancer Drugs

    Directory of Open Access Journals (Sweden)

    Ilaria Lauri

    2016-01-01

    Full Text Available The study of cell lines by nuclear magnetic resonance (NMR spectroscopy metabolomics represents a powerful tool to understand how the local metabolism and biochemical pathways are influenced by external or internal stimuli. In particular, the use of adherent mammalian cells is emerging in the metabolomics field in order to understand the molecular mechanism of disease progression or, for example, the cellular response to drug treatments. Hereto metabolomics investigations for this kind of cells have generally been limited to mass spectrometry studies. This study proposes an optimized protocol for the analysis of the endo-metabolome of human colon cancer cells (HCT116 by NMR. The protocol includes experimental conditions such as washing, quenching and extraction. In order to test the proposed protocol, it was applied to an exploratory study of cancer cells with and without treatment by anti-cancer drugs, such as DNA G-quadruplex binders and Adriamycin (a traditional anti-cancer drug. The exploratory NMR metabolomics analysis resulted in NMR assignment of all endo-metabolites that could be detected and provided preliminary insights about the biological behavior of the drugs tested.

  6. Development of an Optimized Protocol for NMR Metabolomics Studies of Human Colon Cancer Cell Lines and First Insight from Testing of the Protocol Using DNA G-Quadruplex Ligands as Novel Anti-Cancer Drugs.

    Science.gov (United States)

    Lauri, Ilaria; Savorani, Francesco; Iaccarino, Nunzia; Zizza, Pasquale; Pavone, Luigi Michele; Novellino, Ettore; Engelsen, Søren Balling; Randazzo, Antonio

    2016-01-15

    The study of cell lines by nuclear magnetic resonance (NMR) spectroscopy metabolomics represents a powerful tool to understand how the local metabolism and biochemical pathways are influenced by external or internal stimuli. In particular, the use of adherent mammalian cells is emerging in the metabolomics field in order to understand the molecular mechanism of disease progression or, for example, the cellular response to drug treatments. Hereto metabolomics investigations for this kind of cells have generally been limited to mass spectrometry studies. This study proposes an optimized protocol for the analysis of the endo-metabolome of human colon cancer cells (HCT116) by NMR. The protocol includes experimental conditions such as washing, quenching and extraction. In order to test the proposed protocol, it was applied to an exploratory study of cancer cells with and without treatment by anti-cancer drugs, such as DNA G-quadruplex binders and Adriamycin (a traditional anti-cancer drug). The exploratory NMR metabolomics analysis resulted in NMR assignment of all endo-metabolites that could be detected and provided preliminary insights about the biological behavior of the drugs tested.

  7. Modulation of Telomeres in Alternative Lengthening of Telomeres Type I Like Human Cells by the Expression of Werner Protein and Telomerase

    Directory of Open Access Journals (Sweden)

    Aisha Siddiqa

    2012-01-01

    Full Text Available The alternative lengthening of telomeres (ALT is a recombination-based mechanism of telomere maintenance activated in 5–20% of human cancers. In Saccharomyces cerevisiae, survivors that arise after inactivation of telomerase can be classified as type I or type II ALT. In type I, telomeres have a tandem array structure, with each subunit consisting of a subtelomeric Y′ element and short telomere sequence. Telomeres in type II have only long telomere repeats and require Sgs1, the S. cerevisiae RecQ family helicase. We previously described the first human ALT cell line, AG11395, that has a telomere structure similar to type I ALT yeast cells. This cell line lacks the activity of the Werner syndrome protein, a human RecQ helicase. The telomeres in this cell line consist of tandem repeats containing SV40 DNA, including the origin of replication, and telomere sequence. We investigated the role of the SV40 origin of replication and the effects of Werner protein and telomerase on telomere structure and maintenance in AG11395 cells. We report that the expression of Werner protein facilitates the transition in human cells of ALT type I like telomeres to type II like telomeres in some aspects. These findings have implications for the diagnosis and treatment of cancer.

  8. Cancer and aging: The importance of telomeres in genome maintenance

    Energy Technology Data Exchange (ETDEWEB)

    Rodier, Francis; Kim, Sahn-ho; Nijjar, Tarlochan; Yaswen, Paul; Campisi, Judith

    2004-10-01

    Telomeres are the specialized DNA-protein structures that cap the ends of linear chromosomes, thereby protecting them from degradation and fusion by cellular DNA repair processes. In vertebrate cells, telomeres consist of several kilobase pairs of DNA having the sequence TTAGGG, a few hundred base pairs of single-stranded DNA at the 3' end of the telomeric DNA tract, and a host of proteins that organize the telomeric double and single stranded DNA into a protective structure. Functional telomeres are essential for maintaining the integrity and stability of genomes. When combined with loss of cell cycle checkpoint controls, telomere dysfunction can lead to genomic instability, a common cause and hallmark of cancer. Consequently, normal mammalian cells respond to dysfunctional telomeres by undergoing apoptosis (programmed cell death) or cellular senescence (permanent cell cycle arrest), two cellular tumor suppressor mechanisms. These tumor suppressor mechanisms are potent suppressors of cancer, but recent evidence suggests that they can antagonistically also contribute to aging phenotypes. Here, we review what is known about the structure and function of telomeres in mammalian cells, particularly human cells, and how telomere dysfunction may arise and contribute to cancer and aging phenotypes.

  9. Molecular recognition in complexes of TRF proteins with telomeric DNA.

    Directory of Open Access Journals (Sweden)

    Miłosz Wieczór

    Full Text Available Telomeres are specialized nucleoprotein assemblies that protect the ends of linear chromosomes. In humans and many other species, telomeres consist of tandem TTAGGG repeats bound by a protein complex known as shelterin that remodels telomeric DNA into a protective loop structure and regulates telomere homeostasis. Shelterin recognizes telomeric repeats through its two major components known as Telomere Repeat-Binding Factors, TRF1 and TRF2. These two homologous proteins are therefore essential for the formation and normal function of telomeres. Indeed, TRF1 and TRF2 are implicated in a plethora of different cellular functions and their depletion leads to telomere dysfunction with chromosomal fusions, followed by apoptotic cell death. More specifically, it was found that TRF1 acts as a negative regulator of telomere length, and TRF2 is involved in stabilizing the loop structure. Consequently, these proteins are of great interest, not only because of their key role in telomere maintenance and stability, but also as potential drug targets. In the current study, we investigated the molecular basis of telomeric sequence recognition by TRF1 and TRF2 and their DNA binding mechanism. We used molecular dynamics (MD to calculate the free energy profiles for binding of TRFs to telomeric DNA. We found that the predicted binding free energies were in good agreement with experimental data. Further, different molecular determinants of binding, such as binding enthalpies and entropies, the hydrogen bonding pattern and changes in surface area, were analyzed to decompose and examine the overall binding free energies at the structural level. With this approach, we were able to draw conclusions regarding the consecutive stages of sequence-specific association, and propose a novel aspartate-dependent mechanism of sequence recognition. Finally, our work demonstrates the applicability of computational MD-based methods to studying protein-DNA interactions.

  10. Telomere length and depression

    DEFF Research Database (Denmark)

    Wium-Andersen, Marie Kim; Ørsted, David Dynnes; Rode, Line

    2017-01-01

    as prospectively and genetically. METHOD: Telomere length and three polymorphisms, TERT, TERC and OBFC1, were measured in 67 306 individuals aged 20-100 years from the Danish general population and associated with register-based attendance at hospital for depression and purchase of antidepressant medication.......0-21.5). The genetic analyses suggested that telomere length was not causally associated with attendance at hospital for depression or with purchase of antidepressant medication. CONCLUSIONS: Short telomeres were not associated with depression in prospective or in causal, genetic analyses.......BACKGROUND: Depression has been cross-sectionally associated with short telomeres as a measure of biological age. However, the direction and nature of the association is currently unclear. AIMS: We examined whether short telomere length is associated with depression cross-sectionally as well...

  11. Telomere Q-PNA-FISH--reliable results from stochastic signals.

    Directory of Open Access Journals (Sweden)

    Andrea Cukusic Kalajzic

    Full Text Available Structural and functional analysis of telomeres is very important for understanding basic biological functions such as genome stability, cell growth control, senescence and aging. Recently, serious concerns have been raised regarding the reliability of current telomere measurement methods such as Southern blot and quantitative polymerase chain reaction. Since telomere length is associated with age related pathologies, including cardiovascular disease and cancer, both at the individual and population level, accurate interpretation of measured results is a necessity. The telomere Q-PNA-FISH technique has been widely used in these studies as well as in commercial analysis for the general population. A hallmark of telomere Q-PNA-FISH is the wide variation among telomere signals which has a major impact on obtained results. In the present study we introduce a specific mathematical and statistical analysis of sister telomere signals during cell culture senescence which enabled us to identify high regularity in their variations. This phenomenon explains the reproducibility of results observed in numerous telomere studies when the Q-PNA-FISH technique is used. In addition, we discuss the molecular mechanisms which probably underlie the observed telomere behavior.

  12. PRL-3 promotes telomere deprotection and chromosomal instability

    Science.gov (United States)

    Meng, Lin; Yang, Yongyong; Ma, Ting; Xing, Xiaofang; Feng, Qin; Song, Qian; Liu, Caiyun; Tian, Zhihua

    2017-01-01

    Abstract Phosphatase of regenerating liver (PRL-3) promotes cell invasiveness, but its role in genomic integrity remains unknown. We report here that shelterin component RAP1 mediates association between PRL-3 and TRF2. In addition, TRF2 and RAP1 assist recruitment of PRL-3 to telomeric DNA. Silencing of PRL-3 in colon cancer cells does not affect telomere integrity or chromosomal stability, but induces reactive oxygen species-dependent DNA damage response and senescence. However, overexpression of PRL-3 in colon cancer cells and primary fibroblasts promotes structural abnormalities of telomeres, telomere deprotection, DNA damage response, chromosomal instability and senescence. Furthermore, PRL-3 dissociates RAP1 and TRF2 from telomeric DNA in vitro and in cells. PRL-3-promoted telomere deprotection, DNA damage response and senescence are counteracted by disruption of PRL-3–RAP1 complex or expression of ectopic TRF2. Examination of clinical samples showed that PRL-3 status positively correlates with telomere deprotection and senescence. PRL-3 transgenic mice exhibit hallmarks of telomere deprotection and senescence and are susceptible to dextran sodium sulfate-induced colon malignancy. Our results uncover a novel role of PRL-3 in tumor development through its adverse impact on telomere homeostasis. PMID:28482095

  13. Synthesis and Characterization of thermo/pH-responsive Supramolecular G-Quadruplexes for the Construction of Supramolecular Hacky Sacks for Biorelevant Applications

    Science.gov (United States)

    Negron Rios, Luis M.

    The impact of size, shape, and distribution of lipophilic regions on the surfaces of nanoscopic objects that are amphiphilic or patchy (such as proteins) are yet to be fully understood. One of the reasons for this is the lack of an appropriate model systems in which to probe this question. Our group has previously reported 2'-deoxyguanosine (8ArG) derivatives that self-assemble in aqueous media into discrete supramolecular hexadecamers that show the lower critical solution temperature (LCST) phenomenon. The LCST phenomenon is a convenient and rigorous strategy to measure the hydrophobicity of a system. Although these SGQs are potentially attractive for biomedical applications like drug-delivery, the narrow window of physiological temperatures complicates their implementation. This moved us to redesign the constituent 8ArG subunits to incorporate imidazole moieties that would lead to pH-responsive SGQs, working isothermally. Upon reaching a threshold temperature (Lower Critical Solution Temperature, LCST) at pH 7, these dual-responsive SGQs further self-assemble to form nano/micro hydrogel globules that we called them supramolecular hacky sacks (SHS). However, we can isolate kinetically stable versions of these SHS by lowering the ionic strength of the medium (i.e., from the molar to the millimolar range) in a process that we term "fixing the SHS", in which these SHS maintain their integrity (size and shape) and stability without the requirement of crosslinking agents. After structural characterization and in vitro studies of SHS, we performed encapsulation studies of DOX, rhodamine, dsDNA (F26T), thrombin binding aptamer (TBA) and dextran (3 kDa) Texas Red conjugate. Then we performed in vivo studies of cell internalization and drug delivery with neuroblastoma SY-SH5Y. The performed studies will bring new approaches for the development of new biotechnology for fundamental applications and the emerging of novel therapeutic agents for biomedical applications.

  14. Alternative Lengthening of Telomeres: Recurrent Cytogenetic Aberrations and Chromosome Stability under Extreme Telomere Dysfunction

    Directory of Open Access Journals (Sweden)

    Despoina Sakellariou

    2013-11-01

    Full Text Available Human tumors using the alternative lengthening of telomeres (ALT exert high rates of telomere dysfunction. Numerical chromosomal aberrations are very frequent, and structural rearrangements are widely scattered among the genome. This challenging context allows the study of telomere dysfunction-driven chromosomal instability in neoplasia (CIN in a massive scale. We used molecular cytogenetics to achieve detailed karyotyping in 10 human ALT neoplastic cell lines.We identified 518 clonal recombinant chromosomes affected by 649 structural rearrangements. While all human chromosomes were involved in random or clonal, terminal, or pericentromeric rearrangements and were capable to undergo telomere healing at broken ends, a differential recombinatorial propensity of specific genomic regions was noted.We show that ALT cells undergo epigenetic modifications rendering polycentric chromosomes functionally monocentric, and because of increased terminal recombinogenicity, they generate clonal recombinant chromosomes with interstitial telomeric repeats. Losses of chromosomes 13, X, and 22, gains of 2, 3, 5, and 20, and translocation/deletion events involving several common chromosomal fragile sites (CFSs were recurrent. Long-term reconstitution of telomerase activity in ALT cells reduced significantly the rates of random ongoing telomeric and pericentromeric CIN. However, the contribution of CFS in overall CIN remained unaffected, suggesting that in ALT cells whole-genome replication stress is not suppressed by telomerase activation. Our results provide novel insights into ALT-driven CIN, unveiling in parallel specific genomic sites that may harbor genes critical for ALT cancerous cell growth.

  15. Telomeric noncoding RNA TERRA is induced by telomere shortening to nucleate telomerase molecules at short telomeres.

    Science.gov (United States)

    Cusanelli, Emilio; Romero, Carmina Angelica Perez; Chartrand, Pascal

    2013-09-26

    Elongation of a short telomere depends on the action of multiple telomerase molecules, which are visible as telomerase RNA foci or clusters associated with telomeres in yeast and mammalian cells. How several telomerase molecules act on a single short telomere is unknown. Herein, we report that the telomeric noncoding RNA TERRA is involved in the nucleation of telomerase molecules into clusters prior to their recruitment at a short telomere. We find that telomere shortening induces TERRA expression, leading to the accumulation of TERRA molecules into a nuclear focus. Simultaneous time-lapse imaging of telomerase RNA and TERRA reveals spontaneous events of telomerase nucleation on TERRA foci in early S phase, generating TERRA-telomerase clusters. This cluster is subsequently recruited to the short telomere from which TERRA transcripts originate during S phase. We propose that telomere shortening induces noncoding RNA expression to coordinate the recruitment and activity of telomerase molecules at short telomeres. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Human XPF controls TRF2 and telomere length maintenance through distinctive mechanisms.

    Science.gov (United States)

    Wu, Yili; Mitchell, Taylor R H; Zhu, Xu-Dong

    2008-10-01

    XPF-ERCC1, a structure-specific endonuclease, is involved in nucleotide excision repair, crosslink repair and homologous recombination. XPF-ERCC1 is also found to interact with TRF2, a duplex telomeric DNA binding protein. We have previously shown that XPF-ERCC1 is required for TRF2-promoted telomere shortening. However, whether XPF-ERCC1 by itself has a role in telomere length maintenance has not been determined. Here we report that overexpression of XPF induces telomere shortening in XPF-proficient cells whereas XPF complementation suppresses telomere lengthening in XPF-deficient cells. These results suggest that XPF-ERCC1 can function as a negative mediator of telomere length maintenance. In addition, we find that introduction of wild type XPF into XPF-deficient cells leads to over 40% reduction in TRF2 association with telomeric DNA, indicating that XPF-ERCC1 negatively regulates TRF2 binding to telomeric DNA. Furthermore, we show that XPF carrying mutations in the conserved nuclease domain fails to control TRF2 association with telomeric DNA but it is competent for modulating telomere length maintenance. These results imply that XPF-ERCC1 controls TRF2 and telomere length maintenance through two distinctive mechanisms, with the former requiring its nuclease activity. Our results further imply that TRF2 association with telomeres may be deregulated in cells derived from XPF patients.

  17. Telomere-binding proteins of Arabidopsis thaliana.

    Science.gov (United States)

    Zentgraf, U

    1995-02-01

    The nucleoprotein structure of Arabidopsis thaliana telomeres was investigated. A protein specifically binding to telomeric sequences was characterized by gel mobility shift assays with synthetic oligonucleotides consisting of four 7 bp telomeric repeats of Arabidopsis (TTTAGGG) and crude nuclear protein extracts of Arabidopsis leaves. These DNA-protein binding studies revealed that the binding affinity of this telomere-binding protein to the G-rich single-strand as well as to the double-stranded telomeric DNA is much higher than to the C-rich single-strand. The molecular mass of the protein was identified by SDS-PAGE to be 67 kDa. The isoelectric points were determined to be 5.0, 4.85 and 4.7, respectively, indicating that either one protein with different modifications or three slightly different proteins have been isolated. An RNA component, possibly serving as a template for reverse transcription of a plant telomerase, does not mediate the DNA-protein contact because the DNA-protein interactions were not RNAse-sensitive.

  18. Mathematical model of alternative mechanism of telomere length maintenance.

    Science.gov (United States)

    Kollár, Richard; Bod'ová, Katarína; Nosek, Jozef; Tomáška, L'ubomír

    2014-03-01

    Biopolymer length regulation is a complex process that involves a large number of biological, chemical, and physical subprocesses acting simultaneously across multiple spatial and temporal scales. An illustrative example important for genomic stability is the length regulation of telomeres-nucleoprotein structures at the ends of linear chromosomes consisting of tandemly repeated DNA sequences and a specialized set of proteins. Maintenance of telomeres is often facilitated by the enzyme telomerase but, particularly in telomerase-free systems, the maintenance of chromosomal termini depends on alternative lengthening of telomeres (ALT) mechanisms mediated by recombination. Various linear and circular DNA structures were identified to participate in ALT, however, dynamics of the whole process is still poorly understood. We propose a chemical kinetics model of ALT with kinetic rates systematically derived from the biophysics of DNA diffusion and looping. The reaction system is reduced to a coagulation-fragmentation system by quasi-steady-state approximation. The detailed treatment of kinetic rates yields explicit formulas for expected size distributions of telomeres that demonstrate the key role played by the J factor, a quantitative measure of bending of polymers. The results are in agreement with experimental data and point out interesting phenomena: an appearance of very long telomeric circles if the total telomere density exceeds a critical value (excess mass) and a nonlinear response of the telomere size distributions to the amount of telomeric DNA in the system. The results can be of general importance for understanding dynamics of telomeres in telomerase-independent systems as this mode of telomere maintenance is similar to the situation in tumor cells lacking telomerase activity. Furthermore, due to its universality, the model may also serve as a prototype of an interaction between linear and circular DNA structures in various settings.

  19. Telomere attrition due to infection

    National Research Council Canada - National Science Library

    Ilmonen, Petteri; Kotrschal, Alexander; Penn, Dustin J

    2008-01-01

    Telomeres--the terminal caps of chromosomes--become shorter as individuals age, and there is much interest in determining what causes telomere attrition since this process may play a role in biological aging...

  20. CTCF driven TERRA transcription facilitates completion of telomere DNA replication.

    Science.gov (United States)

    Beishline, Kate; Vladimirova, Olga; Tutton, Stephen; Wang, Zhuo; Deng, Zhong; Lieberman, Paul M

    2017-12-13

    Telomere repeat DNA forms a nucleo-protein structure that can obstruct chromosomal DNA replication, especially under conditions of replication stress. Transcription of telomere repeats can initiate at subtelomeric CTCF-binding sites to generate telomere repeat-encoding RNA (TERRA), but the role of transcription, CTCF, and TERRA in telomere replication is not known. Here, we have used CRISPR/Cas9 gene editing to mutate CTCF-binding sites at the putative start site of TERRA transcripts for a class of subtelomeres. Under replication stress, telomeres lacking CTCF-driven TERRA exhibit sister-telomere loss and upon entry into mitosis, exhibit the formation of ultra-fine anaphase bridges and micronuclei. Importantly, these phenotypes could be rescued by the forced transcription of TERRA independent of CTCF binding. Our findings indicate that subtelomeric CTCF facilitates telomeric DNA replication by promoting TERRA transcription. Our findings also demonstrate that CTCF-driven TERRA transcription acts in cis to facilitate telomere repeat replication and chromosome stability.

  1. Quantitative theory of telomere length regulation and cellular senescence.

    Science.gov (United States)

    Rodriguez-Brenes, Ignacio A; Peskin, Charles S

    2010-03-23

    In normal somatic cells, telomere length shortens with each cell replication. This progressive shortening is associated with cellular senescence and apoptosis. Germ cells, stem cells, and the majority of cancer cells express telomerase, an enzyme that extends telomere length and, when expressed at sufficient levels, can immortalize or extend the life span of a cell line. It is believed that telomeres switch between two states: capped and uncapped. The telomere state determines its accessibility to telomerase and also the onset of senescence. One hypothesis is that the t loop, a large lariat-like structure, represents the capped state. In this paper we model a telomere state on the basis of the biophysics of t-loop formation, allowing us to develop a single mathematical model that accounts for two processes: telomere length regulation for telomerase positive cells and cellular senescence in somatic cells. The model predicts the steady-state length distribution for telomerase positive cells, describes the time evolution of telomere length, and computes the life span of a cell line on the basis of the levels of TRF2 and telomerase expression. The model reproduces a wide range of experimental behavior and fits data from immortal cell lines (HeLa S3 and 293T) and somatic cells (human diploid fibroblasts) well. We conclude that the t loop as the capped state is a quantitatively reasonable model of telomere length regulation and cellular senescence.

  2. New insights into transcription fidelity: thermal stability of non-canonical structures in template DNA regulates transcriptional arrest, pause, and slippage.

    Science.gov (United States)

    Tateishi-Karimata, Hisae; Isono, Noburu; Sugimoto, Naoki

    2014-01-01

    The thermal stability and topology of non-canonical structures of G-quadruplexes and hairpins in template DNA were investigated, and the effect of non-canonical structures on transcription fidelity was evaluated quantitatively. We designed ten template DNAs: A linear sequence that does not have significant higher-order structure, three sequences that form hairpin structures, and six sequences that form G-quadruplex structures with different stabilities. Templates with non-canonical structures induced the production of an arrested, a slipped, and a full-length transcript, whereas the linear sequence produced only a full-length transcript. The efficiency of production for run-off transcripts (full-length and slipped transcripts) from templates that formed the non-canonical structures was lower than that from the linear. G-quadruplex structures were more effective inhibitors of full-length product formation than were hairpin structure even when the stability of the G-quadruplex in an aqueous solution was the same as that of the hairpin. We considered that intra-polymerase conditions may differentially affect the stability of non-canonical structures. The values of transcription efficiencies of run-off or arrest transcripts were correlated with stabilities of non-canonical structures in the intra-polymerase condition mimicked by 20 wt% polyethylene glycol (PEG). Transcriptional arrest was induced when the stability of the G-quadruplex structure (-ΔG°37) in the presence of 20 wt% PEG was more than 8.2 kcal mol(-1). Thus, values of stability in the presence of 20 wt% PEG are an important indicator of transcription perturbation. Our results further our understanding of the impact of template structure on the transcription process and may guide logical design of transcription-regulating drugs.

  3. New insights into transcription fidelity: thermal stability of non-canonical structures in template DNA regulates transcriptional arrest, pause, and slippage.

    Directory of Open Access Journals (Sweden)

    Hisae Tateishi-Karimata

    Full Text Available The thermal stability and topology of non-canonical structures of G-quadruplexes and hairpins in template DNA were investigated, and the effect of non-canonical structures on transcription fidelity was evaluated quantitatively. We designed ten template DNAs: A linear sequence that does not have significant higher-order structure, three sequences that form hairpin structures, and six sequences that form G-quadruplex structures with different stabilities. Templates with non-canonical structures induced the production of an arrested, a slipped, and a full-length transcript, whereas the linear sequence produced only a full-length transcript. The efficiency of production for run-off transcripts (full-length and slipped transcripts from templates that formed the non-canonical structures was lower than that from the linear. G-quadruplex structures were more effective inhibitors of full-length product formation than were hairpin structure even when the stability of the G-quadruplex in an aqueous solution was the same as that of the hairpin. We considered that intra-polymerase conditions may differentially affect the stability of non-canonical structures. The values of transcription efficiencies of run-off or arrest transcripts were correlated with stabilities of non-canonical structures in the intra-polymerase condition mimicked by 20 wt% polyethylene glycol (PEG. Transcriptional arrest was induced when the stability of the G-quadruplex structure (-ΔG°37 in the presence of 20 wt% PEG was more than 8.2 kcal mol(-1. Thus, values of stability in the presence of 20 wt% PEG are an important indicator of transcription perturbation. Our results further our understanding of the impact of template structure on the transcription process and may guide logical design of transcription-regulating drugs.

  4. Does oxidative stress shorten telomeres?

    NARCIS (Netherlands)

    Boonekamp, Jelle J.; Bauch, Christina; Mulder, Ellis; Verhulst, Simon

    Oxidative stress shortens telomeres in cell culture, but whether oxidative stress explains variation in telomere shortening in vivo at physiological oxidative stress levels is not well known. We therefore tested for correlations between six oxidative stress markers and telomere attrition in nestling

  5. A loopy view of telomere evolution

    Directory of Open Access Journals (Sweden)

    Titia eDe Lange

    2015-10-01

    Full Text Available About a decade ago, I proposed that t-loops, the lariat structures adopted by many eukaryotic telomeres, could explain how the transition from circular to linear chromosomes was successfully negotiated by early eukaryotes. Here I reconsider this loopy hypothesis in the context of the idea that eukaryotes evolved through a period of genome invasion by Group II introns.

  6. Elevated TRF2 in advanced breast cancers with short telomeres.

    Science.gov (United States)

    Diehl, Malissa C; Idowu, Michael O; Kimmelshue, Katherine N; York, Timothy P; Jackson-Cook, Colleen K; Turner, Kristi C; Holt, Shawn E; Elmore, Lynne W

    2011-06-01

    Telomere repeat binding factor 2 (TRF2) binds directly to telomeres and preserves the structural integrity of chromosome ends. In vitro models suggest that expression of TRF2 protein increases during mammary cancer progression. However, a recent study has reported that TRF2 mRNA levels tend to be lower in clinical specimens of malignant breast tissue. Here, we conduct the first large-scale investigation to assess the levels and cellular localization of the TRF2 protein in normal, pre-malignant and malignant breast tissues. Breast tissue arrays, containing normal, ductal carcinoma in situ (DCIS) and invasive carcinoma specimens, were used to assess the expression and localization of TRF2 protein. Telomere lengths were semi-quantitatively measured using a pantelomeric peptide nucleic acid probe. A mixed effects modeling approach was used to assess the relationship between TRF2 expression and telomeric signal scores across disease states or clinical staging. We demonstrate that TRF2 is exclusively nuclear with a trend toward lower expression with increased malignancy. More case-to-case variability of TRF2 immunostaining intensity was noted amongst the invasive carcinomas than the other disease groups. Invasive carcinomas also displayed variable telomere lengths while telomeres in normal mammary epithelium were generally longer. Statistical analyses revealed that increased TRF2 immunostaining intensity in invasive carcinomas is associated with shorter telomeres and shorter telomeres correlate with a higher TNM stage. All immortalized and cancer cell lines within the array displayed strong, nuclear TRF2 expression. Our data indicate that elevated expression of TRF2 is not a frequent occurrence during the transformation of breast cancer cells in vivo, but higher levels of this telomere-binding protein may be important for protecting advanced cancer cells with critically short telomeres. Our findings also reinforce the concept that serially propagated cancer cells

  7. Characterization of telomeres and telomerase from the single-celled eukaryote Giardia intestinalis.

    Science.gov (United States)

    Uzlíková, Magdalena; Fulnečková, Jana; Weisz, Filip; Sýkorová, Eva; Nohýnková, Eva; Tůmová, Pavla

    2017-01-01

    The ends of linear chromosomes, telomeres, are most commonly maintained by the enzyme telomerase. Our study presents the characteristics of telomeres and telomerase from the single-celled parasitic eukaryote Giardia intestinalis. Using fluorescence in situ hybridization, we localized telomeres during all stages of the trophozoite cell cycle and demonstrated differences in the observed number of telomeric foci, indicating telomere clustering. The length of Giardia telomeres was determined in different cell lines derived from WB clinical isolate using terminal restriction fragment analysis and ranged from 0.5 to 2.5kb; moreover, a BAL-31 digestion experiment did not reveal any long interstitial telomeric sequences in the genome. Despite the absence of the specific T motif in the telomerase catalytic subunit, the presence of an active telomerase enzyme synthesising telomeric repeats in Giardia was proved by a Telomere repeat amplification protocol assay, and its localization in nuclei was determined by the expression of recombinant GiTERT. Except for the Giardia-type TAGGG telomeric repeat, Giardia telomerase was proved to synthesize in vitro also other repeat variants, TAAGG and TAAGGG. In summary, despite its unusual characteristics, including a structurally divergent but active telomerase, unique terminal sequences and relatively short telomeres, the present data support the view that the chromosomal termini in Giardia are maintained in a conservative manner that is common to other eukaryotes. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Break-induced telomere synthesis underlies alternative telomere maintenance.

    Science.gov (United States)

    Dilley, Robert L; Verma, Priyanka; Cho, Nam Woo; Winters, Harrison D; Wondisford, Anne R; Greenberg, Roger A

    2016-11-03

    Homology-directed DNA repair is essential for genome maintenance through templated DNA synthesis. Alternative lengthening of telomeres (ALT) necessitates homology-directed DNA repair to maintain telomeres in about 10-15% of human cancers. How DNA damage induces assembly and execution of a DNA replication complex (break-induced replisome) at telomeres or elsewhere in the mammalian genome is poorly understood. Here we define break-induced telomere synthesis and demonstrate that it utilizes a specialized replisome, which underlies ALT telomere maintenance. DNA double-strand breaks enact nascent telomere synthesis by long-tract unidirectional replication. Proliferating cell nuclear antigen (PCNA) loading by replication factor C (RFC) acts as the initial sensor of telomere damage to establish predominance of DNA polymerase δ (Pol δ) through its POLD3 subunit. Break-induced telomere synthesis requires the RFC-PCNA-Pol δ axis, but is independent of other canonical replisome components, ATM and ATR, or the homologous recombination protein Rad51. Thus, the inception of telomere damage recognition by the break-induced replisome orchestrates homology-directed telomere maintenance.

  9. Post-translational modifications of TRF1 and TRF2 and their roles in telomere maintenance.

    Science.gov (United States)

    Walker, John R; Zhu, Xu-Dong

    2012-06-01

    Telomeres, heterochromatic structures, found at the ends of linear eukaryotic chromosomes, function to protect natural chromosome ends from nucleolytic attack. Human telomeric DNA is bound by a telomere-specific six-subunit protein complex, termed shelterin/telosome. The shelterin subunits TRF1 and TRF2 bind in a sequence-specific manner to double-stranded telomeric DNA, providing a vital platform for recruitment of additional shelterin proteins as well as non-shelterin factors crucial for the maintenance of telomere length and structure. Both TRF1 and TRF2 are engaged in multiple roles at telomeres including telomere protection, telomere replication, sister telomere resolution and telomere length maintenance. Regulation of TRF1 and TRF2 in these various processes is controlled by post-translational modifications, at times in a cell-cycle-dependent manner, affecting key functions such as DNA binding, dimerization, localization, degradation and interactions with other proteins. Here we review the post-translational modifications of TRF1 and TRF2 and discuss the mechanisms by which these modifications contribute to the function of these two proteins. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  10. Telomere dysfunction and chromosome instability

    Energy Technology Data Exchange (ETDEWEB)

    Murnane, John P., E-mail: jmurnane@radonc.ucsf.edu [Department of Radiation Oncology, University of California San Francisco, 2340 Sutter Street, San Francisco, CA 94143-1331 (United States)

    2012-02-01

    The ends of chromosomes are composed of a short repeat sequence and associated proteins that together form a cap, called a telomere, that keeps the ends from appearing as double-strand breaks (DSBs) and prevents chromosome fusion. The loss of telomeric repeat sequences or deficiencies in telomeric proteins can result in chromosome fusion and lead to chromosome instability. The similarity between chromosome rearrangements resulting from telomere loss and those found in cancer cells implicates telomere loss as an important mechanism for the chromosome instability contributing to human cancer. Telomere loss in cancer cells can occur through gradual shortening due to insufficient telomerase, the protein that maintains telomeres. However, cancer cells often have a high rate of spontaneous telomere loss despite the expression of telomerase, which has been proposed to result from a combination of oncogene-mediated replication stress and a deficiency in DSB repair in telomeric regions. Chromosome fusion in mammalian cells primarily involves nonhomologous end joining (NHEJ), which is the major form of DSB repair. Chromosome fusion initiates chromosome instability involving breakage-fusion-bridge (B/F/B) cycles, in which dicentric chromosomes form bridges and break as the cell attempts to divide, repeating the process in subsequent cell cycles. Fusion between sister chromatids results in large inverted repeats on the end of the chromosome, which amplify further following additional B/F/B cycles. B/F/B cycles continue until the chromosome acquires a new telomere, most often by translocation of the end of another chromosome. The instability is not confined to a chromosome that loses its telomere, because the instability is transferred to the chromosome donating a translocation. Moreover, the amplified regions are unstable and form extrachromosomal DNA that can reintegrate at new locations. Knowledge concerning the factors promoting telomere loss and its consequences is

  11. Telomere elongation chooses TERRA ALTernatives.

    Science.gov (United States)

    Arora, Rajika; Azzalin, Claus M

    2015-01-01

    Alternative Lengthening of Telomeres (ALT) mechanisms allow telomerase-negative immortal cells to buffer replicative telomere shortening. ALT is naturally active in a number of human cancers and might be selected upon telomerase inactivation. ALT is thought to operate through homologous recombination (HR) occurring between telomeric repeats from independent chromosome ends. Indeed, suppression of a number of HR factors impairs ALT cell proliferation. Yet, how HR is initiated at ALT telomeres remains elusive. Mounting evidence suggests that the long noncoding telomeric RNA TERRA renders ALT telomeres recombinogenic by forming RNA:DNA hybrids with the telomeric C-rich strand. TERRA and telomeric hybrids act in concert with a number of other factors, including the RNA endoribonuclease RNaseH1 and the single stranded DNA binding protein RPA. The functional interaction network built upon these different players seems indispensable for ALT telomere maintenance, and digging into the molecular details of this previously unappreciated network might open the way to novel avenues for cancer treatments.

  12. Glyceraldehyde 3-phosphate dehydrogenase-telomere association correlates with redox status in Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Ricardo Pariona-Llanos

    Full Text Available Glyceraldehyde 3-phosphate dehydrogenase (GAPDH is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH. We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA.

  13. Telomere-Centromere-Driven Genomic Instability Contributes to Karyotype Evolution in a Mouse Model of Melanoma

    Directory of Open Access Journals (Sweden)

    Amanda Gonçalves dos Santos Silva

    2010-01-01

    Full Text Available Aneuploidy and chromosomal instability (CIN are hallmarks of most solid tumors. These alterations may result from inaccurate chromosomal segregation during mitosis, which can occur through several mechanisms including defective telomere metabolism, centrosome amplification, dysfunctional centromeres, and/or defective spindle checkpoint control. In this work, we used an in vitro murine melanoma model that uses a cellular adhesion blockade as a transforming factor to characterize telomeric and centromeric alterations that accompany melanocyte transformation. To study the timing of the occurrence of telomere shortening in this transformation model, we analyzed the profile of telomere length by quantitative fluorescent in situ hybridization and found that telomere length significantly decreased as additional rounds of cell adhesion blockages were performed. Together with it, an increase in telomere-free ends and complex karyotypic aberrations were also found, which include Robertsonian fusions in 100% of metaphases of the metastatic melanoma cells. These findings are in agreement with the idea that telomere length abnormalities seem to be one of the earliest genetic alterations acquired in the multistep process of malignant transformation and that telomere abnormalities result in telomere aggregation, breakage-bridge-fusion cycles, and CIN. Another remarkable feature of this model is the abundance of centromeric instability manifested as centromere fragments and centromeric fusions. Taken together, our results illustrate for this melanoma model CIN with a structural signature of centromere breakage and telomeric loss.

  14. TERRA-Reinforced Association of LSD1 with MRE11 Promotes Processing of Uncapped Telomeres

    Directory of Open Access Journals (Sweden)

    Antonio Porro

    2014-02-01

    Full Text Available Telomeres protect chromosome ends from being recognized as sites of DNA damage. Upon telomere shortening or telomere uncapping induced by loss of telomeric repeat-binding factor 2 (TRF2, telomeres elicit a DNA-damage response leading to cellular senescence. Here, we show that following TRF2 depletion, the levels of the long noncoding RNA TERRA increase and LSD1, which binds TERRA, is recruited to telomeres. At uncapped telomeres, LSD1 associates with MRE11, one of the nucleases implicated in the processing of 3′ telomeric G overhangs, and we show that LSD1 is required for efficient removal of these structures. The LSD1-MRE11 interaction is reinforced in vivo following TERRA upregulation in TRF2-deficient cells and in vitro by TERRA-mimicking RNA oligonucleotides. Furthermore, LSD1 enhances the nuclease activity of MRE11 in vitro. Our data indicate that recruitment of LSD1 to deprotected telomeres requires MRE11 and is promoted by TERRA. LSD1 stimulates MRE11 catalytic activity and nucleolytic processing of uncapped telomeres.

  15. TERRA-reinforced association of LSD1 with MRE11 promotes processing of uncapped telomeres.

    Science.gov (United States)

    Porro, Antonio; Feuerhahn, Sascha; Lingner, Joachim

    2014-02-27

    Telomeres protect chromosome ends from being recognized as sites of DNA damage. Upon telomere shortening or telomere uncapping induced by loss of telomeric repeat-binding factor 2 (TRF2), telomeres elicit a DNA-damage response leading to cellular senescence. Here, we show that following TRF2 depletion, the levels of the long noncoding RNA TERRA increase and LSD1, which binds TERRA, is recruited to telomeres. At uncapped telomeres, LSD1 associates with MRE11, one of the nucleases implicated in the processing of 3' telomeric G overhangs, and we show that LSD1 is required for efficient removal of these structures. The LSD1-MRE11 interaction is reinforced in vivo following TERRA upregulation in TRF2-deficient cells and in vitro by TERRA-mimicking RNA oligonucleotides. Furthermore, LSD1 enhances the nuclease activity of MRE11 in vitro. Our data indicate that recruitment of LSD1 to deprotected telomeres requires MRE11 and is promoted by TERRA. LSD1 stimulates MRE11 catalytic activity and nucleolytic processing of uncapped telomeres. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  16. The effects of oxidative stress on telomeres and cell life span

    OpenAIRE

    Pańczyszyn, Anna; Boniewska-Bernacka, Ewa

    2016-01-01

    Oxidative stress is associated with excessive amounts of reactive oxygen species (ROSOS) in the body. The sources of ROSOS constitute the respiratory chain, immune system cells and external factors, e.g. smoking. ROSOS may cause damage and faster shortening of nucleoprotein structures called telomeres, which protect chromosome ends. The consequence of faster shortening of telomeres is aging and death of cells. The aim of this paper was to present the impact of ROSOS on the rate of telomere sh...

  17. No Association between Mean Telomere Length and Life Stress Observed in a 30 Year Birth Cohort

    OpenAIRE

    Sarah Jodczyk; Fergusson, David M; John Horwood, L.; Pearson, John F; Martin A Kennedy

    2014-01-01

    Telomeres are specialised structures that cap the ends of chromosomes. They shorten with each cell division and have been proposed as a marker of cellular aging. Previous studies suggest that early life stressors increase the rate of telomere shortening with potential impact on disease states and mortality later in life. This study examined the associations between telomere length and exposure to a number of stressors that arise during development from the antenatal/perinatal period through t...

  18. Predictors of telomere content in dragon lizards

    Science.gov (United States)

    Ballen, Cissy; Healey, Mo; Wilson, Mark; Tobler, Michael; Olsson, Mats

    2012-08-01

    Telomeres shorten as a consequence of DNA replication, in particular in cells with low production of telomerase and perhaps in response to physiological stress from exposure to reactive oxygen species, such as superoxide. This process of telomere attrition is countered by innate antioxidation, such as via the production of superoxide dismutase. We studied the inheritance of telomere length in the Australian painted dragon lizard ( Ctenophorus pictus) and the extent to which telomere length covaries with mass-corrected maternal reproductive investment, which reflects the level of circulating yolk precursor and antioxidant, vitellogenin. Our predictors of offspring telomere length explained 72 % of telomere variation (including interstitial telomeres if such are present). Maternal telomere length and reproductive investment were positively influencing offspring telomere length in our analyses, whereas flow cytometry-estimated superoxide level was negatively impacting offspring telomere length. We suggest that the effects of superoxide on hatchling telomere shortening may be partly balanced by transgenerational effects of vitellogenin antioxidation.

  19. TERRA, hnRNP A1, and DNA-PKcs Interactions at Human Telomeres.

    Science.gov (United States)

    Le, Phuong N; Maranon, David G; Altina, Noelia H; Battaglia, Christine L R; Bailey, Susan M

    2013-01-01

    Maintenance of telomeres, repetitive elements at eukaryotic chromosomal termini, and the end-capping structure and function they provide, are imperative for preserving genome integrity and stability. The discovery that telomeres are transcribed into telomere repeat containing RNA (TERRA) has revolutionized our view of this repetitive, rather unappreciated region of the genome. We have previously shown that the non-homologous end-joining, shelterin associated DNA dependent protein kinase catalytic subunit (DNA-PKcs) participates in mammalian telomeric end-capping, exclusively at telomeres created by leading-strand synthesis. Here, we explore potential roles of DNA-PKcs and its phosphorylation target heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in the localization of TERRA at human telomeres. Evaluation of co-localized foci utilizing RNA-FISH and three-dimensional (3D) reconstruction strategies provided evidence that both inhibition of DNA-PKcs kinase activity and siRNA depletion of hnRNP A1 result in accumulation of TERRA at individual telomeres; depletion of hnRNP A1 also resulted in increased frequencies of fragile telomeres. These observations are consistent with previous demonstrations that decreased levels of the nonsense RNA-mediated decay factors SMG1 and UPF1 increase TERRA at telomeres and interfere with replication of leading-strand telomeres. We propose that hTR mediated stimulation of DNA-PKcs and subsequent phosphorylation of hnRNP A1 influences the cell cycle dependent distribution of TERRA at telomeres by contributing to the removal of TERRA from telomeres, an action important for progression of S-phase, and thereby facilitating efficient telomere replication and end-capping.

  20. Age-dependence of relative telomere length profiles during spermatogenesis in man

    DEFF Research Database (Denmark)

    Jørgensen, Pernille Bach; Fedder, Jens; Koelvraa, Steen

    2013-01-01

    Telomeres, the protective structures at the outmost ends of chromosomes, shorten in all somatic cells with each cell-division and by cumulative oxidative damage. To counteract that these shortened telomeres are passed on to offspring, the telomeres are elongated by the enzyme, telomerase, during...... human spermatogenesis. A few groups have tried to elucidate this process by measuring telomerase activity in the various cell-types during spermatogenesis, but until now, no one has ever measured telomere length (TL) during these different stages in humans. Some groups have measured TL in spermatozoa...... by telomere QFISH. Our data revealed no difference in the TL profile during spermatogenesis between younger and older men. All men had a similar profile which strongly resembled the telomerase expression profile found by others. This indicates that the longer telomeres in older men are not caused by a wider...

  1. Replication stress as a source of telomere recombination during replicative senescence in Saccharomyces cerevisiae.

    Science.gov (United States)

    Simon, Marie-Noëlle; Churikov, Dmitri; Géli, Vincent

    2016-11-01

    Replicative senescence is triggered by short unprotected telomeres that arise in the absence of telomerase. In addition, telomeres are known as difficult regions to replicate due to their repetitive G-rich sequence prone to secondary structures and tightly bound non-histone proteins. Here we review accumulating evidence that telomerase inactivation in yeast immediately unmasks the problems associated with replication stress at telomeres. Early after telomerase inactivation, yeast cells undergo successive rounds of stochastic DNA damages and become dependent on recombination for viability long before the bulk of telomeres are getting critically short. The switch from telomerase to recombination to repair replication stress-induced damage at telomeres creates telomere instability, which may drive further genomic alterations and prepare the ground for telomerase-independent immortalization observed in yeast survivors and in 15% of human cancer. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. The telomere binding protein TRF2 induces chromatin compaction.

    Science.gov (United States)

    Baker, Asmaa M; Fu, Qiang; Hayward, William; Victoria, Samuel; Pedroso, Ilene M; Lindsay, Stuart M; Fletcher, Terace M

    2011-04-19

    Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

  3. p300-mediated acetylation of TRF2 is required for maintaining functional telomeres.

    Science.gov (United States)

    Her, Yoon Ra; Chung, In Kwon

    2013-02-01

    The human telomeric protein TRF2 is required to protect chromosome ends by facilitating their organization into the protective capping structure. Post-translational modifications of TRF2 such as phosphorylation, ubiquitination, SUMOylation, methylation and poly(ADP-ribosyl)ation have been shown to play important roles in telomere function. Here we show that TRF2 specifically interacts with the histone acetyltransferase p300, and that p300 acetylates the lysine residue at position 293 of TRF2. We also report that p300-mediated acetylation stabilizes the TRF2 protein by inhibiting its ubiquitin-dependent proteolysis and is required for efficient telomere binding of TRF2. Furthermore, overexpression of the acetylation-deficient mutant, K293R, induces DNA-damage response foci at telomeres, thereby leading to induction of impaired cell growth, cellular senescence and altered cell cycle distribution. A small but significant number of metaphase chromosomes show no telomeric signals at chromatid ends, suggesting an aberrant telomere structure. These findings demonstrate that acetylation of TRF2 by p300 plays a crucial role in the maintenance of functional telomeres as well as in the regulation of the telomere-associated DNA-damage response, thus providing a new route for modulating telomere protection function.

  4. E-type cyclins modulate telomere integrity in mammalian male meiosis.

    Science.gov (United States)

    Manterola, Marcia; Sicinski, Piotr; Wolgemuth, Debra J

    2016-06-01

    We have shown that E-type cyclins are key regulators of mammalian male meiosis. Depletion of cyclin E2 reduced fertility in male mice due to meiotic defects, involving abnormal pairing and synapsis, unrepaired DNA, and loss of telomere structure. These defects were exacerbated by additional loss of cyclin E1, and complete absence of both E-type cyclins produces a meiotic catastrophe. Here, we investigated the involvement of E-type cyclins in maintaining telomere integrity in male meiosis. Spermatocytes lacking cyclin E2 and one E1 allele (E1+/-E2-/-) displayed a high rate of telomere abnormalities but can progress to pachytene and diplotene stages. We show that their telomeres exhibited an aberrant DNA damage repair response during pachynema and that the shelterin complex proteins TRF2 and RAP2 were significantly decreased in the proximal telomeres. Moreover, the insufficient level of these proteins correlated with an increase of γ-H2AX foci in the affected telomeres and resulted in telomere associations involving TRF1 and telomere detachment in later prophase-I stages. These results suggest that E-type cyclins are key modulators of telomere integrity during meiosis by, at least in part, maintaining the balance of shelterin complex proteins, and uncover a novel role of E-type cyclins in regulating chromosome structure during male meiosis.

  5. Curcusone C induces telomeric DNA-damage response in cancer cells through inhibition of telomeric repeat factor 2.

    Science.gov (United States)

    Wang, Mingxue; Cao, Jiaojiao; Zhu, Jian-Yong; Qiu, Jun; Zhang, Yan; Shu, Bing; Ou, Tian-Miao; Tan, Jia-Heng; Gu, Lian-Quan; Huang, Zhi-Shu; Yin, Sheng; Li, Ding

    2017-11-01

    Telomeric repeat factor 2 (known as TRF2 or TERF2) is a key component of telomere protection protein complex named as Shelterin. TRF2 helps the folding of telomere to form T-loop structure and the suppression of ATM-dependent DNA damage response activation. TRF2 has been recognized as a potentially new therapeutic target for cancer treatment. In our routine screening of small molecule libraries, we found that Curcusone C had significant effect in disrupting the binding between TRF2 and telomeric DNA, with potent antitumor activity against cancer cells. Our result showed that Curcusone C could bind with TRF2 without binding interaction with TRF1 (telomeric repeat factor 1) although these two proteins share high sequence homology, indicating that their binding conformations and biological functions in telomere could be different. Our mechanistic studies showed that Curcusone C bound with TRF2 possibly through its DNA binding site causing blockage of its interaction with telomeric DNA. Further in cellular studies indicated that the interaction of TRF2 with Curcusone C could activate DNA-damage response, inhibit tumor cell proliferation, and cause cell cycle arrest, resulting in tumor cell apoptosis. Our studies showed that Curcusone C could become a promising lead compound for further development for cancer treatment. Here, TRF2 was firstly identified as a target of Curcusone C. It is likely that the anti-cancer activity of some other terpenes and terpenoids are related with their possible effect for telomere protection proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. The Analysis of Pendolino (peo Mutants Reveals Differences in the Fusigenic Potential among Drosophila Telomeres.

    Directory of Open Access Journals (Sweden)

    Giovanni Cenci

    2015-06-01

    Full Text Available Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo cause telomeric fusions (TFs. The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres. Alternatively, it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping.

  7. The Analysis of Pendolino (peo) Mutants Reveals Differences in the Fusigenic Potential among Drosophila Telomeres.

    Science.gov (United States)

    Cenci, Giovanni; Ciapponi, Laura; Marzullo, Marta; Raffa, Grazia D; Morciano, Patrizia; Raimondo, Domenico; Burla, Romina; Saggio, Isabella; Gatti, Maurizio

    2015-06-01

    Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT) rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver) that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo) cause telomeric fusions (TFs). The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres. Alternatively, it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping.

  8. Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans.

    Science.gov (United States)

    Seo, Beomseok; Lee, Junho

    2016-08-04

    Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)(1). Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT(2). Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

  9. TRF1 and TRF2 binding to telomeres is modulated by nucleosomal organization.

    Science.gov (United States)

    Galati, Alessandra; Micheli, Emanuela; Alicata, Claudia; Ingegnere, Tiziano; Cicconi, Alessandro; Pusch, Miriam Caroline; Giraud-Panis, Marie-Josèphe; Gilson, Eric; Cacchione, Stefano

    2015-07-13

    The ends of eukaryotic chromosomes need to be protected from the activation of a DNA damage response that leads the cell to replicative senescence or apoptosis. In mammals, protection is accomplished by a six-factor complex named shelterin, which organizes the terminal TTAGGG repeats in a still ill-defined structure, the telomere. The stable interaction of shelterin with telomeres mainly depends on the binding of two of its components, TRF1 and TRF2, to double-stranded telomeric repeats. Tethering of TRF proteins to telomeres occurs in a chromatin environment characterized by a very compact nucleosomal organization. In this work we show that binding of TRF1 and TRF2 to telomeric sequences is modulated by the histone octamer. By means of in vitro models, we found that TRF2 binding is strongly hampered by the presence of telomeric nucleosomes, whereas TRF1 binds efficiently to telomeric DNA in a nucleosomal context and is able to remodel telomeric nucleosomal arrays. Our results indicate that the different behavior of TRF proteins partly depends on the interaction with histone tails of their divergent N-terminal domains. We propose that the interplay between the histone octamer and TRF proteins plays a role in the steps leading to telomere deprotection. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. A sharp Pif1-dependent threshold separates DNA double-strand breaks from critically short telomeres

    NARCIS (Netherlands)

    Strecker, Jonathan; Stinus, Sonia; Caballero, Mariana Pliego; Szilard, Rachel K.; Chang, Michael; Durocher, Daniel

    2017-01-01

    DNA double-strand breaks (DSBs) and short telomeres are structurally similar, yet have diametrically opposed fates. Cells must repair DSBs while blocking the action of telomerase on these ends. Short telomeres must avoid recognition by the DNA damage response while promoting telomerase recruitment.

  11. Controlled light exposure microscopy reveals dynamic telomere microterritories throughout the cell cycle

    NARCIS (Netherlands)

    de Vos, W.H.; Hoebe, R.A.; Joss, G.H.; Haffmans, W.; Baatout, S.; van Oostveldt, P.; Manders, E.M.M.

    2009-01-01

    Telomeres are complex end structures that confer functional integrity and positional stability to human chromosomes. Despite their critical importance, there is no clear view on telomere organization in cycling human cells and their dynamic behavior throughout the cell cycle. We investigated

  12. TRF1 and TRF2 differentially modulate Rad51-mediated telomeric and nontelomeric displacement loop formation in vitro.

    Science.gov (United States)

    Bower, Brian D; Griffith, Jack D

    2014-09-02

    A growing body of literature suggests that the homologous recombination/repair (HR) pathway cooperates with components of the shelterin complex to promote both telomere maintenance and nontelomeric HR. This may be due to the ability of both HR and shelterin proteins to promote strand invasion, wherein a single-stranded DNA (ssDNA) substrate base pairs with a homologous double-stranded DNA (dsDNA) template displacing a loop of ssDNA (D-loop). Rad51 recombinase catalyzes D-loop formation during HR, and telomere repeat binding factor 2 (TRF2) catalyzes the formation of a telomeric D-loop that stabilizes a looped structure in telomeric DNA (t-loop) that may facilitate telomere protection. We have characterized this functional interaction in vitro using a fluorescent D-loop assay measuring the incorporation of Cy3-labeled 90-nucleotide telomeric and nontelomeric substrates into telomeric and nontelomeric plasmid templates. We report that preincubation of a telomeric template with TRF2 inhibits the ability of Rad51 to promote telomeric D-loop formation upon preincubation with a telomeric substrate. This suggests Rad51 does not facilitate t-loop formation and suggests a mechanism whereby TRF2 can inhibit HR at telomeres. We also report a TRF2 mutant lacking the dsDNA binding domain promotes Rad51-mediated nontelomeric D-loop formation, possibly explaining how TRF2 promotes nontelomeric HR. Finally, we report telomere repeat binding factor 1 (TRF1) promotes Rad51-mediated telomeric D-loop formation, which may facilitate HR-mediated replication fork restart and explain why TRF1 is required for efficient telomere replication.

  13. Inheritance of telomere length in a bird.

    Directory of Open Access Journals (Sweden)

    Thorsten Horn

    Full Text Available Telomere dynamics are intensively studied in human ageing research and epidemiology, with many correlations reported between telomere length and age-related diseases, cancer and death. While telomere length is influenced by environmental factors there is also good evidence for a strong heritable component. In human, the mode of telomere length inheritance appears to be paternal and telomere length differs between sexes, with females having longer telomeres than males. Genetic factors, e.g. sex chromosomal inactivation, and non-genetic factors, e.g. antioxidant properties of oestrogen, have been suggested as possible explanations for these sex-specific telomere inheritance and telomere length differences. To test the influence of sex chromosomes on telomere length, we investigated inheritance and sex-specificity of telomere length in a bird species, the kakapo (Strigops habroptilus, in which females are the heterogametic sex (ZW and males are the homogametic (ZZ sex. We found that, contrary to findings in humans, telomere length was maternally inherited and also longer in males. These results argue against an effect of sex hormones on telomere length and suggest that factors associated with heterogamy may play a role in telomere inheritance and sex-specific differences in telomere length.

  14. Zebrafish as a model system to study the physiological function of telomeric protein TPP1.

    Directory of Open Access Journals (Sweden)

    Yiying Xie

    Full Text Available Telomeres are specialized chromatin structures at the end of chromosomes. Telomere dysfunction can lead to chromosomal abnormalities, DNA damage responses, and even cancer. In mammalian cells, a six-protein complex (telosome/shelterin is assembled on the telomeres through the interactions between various domain structures of the six telomere proteins (POT1, TPP1, TIN2, TRF1, TRF2 and RAP1, and functions in telomere maintenance and protection. Within the telosome, TPP1 interacts directly with POT1 and TIN2 and help to mediate telosome assembly. Mechanisms of telomere regulation have been extensively studied in a variety of model organisms. For example, the physiological roles of telomere-targeted proteins have been assessed in mice through homozygous inactivation. In these cases, early embryonic lethality has prevented further studies of these proteins in embryogenesis and development. As a model system, zebrafish offers unique advantages such as genetic similarities with human, rapid developmental cycles, and ease of manipulation of its embryos. In this report, we detailed the identification of zebrafish homologues of TPP1, POT1, and TIN2, and showed that the domain structures and interactions of these telosome components appeared intact in zebrafish. Importantly, knocking down TPP1 led to multiple abnormalities in zebrafish embryogenesis, including neural death, heart malformation, and caudal defect. And these embryos displayed extensive apoptosis. These results underline the importance of TPP1 in zebrafish embryogenesis, and highlight the feasibility and advantages of investigating the signaling pathways and physiological function of telomere proteins in zebrafish.

  15. Dissecting the telomere-inner nuclear membrane interface formed in meiosis.

    Science.gov (United States)

    Pendlebury, Devon F; Fujiwara, Yasuhiro; Tesmer, Valerie M; Smith, Eric M; Shibuya, Hiroki; Watanabe, Yoshinori; Nandakumar, Jayakrishnan

    2017-12-01

    Tethering telomeres to the inner nuclear membrane (INM) allows homologous chromosome pairing during meiosis. The meiosis-specific protein TERB1 binds the telomeric protein TRF1 to establish telomere-INM connectivity and is essential for mouse fertility. Here we solve the structure of the human TRF1-TERB1 interface to reveal the structural basis for telomere-INM linkage. Disruption of this interface abrogates binding and compromises telomere-INM attachment in mice. An embedded CDK-phosphorylation site within the TRF1-binding region of TERB1 provides a mechanism for cap exchange, a late-pachytene phenomenon involving the dissociation of the TRF1-TERB1 complex. Indeed, further strengthening this interaction interferes with cap exchange. Finally, our biochemical analysis implicates distinct complexes for telomere-INM tethering and chromosome-end protection during meiosis. Our studies unravel the structure, stoichiometry, and physiological implications underlying telomere-INM tethering, thereby providing unprecedented insights into the unique function of telomeres in meiosis.

  16. Long Telomeres Bypass the Requirement for Telomere Maintenance in Human Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Michael A.S. Taboski

    2012-02-01

    Full Text Available Despite the importance of telomere maintenance in cancer cell survival via the elongation of telomeres by telomerase reverse transcriptase (TERT or alternative lengthening of telomeres (ALT, it had not been tested directly whether telomere maintenance is dispensable for human tumorigenesis. We engineered human tumor cells containing loxP-flanked hTERT to enable extensive telomere elongation prior to complete hTERT excision. Despite unabated telomere erosion, hTERT-excised cells formed tumors in mice and proliferated in vitro for up to 1 year. Telomerase reactivation or ALT was not observed, and the eventual loss of telomeric signal coincided with loss of tumorigenic potential and cell viability. Crisis was averted via the reintroduction of active but not inactive hTERT. Thus, telomere maintenance is dispensable for human tumorigenesis when telomere reserves are long. Yet, despite telomere instability and the presence of oncogenic RAS, human tumors remain susceptible to crisis induced by critically short telomeres.

  17. Mathematical model of alternative mechanism of telomere length maintenance

    CERN Document Server

    Kollár, Richard; Nosek, Jozef; Tomaska, Lubomir

    2014-01-01

    Biopolymer length regulation is a complex process that involves a large number of subprocesses acting simultaneously across multiple spatial and temporal scales. An illustrative example important for genomic stability is the length regulation of telomeres---nucleo-protein structures at the ends of linear chromosomes. Maintenance of telomeres is often facilitated by the enzyme telomerase but, particularly in telomerase-free systems, the maintenance of chromosomal termini depends on alternative lengthening of telomeres (ALT) mechanisms mediated by recombination. Various linear and circular DNA structures were identified to participate in ALT, however, dynamics of the whole process is still poorly understood. We propose a chemical kinetics model of ALT with kinetic rates systematically derived from the biophysics of DNA diffusion and looping. The reaction system is reduced to a coagulation-fragmentation system by quasi-steady state approximation. The detailed treatment of kinetic rates yields explicit formulae f...

  18. The Effect of INA [(R)-1-O-(1-Pyrenylmethyl)Glycerol] Insertions on the Structure and Biological Activity of a G-Quadruplex from a Critical Kras G-Rich Sequence

    DEFF Research Database (Denmark)

    Cogoi, Susanna; Paramasivan, Manikandan; Xodo, Luigi E.

    2007-01-01

    Quadruplex-forming oligonucleotides containing INA [(R)-1-O-(1-pyrenylmethyl)glycerol] insertions have been designed and studied for their capacity to inhibit the expression of the KRAS oncogene in pancreatic adenocarcinoma cells. It is found that INA can influence the folding topology of the G...

  19. DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes, a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase

    NARCIS (Netherlands)

    Bharti, S.K.; Sommers, J.A.; Zhou, J.; Kaplan, D.L.; Spelbrink, J.N.; Mergny, J.L.; Brosh, R.M., Jr.

    2014-01-01

    Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective

  20. Using centromere mediated genome elimination to elucidate the functional redundancy of candidate telomere binding proteins in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Karel eRiha

    2016-01-01

    Full Text Available Proteins that bind to telomeric DNA form the key structural and functional constituents of telomeres. While telomere binding proteins have been described in the majority of organisms, their identity in plants remains unknown. Several protein families containing a telomere binding motif known as the telobox have been previously described in Arabidopsis thaliana. Nonetheless, functional evidence for their involvement at telomeres has not been obtained, likely due to functional redundancy. Here we performed genetic analysis on the TRF-like family consisting of six proteins (TRB1, TRP1, TRFL1, TRFL2, TRFL4 and TRF9 which have previously shown to bind telomeric DNA in vitro. We used haploid genetics to create multiple knock-out plants deficient for all six proteins of this gene family. These plants did not exhibit changes in telomere length, or phenotypes associated with telomere dysfunction. This data demonstrates that this telobox protein family is not involved in telomere maintenance in Arabidopsis. Phylogenetic analysis in major plant lineages revealed early diversification of telobox proteins families indicating that telomere function may be associated with other telobox proteins.

  1. DNA-PKcs-interacting protein KIP binding to TRF2 is required for the maintenance of functional telomeres.

    Science.gov (United States)

    Khadka, Prabhat; Lee, Ji Hoon; Baek, Seung Han; Oh, Sue Young; Chung, In Kwon

    2014-10-01

    Human telomeres associate with shelterin, a six-protein complex that protects chromosome ends from being recognized as sites of DNA damage. The shelterin subunit TRF2 (telomeric repeat-binding factor 2) protects telomeres by facilitating their organization into the protective capping structure. We have reported previously that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit)-interacting protein KIP associates with telomerase through an interaction with hTERT (human telomerase reverse transcriptase). In the present study, we identify KIP as a novel interacting partner of TRF2. KIP is able to interact with both TRF2 and DNA-PKcs at telomeres. Because KIP is required for the association between TRF2 and DNA-PKcs, the interplay of these three proteins may provide a mechanism for the recruitment of DNA-PKcs to telomeres. We also show that KIP binding to TRF2 enhances the telomere-binding activity of TRF2, suggesting that KIP acts as a positive regulator of TRF2 function. Furthermore, depletion of KIP induces DNA-damage response foci at telomeres, thereby leading to induction of growth arrest, cellular senescence and altered cell cycle distribution. Collectively, our findings suggest that KIP, in addition to its association with catalytically active telomerase, plays important roles in the maintenance of functional telomeres and the regulation of telomere-associated DNA-damage response. Thus KIP represents a new pathway for modulating telomerase and telomere function in cancer.

  2. Engineered telomere degradation models dyskeratosis congenita

    National Research Council Canada - National Science Library

    Hockemeyer, Dirk; Palm, Wilhelm; Wang, Richard C; Couto, Suzana S; de Lange, Titia

    2008-01-01

    .... However, mice with extensively shortened telomeres due to telomerase deficiency do not develop the characteristics of DC, raising questions about the etiology of DC and/or mouse models for human telomere dysfunction...

  3. Processing of semen by density gradient centrifugation selects spermatozoa with longer telomeres for assisted reproduction techniques.

    Science.gov (United States)

    Yang, Qingling; Zhang, Nan; Zhao, Feifei; Zhao, Wanli; Dai, Shanjun; Liu, Jinhao; Bukhari, Ihtisham; Xin, Hang; Niu, Wenbing; Sun, Yingpu

    2015-07-01

    The ends of eukaryotic chromosomes contain specialized chromatin structures called telomeres, the length of which plays a key role in early human embryonic development. Although the effect of sperm preparation techniques on major sperm characteristics, such as concentration, motility and morphology have been previously documented, the possible status of telomere length and its relation with sperm preparation techniques is not well-known for humans. The aim of this study was to investigate the role of density gradient centrifugation in the selection of spermatozoa with longer telomeres for use in assisted reproduction techniques in 105 samples before and after sperm processing. After density gradient centrifugation, the average telomere length of the sperm was significantly longer (6.51 ± 2.54 versus 5.16 ± 2.29, P technique for selection of sperm with longer telomeres. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  4. TRF2 recruits RTEL1 to telomeres in S phase to promote t-loop unwinding.

    Science.gov (United States)

    Sarek, Grzegorz; Vannier, Jean-Baptiste; Panier, Stephanie; Petrini, John H J; Boulton, Simon J

    2015-02-19

    The helicase RTEL1 promotes t-loop unwinding and suppresses telomere fragility to maintain the integrity of vertebrate telomeres. An interaction between RTEL1 and PCNA is important to prevent telomere fragility, but how RTEL1 engages with the telomere to promote t-loop unwinding is unclear. Here, we establish that the shelterin protein TRF2 recruits RTEL1 to telomeres in S phase, which is required to prevent catastrophic t-loop processing by structure-specific nucleases. We show that the TRF2-RTEL1 interaction is mediated by a metal-coordinating C4C4 motif in RTEL1, which is compromised by the Hoyeraal-Hreidarsson syndrome (HHS) mutation, RTEL1(R1264H). Conversely, we define a TRF2(I124D) substitution mutation within the TRFH domain of TRF2, which eliminates RTEL1 binding and phenocopies the RTEL1(R1264H) mutation, giving rise to aberrant t-loop excision, telomere length heterogeneity, and loss of the telomere as a circle. These results implicate TRF2 in the recruitment of RTEL1 to facilitate t-loop disassembly at telomeres in S phase. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Downregulation of Histone Methyltransferase Genes SUV39H1 and SUV39H2 Increases Telomere Length in Embryonic Stem-like Cells and Embryonic Fibroblasts in Pigs

    OpenAIRE

    DANG-NGUYEN, Thanh Quang; Haraguchi, Seiki; FURUSAWA, Tadashi; SOMFAI, Tamas; Kaneda, Masahiro; Watanabe, Shinya; AKAGI, Satoshi; KIKUCHI, Kazuhiro; Tajima, Atsushi; Nagai, Takashi

    2012-01-01

    Abstract Telomere is a nucleoprotein structure at the ends of chromosomes that helps to protect the ends of chromosomes from being fused with other chromosomes. Knockout of histone methyltransferases Suv39h1 and Suv39h2 increases the telomere length in murine cells, whereas downregulation of SUV39H1 and SUV39H2 genes decreases the telomere length in human cells, suggesting that telomere biology is different among mammalian species. However, epigenetic regulation of the telomere has not been s...

  6. DNA and RNA Quadruplex-Binding Proteins

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    Václav Brázda

    2014-09-01

    Full Text Available Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc., the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGGn, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2 and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

  7. Persistent telomere cohesion triggers a prolonged anaphase

    Science.gov (United States)

    Kim, Mi Kyung; Smith, Susan

    2014-01-01

    Telomeres use distinct mechanisms (not used by arms or centromeres) to mediate cohesion between sister chromatids. However, the motivation for a specialized mechanism at telomeres is not well understood. Here we show, using fluorescence in situ hybridization and live-cell imaging, that persistent sister chromatid cohesion at telomeres triggers a prolonged anaphase in normal human cells and cancer cells. Excess cohesion at telomeres can be induced by inhibition of tankyrase 1, a poly(ADP-ribose) polymerase that is required for resolution of telomere cohesion, or by overexpression of proteins required to establish telomere cohesion, the shelterin subunit TIN2 and the cohesin subunit SA1. Regardless of the method of induction, excess cohesion at telomeres in mitosis prevents a robust and efficient anaphase. SA1- or TIN2-induced excess cohesion and anaphase delay can be rescued by overexpression of tankyrase 1. Moreover, we show that primary fibroblasts, which accumulate excess telomere cohesion at mitosis naturally during replicative aging, undergo a similar delay in anaphase progression that can also be rescued by overexpression of tankyrase 1. Our study demonstrates that there are opposing forces that regulate telomere cohesion. The observation that cells respond to unresolved telomere cohesion by delaying (but not completely disrupting) anaphase progression suggests a mechanism for tolerating excess cohesion and maintaining telomere integrity. This attempt to deal with telomere damage may be ultimately futile for aging fibroblasts but useful for cancer cells. PMID:24173716

  8. Paternal age and telomere length in twins

    DEFF Research Database (Denmark)

    Hjelmborg, Jacob B; Dalgård, Christine; Mangino, Massimo

    2015-01-01

    Telomere length, a highly heritable trait, is longer in offspring of older fathers. This perplexing feature has been attributed to the longer telomeres in sperm of older men and it might be an 'epigenetic' mechanism through which paternal age plays a role in telomere length regulation in humans...

  9. The telomere binding protein TRF2 induces chromatin compaction.

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    Asmaa M Baker

    2011-04-01

    Full Text Available Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

  10. Peroxiredoxin 1 Protects Telomeres from Oxidative Damage and Preserves Telomeric DNA for Extension by Telomerase

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    Eric Aeby

    2016-12-01

    Full Text Available Oxidative damage of telomeres can promote cancer, cardiac failure, and muscular dystrophy. Specific mechanisms protecting telomeres from oxidative damage have not been described. We analyzed telomeric chromatin composition during the cell cycle and show that the antioxidant enzyme peroxiredoxin 1 (PRDX1 is enriched at telomeres during S phase. Deletion of the PRDX1 gene leads to damage of telomeric DNA upon oxidative stress, revealing a protective function of PRDX1 against oxidative damage at telomeres. We also show that the oxidized nucleotide 8-oxo-2′deoxyguanosine-5′-triphosphate (8oxodGTP causes premature chain termination when incorporated by telomerase and that some DNA substrates terminating in 8oxoG prevent extension by telomerase. Thus, PRDX1 safeguards telomeres from oxygen radicals to counteract telomere damage and preserve telomeric DNA for elongation by telomerase.

  11. Telomere length in early life predicts lifespan.

    Science.gov (United States)

    Heidinger, Britt J; Blount, Jonathan D; Boner, Winnie; Griffiths, Kate; Metcalfe, Neil B; Monaghan, Pat

    2012-01-31

    The attrition of telomeres, the ends of eukaryote chromosomes, is thought to play an important role in cell deterioration with advancing age. The observed variation in telomere length among individuals of the same age is therefore thought to be related to variation in potential longevity. Studies of this relationship are hampered by the time scale over which individuals need to be followed, particularly in long-lived species where lifespan variation is greatest. So far, data are based either on simple comparisons of telomere length among different age classes or on individuals whose telomere length is measured at most twice and whose subsequent survival is monitored for only a short proportion of the typical lifespan. Both approaches are subject to bias. Key studies, in which telomere length is tracked from early in life, and actual lifespan recorded, have been lacking. We measured telomere length in zebra finches (n = 99) from the nestling stage and at various points thereafter, and recorded their natural lifespan (which varied from less than 1 to almost 9 y). We found telomere length at 25 d to be a very strong predictor of realized lifespan (P telomeres at all points at which they were measured. Reproduction increased adult telomere loss, but this effect appeared transient and did not influence survival. Our results provide the strongest evidence available of the relationship between telomere length and lifespan and emphasize the importance of understanding factors that determine early life telomere length.

  12. Telomere functions grounding on TERRA firma.

    Science.gov (United States)

    Azzalin, Claus M; Lingner, Joachim

    2015-01-01

    Long noncoding telomeric repeat-containing RNAs - TERRAs - are transcribed in a regulated manner from telomeres throughout eukaryotes. TERRA molecules consist of chromosome end-specific subtelomeric sequences and telomeric repeats at their 3' ends. Recent work suggests that TERRA sustains several important functions at chromosome ends. TERRA can regulate telomere length through modulation of exonuclease 1 and telomerase, it may promote recruitment of chromatin modifiers to damaged telomeres and thereby enable DNA end-processing, and it may promote telomere protein composition changes during cell cycle progression. Furthermore, telomere transcription regulates chromosome-end mobility within the nucleus. We review how TERRA, by regulated expression and by providing a molecular scaffold for various protein enzymes, can support a large variety of vital functions. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Environmental stresses disrupt telomere length homeostasis.

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    Gal Hagit Romano

    Full Text Available Telomeres protect the chromosome ends from degradation and play crucial roles in cellular aging and disease. Recent studies have additionally found a correlation between psychological stress, telomere length, and health outcome in humans. However, studies have not yet explored the causal relationship between stress and telomere length, or the molecular mechanisms underlying that relationship. Using yeast as a model organism, we show that stresses may have very different outcomes: alcohol and acetic acid elongate telomeres, whereas caffeine and high temperatures shorten telomeres. Additional treatments, such as oxidative stress, show no effect. By combining genome-wide expression measurements with a systematic genetic screen, we identify the Rap1/Rif1 pathway as the central mediator of the telomeric response to environmental signals. These results demonstrate that telomere length can be manipulated, and that a carefully regulated homeostasis may become markedly deregulated in opposing directions in response to different environmental cues.

  14. Regulation of homologous recombination at telomeres in budding yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine; Lisby, Michael

    2010-01-01

    Homologous recombination is suppressed at normal length telomere sequences. In contrast, telomere recombination is allowed when telomeres erode in the absence of telomerase activity or as a consequence of nucleolytic degradation or incomplete replication. Here, we review the mechanisms...... that contribute to regulating mitotic homologous recombination at telomeres and the role of these mechanisms in signalling short telomeres in the budding yeast Saccharomyces cerevisiae....

  15. TERRA RNA Antagonizes ATRX and Protects Telomeres.

    Science.gov (United States)

    Chu, Hsueh-Ping; Cifuentes-Rojas, Catherine; Kesner, Barry; Aeby, Eric; Lee, Hun-Goo; Wei, Chunyao; Oh, Hyun Jung; Boukhali, Myriam; Haas, Wilhelm; Lee, Jeannie T

    2017-06-29

    Through an integration of genomic and proteomic approaches to advance understanding of long noncoding RNAs, we investigate the function of the telomeric transcript, TERRA. By identifying thousands of TERRA target sites in the mouse genome, we demonstrate that TERRA can bind both in cis to telomeres and in trans to genic targets. We then define a large network of interacting proteins, including epigenetic factors, telomeric proteins, and the RNA helicase, ATRX. TERRA and ATRX share hundreds of target genes and are functionally antagonistic at these loci: whereas TERRA activates, ATRX represses gene expression. At telomeres, TERRA competes with telomeric DNA for ATRX binding, suppresses ATRX localization, and ensures telomeric stability. Depleting TERRA increases telomerase activity and induces telomeric pathologies, including formation of telomere-induced DNA damage foci and loss or duplication of telomeric sequences. We conclude that TERRA functions as an epigenomic modulator in trans and as an essential regulator of telomeres in cis. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Localization-Dependent and -Independent Roles of SLX4 in Regulating Telomeres

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    Jamie S.J. Wilson

    2013-09-01

    Full Text Available SLX4, a scaffold for structure-specific DNA repair nucleases, is important for several types of DNA repair. Many repair proteins bind to sites of DNA damage, resulting in subnuclear “foci,” but SLX4 forms foci in human cells even without DNA damage. Using several approaches, we show that most, but not all, SLX4 foci localize to telomeres in a range of human cell lines irrespective of the mechanisms used to maintain telomere length. The SLX1 Holliday-junction-processing enzyme is recruited to telomeres by SLX4, and SLX4, in turn, is recruited by a motif that binds to the shelterin subunit TRF2 directly. We also show that TRF2-dependent recruitment of SLX4 prevents telomere damage. Furthermore, SLX4 prevents telomere lengthening and fragility in a manner that appears to be independent of telomere association. These findings reveal that SLX4 plays multiple roles in regulating telomere homeostasis.

  17. Human CST Has Independent Functions during Telomere Duplex Replication and C-Strand Fill-In

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    Feng Wang

    2012-11-01

    Full Text Available Human CST (CTC1-STN1-TEN1 is an RPA-like complex that is needed for efficient replication through the telomere duplex and genome-wide replication restart after fork stalling. Here, we show that STN1/CST has a second function in telomere replication during G-overhang maturation. Analysis of overhang structure after STN1 depletion revealed normal kinetics for telomerase-mediated extension in S phase but a delay in subsequent overhang shortening. This delay resulted from a defect in C-strand fill-in. Short telomeres exhibited the fill-in defect but normal telomere duplex replication, indicating that STN1/CST functions independently in these processes. Our work also indicates that the requirement for STN1/CST in telomere duplex replication correlates with increasing telomere length and replication stress. Our results provide direct evidence that STN1/CST participates in C-strand fill-in. They also demonstrate that STN1/CST participates in two mechanistically separate steps during telomere replication and identify CST as a replication factor that solves diverse replication-associated problems.

  18. Nickel enhances telomeric silencing in Saccharomyces cerevisiae.

    Science.gov (United States)

    Broday, L; Cai, J; Costa, M

    1999-04-06

    Certain nickel compounds including crystalline nickel sulfide (NiS) and subsulfide (Ni3S2) are potent human and animal carcinogens. In Chinese hamster embryo cells, an X-linked senescence gene was inactivated following nickel-induced DNA methylation. Nickel also induced the inactivation of the gpt reporter gene by chromatin condensation and a DNA methylation process in a transgenic gpt+ Chinese hamster cell line (G12), which is located near a heterochromatic region. To determine if nickel can cause gene silencing independently of DNA methylation, based only on the induction of changes in chromatin structure, we measured its effect on gene silencing in Saccharomyces cerevisiae. Growth of yeast in the presence of nickel chloride repressed a telomeric marker gene (URA3) and resulted in a stable epigenetic switch. This phenomenon was dependent on the number of cell doubling prior to selection and also on the distance of the marker gene from the end of the chromosome. The level of TPE (telomeric position effect) increased linearly with elevations of nickel concentration. Addition of magnesium inhibited this effect, but magnesium did not silence the reporter gene by itself. The level of silencing was also assessed following treatment with other transition metals: cobalt, copper and cadmium. In the sublethal range, cobalt induced similar effects as nickel, while copper and cadmium did not change the basal level of gene expression. Silencing by copper and cadmium were evident only at concentrations of those metals where the viability was very low. Copyright 1999 Elsevier Science B.V.

  19. TRF2 promotes, remodels and protects telomeric Holliday junctions.

    Science.gov (United States)

    Poulet, Anaïs; Buisson, Rémi; Faivre-Moskalenko, Cendrine; Koelblen, Mélanie; Amiard, Simon; Montel, Fabien; Cuesta-Lopez, Santiago; Bornet, Olivier; Guerlesquin, Françoise; Godet, Thomas; Moukhtar, Julien; Argoul, Françoise; Déclais, Anne-Cécile; Lilley, David M J; Ip, Stephen C Y; West, Stephen C; Gilson, Eric; Giraud-Panis, Marie-Josèphe

    2009-03-18

    The ability of the telomeric DNA-binding protein, TRF2, to stimulate t-loop formation while preventing t-loop deletion is believed to be crucial to maintain telomere integrity in mammals. However, little is known on the molecular mechanisms behind these properties of TRF2. In this report, we show that TRF2 greatly increases the rate of Holliday junction (HJ) formation and blocks the cleavage by various types of HJ resolving activities, including the newly identified human GEN1 protein. By using potassium permanganate probing and differential scanning calorimetry, we reveal that the basic domain of TRF2 induces structural changes to the junction. We propose that TRF2 contributes to t-loop stabilisation by stimulating HJ formation and by preventing resolvase cleavage. These findings provide novel insights into the interplay between telomere protection and homologous recombination and suggest a general model in which TRF2 maintains telomere integrity by controlling the turnover of HJ at t-loops and at regressed replication forks.

  20. Do Telomeres Adapt to Physiological Stress? Exploring the Effect of Exercise on Telomere Length and Telomere-Related Proteins

    Directory of Open Access Journals (Sweden)

    Andrew T. Ludlow

    2013-01-01

    Full Text Available Aging is associated with a tissue degeneration phenotype marked by a loss of tissue regenerative capacity. Regenerative capacity is dictated by environmental and genetic factors that govern the balance between damage and repair. The age-associated changes in the ability of tissues to replace lost or damaged cells is partly the cause of many age-related diseases such as Alzheimer's disease, cardiovascular disease, type II diabetes, and sarcopenia. A well-established marker of the aging process is the length of the protective cap at the ends of chromosomes, called telomeres. Telomeres shorten with each cell division and with increasing chronological age and short telomeres have been associated with a range of age-related diseases. Several studies have shown that chronic exposure to exercise (i.e., exercise training is associated with telomere length maintenance; however, recent evidence points out several controversial issues concerning tissue-specific telomere length responses. The goals of the review are to familiarize the reader with the current telomere dogma, review the literature exploring the interactions of exercise with telomere phenotypes, discuss the mechanistic research relating telomere dynamics to exercise stimuli, and finally propose future directions for work related to telomeres and physiological stress.

  1. An H2A Histone Isotype, H2ac, Associates with Telomere and Maintains Telomere Integrity.

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    Chia-Hsin Su

    Full Text Available Telomeres are capped at the ends of eukaryotic chromosomes and are composed of TTAGGG repeats bound to the shelterin complex. Here we report that a replication-dependent histone H2A isotype, H2ac, was associated with telomeres in human cells and co-immunoprecipitates with telomere repeat factor 2 (TRF2 and protection of telomeres protein 1 (POT1, whereas other histone H2A isotypes and mutations of H2ac did not bind to telomeres or these two proteins. The amino terminal basic domain of TRF2 was necessary for the association with H2ac and for the recruitment of H2ac to telomeres. Depletion of H2ac led to loss of telomeric repeat sequences, the appearance of dysfunctional telomeres, and chromosomal instability, including chromosomal breaks and anaphase bridges, as well as accumulation of telomere-associated DNA damage factors in H2ac depleted cells. Additionally, knockdown of H2ac elicits an ATM-dependent DNA damage response at telomeres and depletion of XPF protects telomeres against H2ac-deficiency-induced G-strand overhangs loss and DNA damage response, and prevents chromosomal instability. These findings suggest that the H2A isotype, H2ac, plays an essential role in maintaining telomere functional integrity.

  2. Probing the Potential Role of Non-B DNA Structures at Yeast Meiosis-Specific DNA Double-Strand Breaks.

    Science.gov (United States)

    Kshirsagar, Rucha; Khan, Krishnendu; Joshi, Mamata V; Hosur, Ramakrishna V; Muniyappa, K

    2017-05-23

    A plethora of evidence suggests that different types of DNA quadruplexes are widely present in the genome of all organisms. The existence of a growing number of proteins that selectively bind and/or process these structures underscores their biological relevance. Moreover, G-quadruplex DNA has been implicated in the alignment of four sister chromatids by forming parallel guanine quadruplexes during meiosis; however, the underlying mechanism is not well defined. Here we show that a G/C-rich motif associated with a meiosis-specific DNA double-strand break (DSB) in Saccharomyces cerevisiae folds into G-quadruplex, and the C-rich sequence complementary to the G-rich sequence forms an i-motif. The presence of G-quadruplex or i-motif structures upstream of the green fluorescent protein-coding sequence markedly reduces the levels of gfp mRNA expression in S. cerevisiae cells, with a concomitant decrease in green fluorescent protein abundance, and blocks primer extension by DNA polymerase, thereby demonstrating the functional significance of these structures. Surprisingly, although S. cerevisiae Hop1, a component of synaptonemal complex axial/lateral elements, exhibits strong affinity to G-quadruplex DNA, it displays a much weaker affinity for the i-motif structure. However, the Hop1 C-terminal but not the N-terminal domain possesses strong i-motif binding activity, implying that the C-terminal domain has a distinct substrate specificity. Additionally, we found that Hop1 promotes intermolecular pairing between G/C-rich DNA segments associated with a meiosis-specific DSB site. Our results support the idea that the G/C-rich motifs associated with meiosis-specific DSBs fold into intramolecular G-quadruplex and i-motif structures, both in vitro and in vivo, thus revealing an important link between non-B form DNA structures and Hop1 in meiotic chromosome synapsis and recombination. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. TERRA and the state of the telomere.

    Science.gov (United States)

    Rippe, Karsten; Luke, Brian

    2015-11-01

    Long noncoding telomeric repeat-containing RNA (TERRA) has been implicated in telomere maintenance in a telomerase-dependent and a telomerase-independent manner during replicative senescence and cancer. TERRA's proposed activities are diverse, thus making it difficult to pinpoint the critical roles that TERRA may have. We propose that TERRA orchestrates different activities at chromosome ends in a manner that depends on the state of the telomere.

  4. The Leishmania amazonensis TRF (TTAGGG repeat-binding factor homologue binds and co-localizes with telomeres

    Directory of Open Access Journals (Sweden)

    Freitas Lucio de H

    2010-05-01

    Full Text Available Abstract Background Telomeres are specialized structures at the end of chromosomes essential for maintaining genome stability and cell viability. The importance of telomeric proteins for telomere maintenance has increased our interest in the identification of homologues within the genus Leishmania. The mammalian TRF1 and TRF2 proteins, for example, bind double-stranded telomeres via a Myb-like DNA-binding domain and are involved with telomere length regulation and chromosome end protection. In addition, TRF2 can modulate the activity of several enzymes and influence the conformation of telomeric DNA. In this work, we identified and characterized a Leishmania protein (LaTRF homologous to both mammalian TRF1 and TRF2. Results LaTRF was cloned using a PCR-based strategy. ClustalW and bl2seq sequence analysis showed that LaTRF shared sequence identity with the Trypanosoma brucei TRF (TbTRF protein and had the same degree of sequence similarities with the dimerization (TRFH and the canonical DNA-binding Myb-like domains of both mammalian TRFs. LaTRF was predicted to be an 82.5 kDa protein, indicating that it is double the size of the trypanosome TRF homologues. Western blot and indirect immunofluorescence combined with fluorescence in situ hybridization showed that LaTRF, similarly to hTRF2, is a nuclear protein that also associates with parasite telomeres. Native and full length LaTRF and a mutant bearing the putative Myb-like domain expressed in bacteria bound double-stranded telomeric DNA in vitro. Chromatin immunoprecipitation showed that LaTRF interacted specifically with telomeres in vivo. Conclusion The nuclear localization of LaTRF, its association and co-localization with parasite telomeres and its high identity with TbTRF protein, support the hypothesis that LaTRF is a Leishmania telomeric protein.

  5. Evolutionary ecology of telomeres: a review.

    Science.gov (United States)

    Olsson, Mats; Wapstra, Erik; Friesen, Christopher R

    2017-10-06

    Telomere-induced selection could take place if telomere-associated disease risk shortens reproductive life span and differently reduces relative fitness among individuals. Some of these diseases first appear before reproductive senescence and could thus influence ongoing selection. We ask whether we can estimate the components of the breeder's equation for telomeres, in which the response to selection (R, by definition "evolution") is the product of ongoing selection (S) and heritability (h2 ). However, telomere inheritance is a conundrum: in quantitative genetics, traits can usually be allocated to categories with relatively high or low heritability, depending on their association with relative fitness. Telomere traits, however, show wide variation in heritability from zero to one, across taxa, gender, ethnicity, age, and disease status. In spite of this, there is divergence in telomere length among populations, supporting past and ongoing telomere evolution. Rates of telomere attrition and elongation vary among taxa with some, but not complete, taxonomic coherence. For example, telomerase is commonly referred to as "restricted to the germ line in mammals," but inbred mice and beavers have telomerase upregulation in somatic tissue, as do many ectotherms. These observations provoke a simplistic understanding of telomere evolutionary biology-clearly much is yet to be discovered. © 2017 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals, Inc. on behalf of The New York Academy of Sciences.

  6. Loss of telomere protection: consequences and opportunities.

    Directory of Open Access Journals (Sweden)

    Jacqueline Johanna Leonarda Jacobs

    2013-04-01

    Full Text Available Telomeres are repetitive sequences at the natural ends of linear eukaryotic chromosomes that protect these from recognition as chromosome breaks. Their ability to do so critically depends on the binding of sufficient quantities of functional shelterin, a six-unit protein complex with specific and crucial roles in telomere maintenance and function. Insufficient telomere length, leading to insufficient concentration of shelterin at chromosome ends, or otherwise crippled shelterin function, causes telomere deprotection. While contributing to aging-related pathologies, loss of telomere protection can act as a barrier to tumorigenesis, as dysfunctional telomeres activate DNA-damage-like checkpoint responses that halt cell proliferation or trigger cell death. In addition, dysfunctional telomeres affect cancer development and progression by being a source of genomic instability. Reviewed here are the different approaches that are being undertaken to investigate the mammalian cellular response to telomere dysfunction and its consequences for cancer. Furthermore, it is discussed how current and future knowledge about the mechanisms underlying telomere damage responses might be applied for diagnostic purposes or therapeutic intervention.

  7. Leukocyte telomere dynamics in the elderly

    DEFF Research Database (Denmark)

    Steenstrup, Troels; Hjelmborg, Jacob V B; Mortensen, Laust Hvas

    2013-01-01

    Limited data suggest that leukocytes of the elderly display ultra-short telomeres. It was reported that in some elderly persons leukocyte telomere length (LTL) shows age-dependent elongation. Using cross-sectional and longitudinal models, we characterized LTL dynamics in participants......, assuming a 340 bp attrition during this period. This was not significantly different from the empirical observation of 7.5 % of individuals showing LTL elongation. We conclude that accumulation of ultra-short telomeres in leukocytes of the elderly reflects a shift toward shorter telomeres in the entire...

  8. Genomic Organization of the Drosophila Telomere RetrotransposableElements

    Energy Technology Data Exchange (ETDEWEB)

    George, J.A.; DeBaryshe, P.G.; Traverse, K.L.; Celniker, S. E.; Pardue, M-L.

    2006-10-16

    The emerging sequence of the heterochromatic portion of the Drosophila melanogaster genome, with the most recent update of euchromatic sequence, gives the first genome-wide view of the chromosomal distribution of the telomeric retrotransposons, HeT-A, TART, and Tahre. As expected, these elements are entirely excluded from euchromatin, although sequence fragments of HeT-A and TART 3 untranslated regions are found in nontelomeric heterochromatin on the Y chromosome. The proximal ends of HeT-A/TART arrays appear to be a transition zone because only here do other transposable elements mix in the array. The sharp distinction between the distribution of telomeric elements and that of other transposable elements suggests that chromatin structure is important in telomere element localization. Measurements reported here show (1) D. melanogaster telomeres are very long, in the size range reported for inbred mouse strains (averaging 46 kb per chromosome end in Drosophila stock 2057). As in organisms with telomerase, their length varies depending on genotype. There is also slight under-replication in polytene nuclei. (2) Surprisingly, the relationship between the number of HeT-A and TART elements is not stochastic but is strongly correlated across stocks, supporting the idea that the two elements are interdependent. Although currently assembled portions of the HeT-A/TART arrays are from the most-proximal part of long arrays, {approx}61% of the total HeT-A sequence in these regions consists of intact, potentially active elements with little evidence of sequence decay, making it likely that the content of the telomere arrays turns over more extensively than has been thought.

  9. Nucleolar organization, ribosomal DNA array stability, and acrocentric chromosome integrity are linked to telomere function.

    Directory of Open Access Journals (Sweden)

    Kaitlin M Stimpson

    Full Text Available The short arms of the ten acrocentric human chromosomes share several repetitive DNAs, including ribosomal RNA genes (rDNA. The rDNA arrays correspond to nucleolar organizing regions that coalesce each cell cycle to form the nucleolus. Telomere disruption by expressing a mutant version of telomere binding protein TRF2 (dnTRF2 causes non-random acrocentric fusions, as well as large-scale nucleolar defects. The mechanisms responsible for acrocentric chromosome sensitivity to dysfunctional telomeres are unclear. In this study, we show that TRF2 normally associates with the nucleolus and rDNA. However, when telomeres are crippled by dnTRF2 or RNAi knockdown of TRF2, gross nucleolar and chromosomal changes occur. We used the controllable dnTRF2 system to precisely dissect the timing and progression of nucleolar and chromosomal instability induced by telomere dysfunction, demonstrating that nucleolar changes precede the DNA damage and morphological changes that occur at acrocentric short arms. The rDNA repeat arrays on the short arms decondense, and are coated by RNA polymerase I transcription binding factor UBF, physically linking acrocentrics to one another as they become fusogenic. These results highlight the importance of telomere function in nucleolar stability and structural integrity of acrocentric chromosomes, particularly the rDNA arrays. Telomeric stress is widely accepted to cause DNA damage at chromosome ends, but our findings suggest that it also disrupts chromosome structure beyond the telomere region, specifically within the rDNA arrays located on acrocentric chromosomes. These results have relevance for Robertsonian translocation formation in humans and mechanisms by which acrocentric-acrocentric fusions are promoted by DNA damage and repair.

  10. Problem-Solving Test: Telomere Replication

    Science.gov (United States)

    Szeberenyi, Jozsef

    2010-01-01

    The Nobel Prize in Physiology or Medicine in 2009 was awarded to Elizabeth H. Blackburn, Carol W. Greider, and Jack W. Szostak for the discovery of "how chromosomes are protected by telomeres and the enzyme telomerase." The discovery has important implications in the processes of cellular aging and carcinogenesis. Telomeres are satellite DNA…

  11. TERRA: telomeric repeat-containing RNA.

    Science.gov (United States)

    Luke, Brian; Lingner, Joachim

    2009-09-02

    Telomeres, the physical ends of eukaryotic chromosomes, consist of tandem arrays of short DNA repeats and a large set of specialized proteins. A recent analysis has identified telomeric repeat-containing RNA (TERRA), a large non-coding RNA in animals and fungi, which forms an integral component of telomeric heterochromatin. TERRA transcription occurs at most or all chromosome ends and it is regulated by RNA surveillance factors and in response to changes in telomere length. TERRA functions that are emerging suggest important roles in the regulation of telomerase and in orchestrating chromatin remodelling throughout development and cellular differentiation. The accumulation of TERRA at telomeres can also interfere with telomere replication, leading to a sudden loss of telomere tracts. Such a phenotype can be observed upon impairment of the RNA surveillance machinery or in cells from ICF (Immunodeficiency, Centromeric region instability, Facial anomalies) patients, in which TERRA is upregulated because of DNA methylation defects in the subtelomeric region. Thus, TERRA may mediate several crucial functions at the telomeres, a region of the genome that had been considered to be transcriptionally silent.

  12. Telomere biology in healthy aging and disease

    NARCIS (Netherlands)

    Oeseburg, Hisko; de Boer, Rudolf A.; van Gilst, Wiek H.; van der Harst, Pim

    Aging is a biological process that affects most cells, organisms and species. Telomeres have been postulated as a universal biological clock that shortens in parallel with aging in cells. Telomeres are located at the end of the chromosomes and consist of an evolutionary conserved repetitive

  13. Disruptor of telomeric silencing 1-like (DOT1L): disclosing a new class of non-nucleoside inhibitors by means of ligand-based and structure-based approaches

    Science.gov (United States)

    Sabatino, Manuela; Rotili, Dante; Patsilinakos, Alexandros; Forgione, Mariantonietta; Tomaselli, Daniela; Alby, Fréderic; Arimondo, Paola B.; Mai, Antonello; Ragno, Rino

    2018-01-01

    Chemical inhibition of chromatin-mediated signaling involved proteins is an established strategy to drive expression networks and alter disease progression. Protein methyltransferases are among the most studied proteins in epigenetics and, in particular, disruptor of telomeric silencing 1-like (DOT1L) lysine methyltransferase plays a key role in MLL-rearranged acute leukemia Selective inhibition of DOT1L is an established attractive strategy to breakdown aberrant H3K79 methylation and thus overexpression of leukemia genes, and leukemogenesis. Although numerous DOT1L inhibitors have been several structural data published no pronounced computational efforts have been yet reported. In these studies a first tentative of multi-stage and LB/SB combined approach is reported in order to maximize the use of available data. Using co-crystallized ligand/DOT1L complexes, predictive 3-D QSAR and COMBINE models were built through a python implementation of previously reported methodologies. The models, validated by either modeled or experimental external test sets, proved to have good predictive abilities. The application of these models to an internal library led to the selection of two unreported compounds that were found able to inhibit DOT1L at micromolar level. To the best of our knowledge this is the first report of quantitative LB and SB DOT1L inhibitors models and their application to disclose new potential epigenetic modulators.

  14. Telomeres and telomerase in prostate cancer development and therapy.

    Science.gov (United States)

    Graham, Mindy Kim; Meeker, Alan

    2017-10-01

    Aberrations in telomere biology are among the earliest events in prostate cancer tumorigenesis and continue during tumour progression. Substantial telomere shortening occurs in prostate cancer cells and high-grade prostatic intraepithelial neoplasia. Not all mechanisms of telomere shortening are understood, but oxidative stress from local inflammation might accelerate prostatic telomere loss. Critically short telomeres can drive the accumulation of tumour-promoting genomic alterations; however, continued telomere erosion is unsustainable and must be mitigated to ensure cancer cell survival and unlimited replication potential. Prostate cancers predominantly maintain telomeres by activating telomerase, but alternative mechanisms of telomere extension can occur in metastatic disease. Telomerase activity and telomere length assessment might be useful in prostate cancer diagnosis and prognosis. Telomere shortening in normal stromal cells has been associated with prostate cancer, whereas variable telomere lengths in prostate cancer cells and telomere shortening in cancer-associated stromal cells correlated with lethal disease. Single-agent telomerase-targeted treatments for solid cancers were ineffective in clinical trials but have not been investigated in prostate cancer and might be useful in combination with established regimens. Telomere-directed strategies have not been explored as extensively. Telomere deprotection strategies have the advantage of being effective in both telomerase-dependent and telomerase-independent cancers. Disruption of androgen receptor function in prostate cancer cells results in telomere dysfunction, indicating telomeres and telomerase as potential therapeutic targets in prostate cancer.

  15. Characterization of two Arabidopsis thaliana myb-like proteins showing affinity to telomeric DNA sequence.

    Science.gov (United States)

    Schrumpfová, Petra; Kuchar, Milan; Miková, Gabriela; Skrísovská, Lenka; Kubicárová, Tatiana; Fajkus, Jirí

    2004-04-01

    Telomere-binding proteins participate in forming a functional nucleoprotein structure at chromosome ends. Using a genomic approach, two Arabidopsis thaliana genes coding for candidate Myb-like telomere binding proteins were cloned and expressed in E. coli. Both proteins, termed AtTBP2 (accession Nos. T46051 (protein database) and GI:638639 (nucleotide database); 295 amino acids, 32 kDa, pI 9.53) and AtTBP3 (BAB08466, GI:9757879; 299 amino acids, 33 kDa, pI 9.88), contain a single Myb-like DNA-binding domain at the N-terminus, and a histone H1/H5-like DNA-binding domain in the middle of the protein sequence. Both proteins are expressed in various A. thaliana tissues. Using the two-hybrid system interaction between the proteins AtTBP2 and AtTBP3 and self interactions of each of the proteins were detected. Gel-retardation assays revealed that each of the two proteins is able to bind the G-rich strand and double-stranded DNA of plant telomeric sequence with an affinity proportional to a number of telomeric repeats. Substrates bearing a non-telomeric DNA sequence positioned between two telomeric repeats were bound with an efficiency depending on the length of interrupting sequence. The ability to bind variant telomere sequences decreased with sequence divergence from the A. thaliana telomeric DNA. None of the proteins alone or their mixture affects telomerase activity in vitro. Correspondingly, no interaction was observed between any of two proteins and the Arabidopsis telomerase reverse transcriptase catalytic subunit TERT (accession No. AF172097) using two-hybrid assay.

  16. RNaseH1 regulates TERRA-telomeric DNA hybrids and telomere maintenance in ALT tumour cells

    Science.gov (United States)

    Arora, Rajika; Lee, Yongwoo; Wischnewski, Harry; Brun, Catherine M.; Schwarz, Tobias; Azzalin, Claus M.

    2014-01-01

    A fraction of cancer cells maintain telomeres through the telomerase-independent, ‘Alternative Lengthening of Telomeres’ (ALT) pathway. ALT relies on homologous recombination (HR) between telomeric sequences; yet, what makes ALT telomeres recombinogenic remains unclear. Here we show that the RNA endonuclease RNaseH1 regulates the levels of RNA–DNA hybrids between telomeric DNA and the long noncoding RNA TERRA, and is a key mediator of telomere maintenance in ALT cells. RNaseH1 associated to telomeres specifically in ALT cells and its depletion led to telomeric hybrid accumulation, exposure of single-stranded telomeric DNA, activation of replication protein A at telomeres and abrupt telomere excision. Conversely, overexpression of RNaseH1 weakened the recombinogenic nature of ALT telomeres and led to telomere shortening. Altering cellular RNaseH1 levels did not perturb telomere homoeostasis in telomerase-positive cells. RNaseH1 maintains regulated levels of telomeric RNA–DNA hybrids at ALT telomeres to trigger HR without compromising telomere integrity too severely. PMID:25330849

  17. Telomeres and telomerase in prostate cancer development and therapy

    OpenAIRE

    Graham, Mindy Kim; Meeker, Alan

    2017-01-01

    Aberrations in telomere biology are among the earliest events in prostate cancer tumorigenesis and continue during tumour progression. Substantial telomere shortening occurs in prostate cancer cells and high-grade prostatic intraepithelial neoplasia. Not all mechanisms of telomere shortening are understood, but oxidative stress from local inflammation might accelerate prostatic telomere loss. Critically short telomeres can drive the accumulation of tumour-promoting genomic alterations; howeve...

  18. Telomere Length in Elite Athletes.

    Science.gov (United States)

    Muniesa, Carlos A; Verde, Zoraida; Diaz-Ureña, Germán; Santiago, Catalina; Gutiérrez, Fernando; Díaz, Enrique; Gómez-Gallego, Félix; Pareja-Galeano, Helios; Soares-Miranda, Luisa; Lucia, Alejandro

    2017-08-01

    Growing evidence suggests that regular moderate-intensity physical activity is associated with an attenuation of leukocyte telomere length (LTL) shortening. However, more controversy exists regarding higher exercise loads such as those imposed by elite-sport participation. The authors investigated LTL differences between young elite athletes (n = 61, 54% men, age [mean ± SD] 27.2 ± 4.9 y) and healthy nonsmoker, physically inactive controls (n = 64, 52% men, 28.9 ± 6.3 y) using analysis of variance (ANOVA). Elite athletes had, on average, higher LTL than control subjects, 0.89 ± 0.26 vs 0.78 ± 0.31, P = .013 for the group effect, with no significant sex (P = .995) or age effect (P = .114). The results suggest that young elite athletes have longer telomeres than their inactive peers. Further research might assess the LTL of elite athletes of varying ages compared with both age-matched active and inactive individuals.

  19. Accelerated Telomere Shortening in Acromegaly; IGF-I Induces Telomere Shortening and Cellular Senescence

    National Research Council Canada - National Science Library

    Matsumoto, Ryusaku; Fukuoka, Hidenori; Iguchi, Genzo; Odake, Yukiko; Yoshida, Kenichi; Bando, Hironori; Suda, Kentaro; Nishizawa, Hitoshi; Takahashi, Michiko; Yamada, Shozo; Ogawa, Wataru; Takahashi, Yutaka

    2015-01-01

    .... However, the underlying mechanism has not been fully elucidated. Telomere shortening is reportedly associated with reduced life expectancy and increased prevalence of these age-related diseases...

  20. Accelerated Telomere Shortening in Acromegaly; IGF-I Induces Telomere Shortening and Cellular Senescence: e0140189

    National Research Council Canada - National Science Library

    Ryusaku Matsumoto; Hidenori Fukuoka; Genzo Iguchi; Yukiko Odake; Kenichi Yoshida; Hironori Bando; Kentaro Suda; Hitoshi Nishizawa; Michiko Takahashi; Shozo Yamada; Wataru Ogawa; Yutaka Takahashi

    2015-01-01

    .... However, the underlying mechanism has not been fully elucidated. Telomere shortening is reportedly associated with reduced life expectancy and increased prevalence of these age-related diseases...

  1. HYPOTHESIS: PARALOG FORMATION FROM PROGENITOR PROTEINS AND PARALOG MUTAGENESIS SPUR THE RAPID EVOLUTION OF TELOMERE BINDING PROTEINS

    Directory of Open Access Journals (Sweden)

    Arthur J Lustig

    2016-02-01

    Full Text Available Through elegant studies in fungal cells and complex organisms, we propose a unifying paradigm for the rapid evolution of telomere binding proteins (TBPs that associate with either (or both telomeric DNA and telomeric proteins. TBPs protect and regulate telomere structure and function. Four critical factors are involved. First, TBPs that commonly bind to telomeric DNA include the c-Myb binding proteins, OB-fold single-stranded binding proteins, and G-G base paired Hoogsteen structure (G4 binding proteins. Each contributes independently or, in some cases, cooperatively, to provide a minimum level of telomere function. As a result of these minimal requirements and the great abundance of homologs of these motifs in the proteome, DNA telomere-binding activity may be generated more easily than expected. Second, telomere dysfunction gives rise to genome instability, through the elevation of recombination rates, genome ploidy, and the frequency of gene mutations. The formation of paralogs that diverge from their progenitor proteins ultimately can form a high frequency of altered TBPs with altered functions. Third, TBPs that assemble into complexes (e.g. mammalian shelterin derive benefits from the novel emergent functions. Fourth, a limiting factor in the evolution of TBP complexes is the formation of mutually compatible interaction surfaces amongst the TBPs. These factors may have different degrees of importance in the evolution of different phyla, illustrated by the apparently simpler telomeres in complex plants. Selective pressures that can utilize the mechanisms of paralog formation and mutagenesis to drive TBP evolution along routes dependent on the requisite physiologic changes.

  2. Telomeric Repeat Containing RNA (TERRA): Aging and Cancer.

    Science.gov (United States)

    Sinha, Sonam; Shukla, Samriddhi; Khan, Sajid; Farhan, Mohammad; Kamal, Mohammad Amjad; Meeran, Syed Musthapa

    2015-01-01

    Telomeric repeat containing RNAs (TERRA) are small RNA molecules synthesized from telomeric regions which were previously considered as silent genomic domains. In normal cells, these RNAs are transcribed in a direction from subtelomeric region towards the chromosome ends, but in case of cancer cells, their expression remains limited or absent. Telomerase is a rate limiting enzyme for cellular senescence, cancer and aging. Most of the studies deal with the manipulation of telomerase enzyme in cancer and aging either by synthetic oligonucleotide or by natural phytochemicals. Here, we collected evidences and discussed intensely about the bio-molecular structure of TERRA, naturally occurring ligands of telomerase, and their genetic and epigenetic regulations in aging associated diseases. Due to their capability to act as naturally occurring ligands of telomerase, these RNAs can overcome the limitations possessed by synthetic oligonucleotides, which are aimed against telomerase. Drugs specifically targeting TERRA molecules could modulate telomerase-mediated telomere lengthening. Thus, targeting TERRA-mediated regulation of telomerase would be a promising therapeutic strategy against cancer and age-associated diseases.

  3. Short Telomere Length and Ischemic Heart Disease

    DEFF Research Database (Denmark)

    Madrid, Alexander Scheller; Rode, Line; Nordestgaard, Børge Grønne

    2016-01-01

    BACKGROUND: Short telomeres are associated with aging and have been associated with a high risk of ischemic heart disease in observational studies; however, the latter association could be due to residual confounding and/or reverse causation. We wanted to test the hypothesis that short telomeres...... are associated with high risk of ischemic heart disease using a Mendelian randomization approach free of reverse causation and of most confounding. METHODS: We genotyped 3 genetic variants in OBFC1 (oligonucleotide/oligosaccharide binding fold containing 1), TERT (telomerase reverse transcriptase), and TERC...... (telomerase RNA component), which code for proteins and RNA involved in telomere maintenance. We studied 105 055 individuals from Copenhagen; 17 235 of these individuals were diagnosed with ischemic heart disease between 1977 and 2013, and 66 618 had telomere length measured. For genetic studies, we further...

  4. Telomere Maintenance in the Absence of Telomerase

    National Research Council Canada - National Science Library

    Lundblad, Vicki

    2000-01-01

    .... In the budding yeasts S. cerevisiae and K. lactis, telomerase- independent survival is mediated via RAD52-dependent recombination which results in amplification of telomeric and subtelomeric repeat sequences...

  5. Stn1-Ten1 is an Rpa2-Rpa3-like complex at telomeres

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Jia; Yu, Eun Young; Yang, Yuting; Confer, Laura A; Sun, Steven H; Wan, Ke; Lue, Neal F; Lei, Ming; (Weill); (Michigan-Med)

    2010-09-02

    In budding yeast, Cdc13, Stn1, and Ten1 form a heterotrimeric complex (CST) that is essential for telomere protection and maintenance. Previous bioinformatics analysis revealed a putative oligonucleotide/oligosaccharide-binding (OB) fold at the N terminus of Stn1 (Stn1N) that shows limited sequence similarity to the OB fold of Rpa2, a subunit of the eukaryotic ssDNA-binding protein complex replication protein A (RPA). Here we present functional and structural analyses of Stn1 and Ten1 from multiple budding and fission yeast. The crystal structure of the Candida tropicalis Stn1N complexed with Ten1 demonstrates an Rpa2N-Rpa3-like complex. In both structures, the OB folds of the two components pack against each other through interactions between two C-terminal helices. The structure of the C-terminal domain of Saccharomyces cerevisiae Stn1 (Stn1C) was found to comprise two related winged helix-turn-helix (WH) motifs, one of which is most similar to the WH motif at the C terminus of Rpa2, again supporting the notion that Stn1 resembles Rpa2. The crystal structure of the fission yeast Schizosaccharomyces pombe Stn1N-Ten1 complex exhibits a virtually identical architecture as the C. tropicalis Stn1N-Ten1. Functional analyses of the Candida albicans Stn1 and Ten1 proteins revealed critical roles for these proteins in suppressing aberrant telomerase and recombination activities at telomeres. Mutations that disrupt the Stn1-Ten1 interaction induce telomere uncapping and abolish the telomere localization of Ten1. Collectively, our structural and functional studies illustrate that, instead of being confined to budding yeast telomeres, the CST complex may represent an evolutionarily conserved RPA-like telomeric complex at the 3' overhangs that works in parallel with or instead of the well-characterized POT1-TPP1/TEBP{alpha}-{beta} complex.

  6. Telomere length in early life predicts lifespan

    OpenAIRE

    Heidinger, B. J.; Blount, J.D.; Boner, W.; Griffiths, K.; Metcalfe, N.B.; Monaghan, P.

    2012-01-01

    The attrition of telomeres, the ends of eukaryote chromosomes, is thought to play an important role in cell deterioration with advancing age. The observed variation in telomere length among individuals of the same age is therefore thought to be related to variation in potential longevity. Studies of this relationship are hampered by the time scale over which individuals need to be followed, particularly in long-lived species where lifespan variation is greatest. So far, data are based either ...

  7. Telomeres and the ethics of human cloning.

    Science.gov (United States)

    Allhoff, Fritz

    2004-01-01

    In search of a potential problem with cloning, I investigate the phenomenon of telomere shortening which is caused by cell replication; clones created from somatic cells will have shortened telomeres and therefore reach a state of senescence more rapidly. While genetic intervention might fix this problem at some point in the future, I ask whether, absent technological advances, this biological phenomenon undermines the moral permissibility of cloning.

  8. Super-resolution fluorescence imaging of telomeres reveals TRF2-dependent T-loop formation.

    Science.gov (United States)

    Doksani, Ylli; Wu, John Y; de Lange, Titia; Zhuang, Xiaowei

    2013-10-10

    We have applied a super-resolution fluorescence imaging method, stochastic optical reconstruction microscopy (STORM), to visualize the structure of functional telomeres and telomeres rendered dysfunctional through removal of shelterin proteins. The STORM images showed that functional telomeres frequently exhibit a t-loop configuration. Conditional deletion of individual components of shelterin showed that TRF2 was required for the formation and/or maintenance of t-loops, whereas deletion of TRF1, Rap1, or the POT1 proteins (POT1a and POT1b) had no effect on the frequency of t-loop occurrence. Within the shelterin complex, TRF2 uniquely serves to protect telomeres from two pathways that are initiated on free DNA ends: classical nonhomologous end-joining (NHEJ) and ATM-dependent DNA damage signaling. The TRF2-dependent remodeling of telomeres into t-loop structures, which sequester the ends of chromosomes, can explain why NHEJ and the ATM signaling pathway are repressed when TRF2 is present. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Telomere--the twilight to immortality.

    Science.gov (United States)

    Shukla, Samarth; Acharya, Sourya; Rajput, Devendra; Vagha, S; Grover, Shobha

    2010-09-01

    Besides forming a very important component of the chromosome, the telomeres have extremely significant modes of action and functions, right from maintaining a basic infrastructure and integrity of the chromosome vis a vis the other chromosomes, telomeres are responsible for the cell divisions and replicative senescence of the cell. The number of mitotic divisions which a cell will go through in its life span while passing through the cell cycle is governed inturn by these telomeres, the crux of the entire functioning of these chromosomal components suggests that they are the ticking clocks of the cell and when they diminish or are worn out so does the cell reach it's senility at the fag end of it's replicative life--resulting fate being--the cell is sent to it's grave yard (the final destination). Clinical implications include--regulation of cell life spans, regulating the cell's replicative behavior and it's utility in forming cells which usually are impossible to divide or replicate, telomeres regulate the cloning process,the telomeres play a major role in predicting the fate of a neoplastic cell and finally enhancing the life span of a single cell, the organ, the body as a whole by enzymes which expand the telomeres--the telomerase.

  10. Effects of G-CSF on telomere lengths in PBMCs from human immunodeficiency virus-infected patients

    DEFF Research Database (Denmark)

    Aladdin, H; Ullum, H; Schjerling, P

    2000-01-01

    Telomeres are unique terminal chromosomal structures, the length of which has been shown to decrease with cell division in vitro and with increased age in vivo for human somatic cells. In human immunodeficiency virus (HIV)-1 infection, decrease of telomere length is primarily found in CD8+ T cell...... that during G-CSF treatment lymphocytes are recruited by a combination of central and peripheral proliferation....

  11. XPF with mutations in its conserved nuclease domain is defective in DNA repair but functions in TRF2-mediated telomere shortening.

    Science.gov (United States)

    Wu, Yili; Zacal, Natalie J; Rainbow, Andrew J; Zhu, Xu-Dong

    2007-02-04

    TRF2, a telomere-binding protein, is a crucial player in telomere length maintenance. Overexpression of TRF2 results in telomere shortening in both normal primary fibroblasts and telomerase-positive cancer cells. TRF2 is found to be associated with XPF-ERCC1, a structure-specific endonuclease involved in nucleotide excision repair, crosslink repair and DNA recombination. XPF-ERCC1 is implicated in TRF2-dependent telomere loss in mouse keratinocytes, however, whether XPF-ERCC1 and its nuclease activity are required for TRF2-mediated telomere shortening in human cells is unknown. Here we report that TRF2-induced telomere shortening is abrogated in human cells deficient in XPF, demonstrating that XPF-ERCC1 is required for TRF2-promoted telomere shortening. To further understand the role of XPF in TRF2-dependent telomere shortening, we generated constructs containing either wild type XPF or mutant XPF proteins carrying amino acid substitutions in its conserved nuclease domain. We show that wild type XPF can complement XPF-deficient cells for repair of UV-induced DNA damage whereas the nuclease-inactive XPF proteins fail to do so, indicating that the nuclease activity of XPF is essential for nucleotide excision repair. In contrast, both wild type XPF and nuclease-inactive XPF proteins, when expressed in XPF-deficient cells, are able to rescue TRF2-mediated telomere shortening. Thus, our results suggest that the function of XPF in TRF2-mediated telomere shortening is conserved between mouse and human. Furthermore, our findings reveal an unanticipated nuclease-independent function of XPF in TRF2-mediated telomere shortening.

  12. XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of Telomeres in Tumor Cells

    Science.gov (United States)

    Taylor‐Kashton, Cheryl; Lichtensztejn, Daniel; Baloglu, Erkan; Senapedis, William; Shacham, Sharon; Kauffman, Michael G.; Kotb, Rami

    2016-01-01

    Previous work has shown that the three‐dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT‐185, KPT‐330/selinexor, and KPT‐8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment‐naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non‐lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. J. Cell. Physiol. 231: 2711–2719, 2016. © 2016 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc. PMID:26991404

  13. XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of Telomeres in Tumor Cells.

    Science.gov (United States)

    Taylor-Kashton, Cheryl; Lichtensztejn, Daniel; Baloglu, Erkan; Senapedis, William; Shacham, Sharon; Kauffman, Michael G; Kotb, Rami; Mai, Sabine

    2016-12-01

    Previous work has shown that the three-dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT-185, KPT-330/selinexor, and KPT-8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment-naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non-lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. J. Cell. Physiol. 231: 2711-2719, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. Convergence of The Nobel Fields of Telomere Biology and DNA Repair.

    Science.gov (United States)

    Fouquerel, Elise; Opresko, Patricia L

    2017-01-01

    The fields of telomere biology and DNA repair have enjoyed a great deal of cross-fertilization and convergence in recent years. Telomeres function at chromosome ends to prevent them from being falsely recognized as chromosome breaks by the DNA damage response and repair machineries. Conversely, both canonical and nonconical functions of numerous DNA repair proteins have been found to be critical for preserving telomere structure and function. In 2009, Elizabeth Blackburn, Carol Greider and Jack Szostak were awarded the Nobel prize in Physiology or Medicine for the discovery of telomeres and telomerase. Four years later, pioneers in the field of DNA repair, Aziz Sancar, Tomas Lindahl and Paul Modrich were recognized for their seminal contributions by being awarded the Nobel Prize in Chemistry. This review is part of a special issue meant to celebrate this amazing achievement, and will focus in particular on the convergence of nucleotide excision repair and telomere biology, and will discuss the profound implications for human health. © 2016 The American Society of Photobiology.

  15. Telomeres and Telomerase in The Aging Heart

    Directory of Open Access Journals (Sweden)

    Anna Meiliana

    2017-12-01

    Full Text Available BACKGROUND: Aging per se is a risk factor for reduced cardiac function and heart diseases, even when adjusted for aging-associated cardiovascular risk factors. Accordingly, aging-related biochemical and cell-biological changes lead to pathophysiological conditions, especially reduced heart function and heart disease. CONTENT: Telomere dysfunction induces a profound p53-dependent repression of the master regulators of mitochondrial biogenesis and function, peroxisome proliferator-activated receptor gamma coactivator (PGC-1a and PGC-1b in the heart, which leads to bioenergetic compromise due to impaired oxidative phosphorylation and ATP generation. This telomere-p53-PGC mitochondrial/metabolic axis integrates many factors linked to heart aging including increased DNA damage, p53 activation, mitochondrial, and metabolic dysfunction and provides a molecular basis of how dysfunctional telomeres can compromise cardiomyocytes and stem cell compartments in the heart to precipitate cardiac aging. SUMMARY: The aging myocardium with telomere shortening and accumulation of senescent cells restricts the tissue regenerative ability, which contributes to systolic or diastolic heart failure. Moreover, patients with ion-channel defects might have genetic imbalance caused by oxidative stress-related accelerated telomere shortening, which may subsequently cause sudden cardiac death. Telomere length can serve as a marker for the biological status of previous cell divisions and DNA damage with inflammation and oxidative stress. It can be integrated into current risk prediction and stratification models for cardiovascular diseases and can be used in precise personalized treatments. KEYWORDS: aging, telomere, telomerase, aging heart, mitochondria, cardiac stem cell

  16. Renal failure induces telomere shortening in the rat heart

    NARCIS (Netherlands)

    Wong, L. S.; Windt, W. A.; Roks, A. J.; van Dokkum, R. P.; Schoemaker, R. G.; de Zeeuw, D.; Henning, R. H.

    Background. Renal failure aggravates pathological cardiac remodelling induced by myocardial infarction (MI). Cardiac remodelling is associated with telomere shortening, a marker for biological ageing. We investigated whether mild and severe renal failure shorten cardiac telomeres and excessively

  17. Mice with Pulmonary Fibrosis Driven by Telomere Dysfunction

    Directory of Open Access Journals (Sweden)

    Juan M. Povedano

    2015-07-01

    Full Text Available Idiopathic pulmonary fibrosis (IPF is a degenerative disease of the lungs with an average survival post-diagnosis of 2–3 years. New therapeutic targets and treatments are necessary. Mutations in components of the telomere-maintenance enzyme telomerase or in proteins important for telomere protection are found in both familial and sporadic IPF cases. However, the lack of mouse models that faithfully recapitulate the human disease has hampered new advances. Here, we generate two independent mouse models that develop IPF owing to either critically short telomeres (telomerase-deficient mice or severe telomere dysfunction in the absence of telomere shortening (mice with Trf1 deletion in type II alveolar cells. We show that both mouse models develop pulmonary fibrosis through induction of telomere damage, thus providing proof of principle of the causal role of DNA damage stemming from dysfunctional telomeres in IPF development and identifying telomeres as promising targets for new treatments.

  18. Tumor viruses and replicative immortality--avoiding the telomere hurdle.

    Science.gov (United States)

    Chen, Xinsong; Kamranvar, Siamak Akbari; Masucci, Maria G

    2014-06-01

    Tumor viruses promote cell proliferation in order to gain access to an environment suitable for persistence and replication. The expression of viral products that promote growth transformation is often accompanied by the induction of multiple signs of telomere dysfunction, including telomere shortening, damage of telomeric DNA and chromosome instability. Long-term survival and progression to full malignancy require the bypassing of senescence programs that are triggered by the damaged telomeres. Here we review different strategies by which tumor viruses interfere with telomere homeostasis during cell transformation. This frequently involves the activation of telomerase, which assures both the integrity and functionality of telomeres. In addition, recent evidence suggests that oncogenic viruses may activate a recombination-based mechanism for telomere elongation known as Alternative Lengthening of Telomeres (ALT). This error-prone strategy promotes genomic instability and could play an important role in viral oncogenesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Linking telomere loss and mitochondrial dysfunction in chronic disease

    DEFF Research Database (Denmark)

    Gonzalez-Ebsen, Ana Carlota; Gregersen, Niels; Olsen, Rikke Kj

    2017-01-01

    Telomeres and mitochondria are known to deteriorate over time. Telomere shortening is associated with aging, early senescence, and premature cell death. Mitochondrial dysfunction produces indiscriminate amounts of reactive oxygen species that may lead to oxidative damage to cellular constituents,...

  20. Telomere stability and telomerase in mesenchymal stem cells

    DEFF Research Database (Denmark)

    Serakinci, Nedime; Graakjaer, Jesper; Kølvrå, Steen

    2008-01-01

    Telomeres are repetitive genetic material that cap and thereby protect the ends of chromosomes. Each time a cell divides, telomeres get shorter. Telomere length is mainly maintained by telomerase. This enzyme is present in high concentrations in the embryonic stem cells and in fast growing...... embryonic cells, and declines with age. It is still unclear to what extent there is telomerase in adult stem cells, but since these are the founder cells of cells of all the tissues in the body, understanding the telomere dynamics and expression of telomerase in adult stem cells is very important....... In the present communication we focus on telomere expression and telomere length in stem cells, with a special focus on mesenchymal stem cells. We consider different mechanisms by which stem cells can maintain telomeres and also focus on the dynamics of telomere length in mesenchymal stem cells, both the overall...

  1. SMARCAL1 Resolves Replication Stress at ALT Telomeres

    Directory of Open Access Journals (Sweden)

    Kelli E. Cox

    2016-02-01

    Full Text Available Cancer cells overcome replicative senescence by exploiting mechanisms of telomere elongation, a process often accomplished by reactivation of the enzyme telomerase. However, a subset of cancer cells lack telomerase activity and rely on the alternative lengthening of telomeres (ALT pathway, a recombination-based mechanism of telomere elongation. Although the mechanisms regulating ALT are not fully defined, chronic replication stress at telomeres might prime these fragile regions for recombination. Here, we demonstrate that the replication stress response protein SMARCAL1 is a critical regulator of ALT activity. SMARCAL1 associates with ALT telomeres to resolve replication stress and ensure telomere stability. In the absence of SMARCAL1, persistently stalled replication forks at ALT telomeres deteriorate into DNA double-strand breaks promoting the formation of chromosome fusions. Our studies not only define a role for SMARCAL1 in ALT telomere maintenance, but also demonstrate that resolution of replication stress is a crucial step in the ALT mechanism.

  2. Downregulation of histone methyltransferase genes SUV39H1 and SUV39H2 increases telomere length in embryonic stem-like cells and embryonic fibroblasts in pigs.

    Science.gov (United States)

    Dang-Nguyen, Thanh Quang; Haraguchi, Seiki; Furusawa, Tadashi; Somfai, Tamas; Kaneda, Masahiro; Watanabe, Shinya; Akagi, Satoshi; Kikuchi, Kazuhiro; Tajima, Atsushi; Nagai, Takashi

    2013-01-01

    Telomere is a nucleoprotein structure at the ends of chromosomes that helps to protect the ends of chromosomes from being fused with other chromosomes. Knockout of histone methyltransferases Suv39h1 and Suv39h2 increases the telomere length in murine cells, whereas downregulation of SUV39H1 and SUV39H2 genes decreases the telomere length in human cells, suggesting that telomere biology is different among mammalian species. However, epigenetic regulation of the telomere has not been studied in mammals other than the human and mouse. In the present study, the effect of knockdown of SUV39H1 and SUV39H2 genes on telomere length was examined in porcine embryonic stem-like cells (pESLCs) and porcine embryonic fibroblasts (PEFs). The telomeres in SUV39H1 and SUV39H2 knockdown (SUV39KD) pESLCs (37.1 ± 0.9 kb) were longer (Ptelomeres (22.1 ± 0.4 kb; Ptelomere elongation in SUV39KD pESLCs and SUV39KD PEFs. Relative levels of trimethylation of histone H3 lysine 9 and expressions of DNMT1, DNMT3A and DNMT3B were decreased in SUV39KD cells, suggesting that telomere lengthening in SUV39KD pESLCs and SUV39KD PEFs might be not only related to the loss of histone modification marks but also linked to the decrease in DNA methyltransferase in pigs.

  3. Telomere reprogramming and maintenance in porcine iPS cells.

    Directory of Open Access Journals (Sweden)

    Guangzhen Ji

    Full Text Available Telomere reprogramming and silencing of exogenous genes have been demonstrated in mouse and human induced pluripotent stem cells (iPS cells. Pigs have the potential to provide xenotransplant for humans, and to model and test human diseases. We investigated the telomere length and maintenance in porcine iPS cells generated and cultured under various conditions. Telomere lengths vary among different porcine iPS cell lines, some with telomere elongation and maintenance, and others telomere shortening. Porcine iPS cells with sufficient telomere length maintenance show the ability to differentiate in vivo by teratoma formation test. IPS cells with short or dysfunctional telomeres exhibit reduced ability to form teratomas. Moreover, insufficient telomerase and incomplete telomere reprogramming and/or maintenance link to sustained activation of exogenous genes in porcine iPS cells. In contrast, porcine iPS cells with reduced expression of exogenous genes or partial exogene silencing exhibit insufficient activation of endogenous pluripotent genes and telomerase genes, accompanied by telomere shortening with increasing passages. Moreover, telomere doublets, telomere sister chromatid exchanges and t-circles that presumably are involved in telomere lengthening by recombination also are found in porcine iPS cells. These data suggest that both telomerase-dependent and telomerase-independent mechanisms are involved in telomere reprogramming during induction and passages of porcine iPS cells, but these are insufficient, resulting in increased telomere damage and shortening, and chromosomal instability. Active exogenes might compensate for insufficient activation of endogenous genes and incomplete telomere reprogramming and maintenance of porcine iPS cells. Further understanding of telomere reprogramming and maintenance may help improve the quality of porcine iPS cells.

  4. Telomeres, workload and life-history in great tits

    OpenAIRE

    Atema, Els

    2017-01-01

    Ageing and the effects of increased workload in great tits A new measurement to quantify variation in quality and rate of ageing between individuals is telomere length. Telomeres are a piece of DNA at the end of chromosomes, and they protect the other DNA. In many species shortening of telomere length with increasing age was demonstrated. This shortening is accelerated by processes that also decrease life expectancy. In this project we discovered that telomeres of great tits differ from telom...

  5. Telomere reprogramming and maintenance in porcine iPS cells.

    Science.gov (United States)

    Ji, Guangzhen; Ruan, Weimin; Liu, Kai; Wang, Fang; Sakellariou, Despoina; Chen, Jijun; Yang, Yang; Okuka, Maja; Han, Jianyong; Liu, Zhonghua; Lai, Liangxue; Gagos, Sarantis; Xiao, Lei; Deng, Hongkui; Li, Ning; Liu, Lin

    2013-01-01

    Telomere reprogramming and silencing of exogenous genes have been demonstrated in mouse and human induced pluripotent stem cells (iPS cells). Pigs have the potential to provide xenotransplant for humans, and to model and test human diseases. We investigated the telomere length and maintenance in porcine iPS cells generated and cultured under various conditions. Telomere lengths vary among different porcine iPS cell lines, some with telomere elongation and maintenance, and others telomere shortening. Porcine iPS cells with sufficient telomere length maintenance show the ability to differentiate in vivo by teratoma formation test. IPS cells with short or dysfunctional telomeres exhibit reduced ability to form teratomas. Moreover, insufficient telomerase and incomplete telomere reprogramming and/or maintenance link to sustained activation of exogenous genes in porcine iPS cells. In contrast, porcine iPS cells with reduced expression of exogenous genes or partial exogene silencing exhibit insufficient activation of endogenous pluripotent genes and telomerase genes, accompanied by telomere shortening with increasing passages. Moreover, telomere doublets, telomere sister chromatid exchanges and t-circles that presumably are involved in telomere lengthening by recombination also are found in porcine iPS cells. These data suggest that both telomerase-dependent and telomerase-independent mechanisms are involved in telomere reprogramming during induction and passages of porcine iPS cells, but these are insufficient, resulting in increased telomere damage and shortening, and chromosomal instability. Active exogenes might compensate for insufficient activation of endogenous genes and incomplete telomere reprogramming and maintenance of porcine iPS cells. Further understanding of telomere reprogramming and maintenance may help improve the quality of porcine iPS cells.

  6. Mechanisms of telomere loss and their consequences for chromosome instability

    Directory of Open Access Journals (Sweden)

    Keiko eMuraki

    2012-10-01

    Full Text Available The ends of chromosomes in mammals, called telomeres, are composed of a 6 base pair repeat sequence, TTAGGG, which is added on by the enzyme telomerase. In combination with a protein complex called shelterin, these telomeric repeat sequences form a cap that protects the ends of chromosomes. Due to insufficient telomerase expression, telomeres shorten gradually with each cell division in human somatic cells, which limits the number of times they can divide. The extensive cell division involved in cancer cell progression therefore requires that cancer cells must acquire the ability to maintain telomeres, either through expression of telomerase, or through an alternative mechanism involving recombination. It is commonly thought that the source of many chromosome rearrangements in cancer cells is a result of the extensive telomere shortening that occurs prior to the expression of telomerase. However, despite the expression of telomerase, tumor cells can continue to show chromosome instability due to telomere loss. Dysfunctional telomeres in cancer cells can result from oncogene-induced replication stress, which results in double-strand breaks (DSBs at fragile sites, including telomeres. DSBs near telomeres are especially prone to chromosome rearrangements, because telomeric regions are deficient in DSB repair. The deficiency in DSB repair near telomeres is also an important mechanism for ionizing radiation-induced replicative senescence in normal human cells. In addition, DSBs near telomeres can result in chromosome instability in mouse embryonic stem cells, suggesting that telomere loss can contribute to heritable chromosome rearrangements. Consistent with this possibility, telomeric regions in humans are highly heterogeneous, and chromosome rearrangements near telomeres are commonly involved in human genetic disease. Understanding the mechanisms of telomere loss will therefore provide important insights into both human cancer and genetic disease.

  7. Characterization of oxidative guanine damage and repair in mammalian telomeres.

    Directory of Open Access Journals (Sweden)

    Zhilong Wang

    2010-05-01

    Full Text Available 8-oxo-7,8-dihydroguanine (8-oxoG and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG are among the most common oxidative DNA lesions and are substrates for 8-oxoguanine DNA glycosylase (OGG1-initiated DNA base excision repair (BER. Mammalian telomeres consist of triple guanine repeats and are subject to oxidative guanine damage. Here, we investigated the impact of oxidative guanine damage and its repair by OGG1 on telomere integrity in mice. The mouse cells were analyzed for telomere integrity by telomere quantitative fluorescence in situ hybridization (telomere-FISH, by chromosome orientation-FISH (CO-FISH, and by indirect immunofluorescence in combination with telomere-FISH and for oxidative base lesions by Fpg-incision/Southern blot assay. In comparison to the wild type, telomere lengthening was observed in Ogg1 null (Ogg1(-/- mouse tissues and primary embryonic fibroblasts (MEFs cultivated in hypoxia condition (3% oxygen, whereas telomere shortening was detected in Ogg1(-/- mouse hematopoietic cells and primary MEFs cultivated in normoxia condition (20% oxygen or in the presence of an oxidant. In addition, telomere length abnormalities were accompanied by altered telomere sister chromatid exchanges, increased telomere single- and double-strand breaks, and preferential telomere lagging- or G-strand losses in Ogg1(-/- mouse cells. Oxidative guanine lesions were increased in telomeres in Ogg1(-/- mice with aging and primary MEFs cultivated in 20% oxygen. Furthermore, oxidative guanine lesions persisted at high level in Ogg1(-/- MEFs after acute exposure to hydrogen peroxide, while they rapidly returned to basal level in wild-type MEFs. These findings indicate that oxidative guanine damage can arise in telomeres where it affects length homeostasis, recombination, DNA replication, and DNA breakage repair. Our studies demonstrate that BER pathway is required in repairing oxidative guanine damage in telomeres and maintaining telomere integrity

  8. Telomeres, workload and life-history in great tits

    NARCIS (Netherlands)

    Atema, Els

    2017-01-01

    Ageing and the effects of increased workload in great tits A new measurement to quantify variation in quality and rate of ageing between individuals is telomere length. Telomeres are a piece of DNA at the end of chromosomes, and they protect the other DNA. In many species shortening of telomere

  9. Acute coronary syndrome: Role of the telomere dynamic | Behjati ...

    African Journals Online (AJOL)

    Telomeres, or historically named "terminal genes" are first discovered by Muller working on fruit fly in 1930s. Since then, the great progress was made in understanding the consequences of telomere erosion on the human health and disease states, as age related vascular diseases. The overlapping links between telomere ...

  10. Approaching TERRA Firma: Genomic Functions of Telomeric Noncoding RNA.

    Science.gov (United States)

    Roake, Caitlin M; Artandi, Steven E

    2017-06-29

    Functions of the telomeric repeat-containing RNA (TERRA), the long noncoding RNA (lncRNA) transcribed from telomeres, have eluded researchers. In this issue of Cell, Graf el al. and Chu et al. uncover new regulatory roles for TERRA at the telomere and at distant genomic sites. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Telomere Length Correlates with Life Span of Dog Breeds

    Directory of Open Access Journals (Sweden)

    Laura J. Fick

    2012-12-01

    Full Text Available Telomeric DNA repeats are lost as normal somatic cells replicate. When telomeres reach a critically short length, a DNA damage signal is initiated, inducing cell senescence. Some studies have indicated that telomere length correlates with mortality, suggesting that telomere length contributes to human life span; however, other studies report no correlation, and thus the issue remains controversial. Domestic dogs show parallels in telomere biology to humans, with similar telomere length, telomere attrition, and absence of somatic cell telomerase activity. Using this model, we find that peripheral blood mononuclear cell (PBMC telomere length is a strong predictor of average life span among 15 different breeds (p < 0.0001, consistent with telomeres playing a role in life span determination. Dogs lose telomeric DNA ∼10-fold faster than humans, which is similar to the ratio of average life spans between these species. Breeds with shorter mean telomere lengths show an increased probability of death from cardiovascular disease, which was previously correlated with short telomere length in humans.

  12. An evolutionary review of human telomere biology: the thrifty telomere hypothesis and notes on potential adaptive paternal effects.

    Science.gov (United States)

    Eisenberg, Dan T A

    2011-01-01

    Telomeres, repetitive DNA sequences found at the ends of linear chromosomes, play a role in regulating cellular proliferation, and shorten with increasing age in proliferating human tissues. The rate of age-related shortening of telomeres is highest early in life and decreases with age. Shortened telomeres are thought to limit the proliferation of cells and are associated with increased morbidity and mortality. Although natural selection is widely assumed to operate against long telomeres because they entail increased cancer risk, the evidence for this is mixed. Instead, here it is proposed that telomere length is primarily limited by energetic constraints. Cell proliferation is energetically expensive, so shorter telomeres should lead to a thrifty phenotype. Shorter telomeres are proposed to restrain adaptive immunity as an energy saving mechanism. Such a limited immune system, however, might also result in chronic infections, inflammatory stress, premature aging, and death--a more "disposable soma." With an increased reproductive lifespan, the fitness costs of premature aging are higher and longer telomeres will be favored by selection. Telomeres exhibit a paternal effect whereby the offspring of older fathers have longer telomeres due to increased telomere lengths of sperm with age. This paternal effect is proposed to be an adaptive signal of the expected age of male reproduction in the environment offspring are born into. The offspring of lineages of older fathers will tend to have longer, and thereby less thrifty, telomeres, better preparing them for an environment with higher expected ages at reproduction. Copyright © 2010 Wiley-Liss, Inc.

  13. Telomere length is shorter in healthy offspring of subjects with coronary artery disease : support for the telomere hypothesis

    NARCIS (Netherlands)

    Brouilette, S. W.; Whittaker, A.; Stevens, S. E.; van der Harst, P.; Goodall, A. H.; Samani, N. J.

    Background: Telomeres are shorter in subjects with coronary artery disease (CAD) and may indicate premature biological ageing. However, whether shorter telomeres are a primary abnormality or secondary to the disease is unclear. Objective: To investigate whether shorter telomeres are a primary

  14. Telomere length alterations unique to invasive lobular carcinoma.

    Science.gov (United States)

    Heaphy, Christopher M; Asch-Kendrick, Rebecca; Argani, Pedram; Meeker, Alan K; Cimino-Mathews, Ashley

    2015-08-01

    Telomeres are nucleoprotein complexes located at the extreme ends of eukaryotic chromosomes and protect chromosomal ends from degradation and recombination. Dysfunctional telomeres contribute to genomic instability, promote tumorigenesis, and, in breast cancer, have been associated with increased cancer risk and poor prognosis. Short telomere lengths have been previously associated with triple-negative and human epidermal growth factor receptor (Her2)--positive ductal carcinomas. However, these investigations have not specifically assessed invasive lobular carcinomas (ILCs), which accounts for 5% to 15% of all invasive breast cancers. Here, we evaluate telomere lengths within 48 primary ILCs with complete characterization of estrogen receptor (ER), progesterone receptor (PR), and Her2 status, including 32 luminal/Her2- (ER+/PR+/Her2-), 8 luminal/Her2+ (ER+/PR+/Her2+), 3 Her2+ (ER-/PR-/Her2+), and 5 triple-negative (ER-/PR-/Her2-) carcinomas. A telomere-specific fluorescence in situ hybridization assay, which provides single-cell telomere length resolution, was used to evaluate telomere lengths and compare with standard clinicopathological markers. In contrast to breast ductal carcinoma, in which more than 85% of cases display abnormally short telomeres, approximately half (52%) of the ILCs displayed either normal or long telomeres. Short telomere length was associated with older patient age. Interestingly, 3 cases (6%) displayed a unique telomere pattern consisting of 1 or 2 bright telomere spots among the normal telomere signals within each individual cancer cell, a phenotype that has not been previously described. Additional studies are needed to further evaluate the significance of the unique bright telomere spot phenotype and the potential utility of telomere length as a prognostic marker in ILC. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Telomere Length Dynamics and the Evolution of Cancer Genome Architecture

    Directory of Open Access Journals (Sweden)

    Kez Cleal

    2018-02-01

    Full Text Available Telomeres are progressively eroded during repeated rounds of cell division due to the end replication problem but also undergo additional more substantial stochastic shortening events. In most cases, shortened telomeres induce a cell-cycle arrest or trigger apoptosis, although for those cells that bypass such signals during tumour progression, a critical length threshold is reached at which telomere dysfunction may ensue. Dysfunction of the telomere nucleoprotein complex can expose free chromosome ends to the DNA double-strand break (DSB repair machinery, leading to telomere fusion with both telomeric and non-telomeric loci. The consequences of telomere fusions in promoting genome instability have long been appreciated through the breakage–fusion–bridge (BFB cycle mechanism, although recent studies using high-throughput sequencing technologies have uncovered evidence of involvement in a wider spectrum of genomic rearrangements including chromothripsis. A critical step in cancer progression is the transition of a clone to immortality, through the stabilisation of the telomere repeat array. This can be achieved via the reactivation of telomerase, or the induction of the alternative lengthening of telomeres (ALT pathway. Whilst telomere dysfunction may promote genome instability and tumour progression, by limiting the replicative potential of a cell and enforcing senescence, telomere shortening can act as a tumour suppressor mechanism. However, the burden of senescent cells has also been implicated as a driver of ageing and age-related pathology, and in the promotion of cancer through inflammatory signalling. Considering the critical role of telomere length in governing cancer biology, we review questions related to the prognostic value of studying the dynamics of telomere shortening and fusion, and discuss mechanisms and consequences of telomere-induced genome rearrangements.

  16. Tetrafluoroethylene telomerization initiated by benzoyl peroxide

    Science.gov (United States)

    Bolshakov, A. I.; Kuzina, S. I.; Kiryukhin, D. P.

    2017-03-01

    The radical telomerization of tetrafluoroethylene initiated by benzoyl peroxide (BP) photolysis at λ ≥ 365 nm is studied in acetone, dichloromethane, carbon tetrachloride, and Freon 114B2 at 25°C. The products of synthesis are a mixture of telomers of different molar masses, segregated into soluble and insoluble fractions. To characterize the radicals initiating telomerization, crystalline BP and its solution in ethanol are subjected to low-temperature (77 K) photolysis, with the liquid system serving as a model for BP behavior in solutions of telogens. It is established that radicals are not only initiators but also participate in chain termination reactions, lowering the telomers' molar mass and thus raising the proportion of the soluble fraction. Telomerization initiated by an initiator compound versus initiation by gamma radiation are compared and discussed.

  17. Mre11 and Blm-Dependent Formation of ALT-Like Telomeres in Ku-Deficient Ustilago maydis.

    Science.gov (United States)

    Yu, Eun Young; Pérez-Martín, José; Holloman, William K; Lue, Neal F

    2015-10-01

    A subset of human cancer cells uses a specialized, aberrant recombination pathway known as ALT to maintain telomeres, which in these cells are characterized by complex aberrations including length heterogeneity, high levels of unpaired C-strand, and accumulation of extra-chromosomal telomere repeats (ECTR). These phenotypes have not been recapitulated in any standard budding or fission yeast mutant. We found that eliminating Ku70 or Ku80 in the yeast-like fungus Ustilago maydis results initially in all the characteristic telomere aberrations of ALT cancer cells, including C-circles, a highly specific marker of ALT. Subsequently the ku mutants experience permanent G2 cell cycle arrest, accompanied by loss of telomere repeats from chromosome ends and even more drastic accumulation of very short ECTRs (vsECTRs). The deletion of atr1 or chk1 rescued the lethality of the ku mutant, and "trapped" the telomere aberrations in the early ALT-like stage. Telomere abnormalities are telomerase-independent, but dramatically suppressed by deletion of mre11 or blm, suggesting major roles for these factors in the induction of the ALT pathway. In contrast, removal of other DNA damage response and repair factors such as Rad51 has disparate effects on the ALT phenotypes, suggesting that these factors process ALT intermediates or products. Notably, the antagonism of Ku and Mre11 in the induction of ALT is reminiscent of their roles in DSB resection, in which Blm is also known to play a key role. We suggest that an aberrant resection reaction may constitute an early trigger for ALT telomeres, and that the outcomes of ALT are distinct from DSB because of the unique telomere nucleoprotein structure.

  18. Mre11 and Blm-Dependent Formation of ALT-Like Telomeres in Ku-Deficient Ustilago maydis.

    Directory of Open Access Journals (Sweden)

    Eun Young Yu

    2015-10-01

    Full Text Available A subset of human cancer cells uses a specialized, aberrant recombination pathway known as ALT to maintain telomeres, which in these cells are characterized by complex aberrations including length heterogeneity, high levels of unpaired C-strand, and accumulation of extra-chromosomal telomere repeats (ECTR. These phenotypes have not been recapitulated in any standard budding or fission yeast mutant. We found that eliminating Ku70 or Ku80 in the yeast-like fungus Ustilago maydis results initially in all the characteristic telomere aberrations of ALT cancer cells, including C-circles, a highly specific marker of ALT. Subsequently the ku mutants experience permanent G2 cell cycle arrest, accompanied by loss of telomere repeats from chromosome ends and even more drastic accumulation of very short ECTRs (vsECTRs. The deletion of atr1 or chk1 rescued the lethality of the ku mutant, and "trapped" the telomere aberrations in the early ALT-like stage. Telomere abnormalities are telomerase-independent, but dramatically suppressed by deletion of mre11 or blm, suggesting major roles for these factors in the induction of the ALT pathway. In contrast, removal of other DNA damage response and repair factors such as Rad51 has disparate effects on the ALT phenotypes, suggesting that these factors process ALT intermediates or products. Notably, the antagonism of Ku and Mre11 in the induction of ALT is reminiscent of their roles in DSB resection, in which Blm is also known to play a key role. We suggest that an aberrant resection reaction may constitute an early trigger for ALT telomeres, and that the outcomes of ALT are distinct from DSB because of the unique telomere nucleoprotein structure.

  19. Identification of two human nuclear proteins that recognise the cytosine-rich strand of human telomeres in vitro

    Science.gov (United States)

    Lacroix, Laurent; Liénard, Hélène; Labourier, Emmanuel; Djavaheri-Mergny, Mojgan; Lacoste, Jérôme; Leffers, Henrik; Tazi, Jamal; Hélène, Claude; Mergny, Jean-Louis

    2000-01-01

    Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)n repeat] with high affinity and bind double-stranded telomeric DNA with a 100× reduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66–64, 45 and 35 kDa, respectively. To identify these polypeptides we screened a λgt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro. PMID:10710423

  20. The Drosophila HOAP protein is required for telomere capping.

    Science.gov (United States)

    Cenci, Giovanni; Siriaco, Giorgia; Raffa, Grazia D; Kellum, Rebecca; Gatti, Maurizio

    2003-01-01

    HOAP (HP1/ORC-associated protein) has recently been isolated from Drosophila melanogaster embryos as part of a cytoplasmic complex that contains heterochromatin protein 1 (HP1) and the origin recognition complex subunit 2 (ORC2). Here, we show that caravaggio, a mutation in the HOAP-encoding gene, causes extensive telomere-telomere fusions in larval brain cells, indicating that HOAP is required for telomere capping. Our analyses indicate that HOAP is specifically enriched at mitotic chromosome telomeres, and strongly suggest that HP1 and HOAP form a telomere-capping complex that does not contain ORC2.

  1. Telomeres and the natural lifespan limit in humans

    DEFF Research Database (Denmark)

    Steenstrup, Troels; Kark, Jeremy D; Verhulst, Simon

    2017-01-01

    An ongoing debate in demography has focused on whether the human lifespan has a maximal natural limit. Taking a mechanistic perspective, and knowing that short telomeres are associated with diminished longevity, we examined whether telomere length dynamics during adult life could set a maximal...... natural lifespan limit. We define leukocyte telomere length of 5 kb as the 'telomeric brink', which denotes a high risk of imminent death. We show that a subset of adults may reach the telomeric brink within the current life expectancy and more so for a 100-year life expectancy. Thus, secular trends...

  2. No Association between Mean Telomere Length and Life Stress Observed in a 30 Year Birth Cohort

    Science.gov (United States)

    Jodczyk, Sarah; Fergusson, David M.; Horwood, L. John; Pearson, John F.; Kennedy, Martin A.

    2014-01-01

    Telomeres are specialised structures that cap the ends of chromosomes. They shorten with each cell division and have been proposed as a marker of cellular aging. Previous studies suggest that early life stressors increase the rate of telomere shortening with potential impact on disease states and mortality later in life. This study examined the associations between telomere length and exposure to a number of stressors that arise during development from the antenatal/perinatal period through to young adulthood. Participants were from the Christchurch Health and Development Study (CHDS), a New Zealand longitudinal birth cohort which has followed participants from birth until age 30. Telomere length was obtained on DNA from peripheral blood samples collected from consenting participants (n = 677) at age 28–30, using a quantitative PCR assay. These data were assessed for associations with 26 measures of life course adversity or stress which occurred prior to 25 years of age. No associations were found between telomere length measured at age 28–30 years and life course adversity or stress for specific measures and for the summary risk scores for each developmental domain. The correlations were very small ranging from −0.06 to 0.06 with a median of 0.01, and none were statistically significant. Our results in this well-studied birth cohort do not support prior reports of such associations, and underscore the need for more extensive replication of proposed links between stress and telomere biology in larger cohorts with appropriate phenotypic data. PMID:24816913

  3. No association between mean telomere length and life stress observed in a 30 year birth cohort.

    Directory of Open Access Journals (Sweden)

    Sarah Jodczyk

    Full Text Available Telomeres are specialised structures that cap the ends of chromosomes. They shorten with each cell division and have been proposed as a marker of cellular aging. Previous studies suggest that early life stressors increase the rate of telomere shortening with potential impact on disease states and mortality later in life. This study examined the associations between telomere length and exposure to a number of stressors that arise during development from the antenatal/perinatal period through to young adulthood. Participants were from the Christchurch Health and Development Study (CHDS, a New Zealand longitudinal birth cohort which has followed participants from birth until age 30. Telomere length was obtained on DNA from peripheral blood samples collected from consenting participants (n = 677 at age 28-30, using a quantitative PCR assay. These data were assessed for associations with 26 measures of life course adversity or stress which occurred prior to 25 years of age. No associations were found between telomere length measured at age 28-30 years and life course adversity or stress for specific measures and for the summary risk scores for each developmental domain. The correlations were very small ranging from -0.06 to 0.06 with a median of 0.01, and none were statistically significant. Our results in this well-studied birth cohort do not support prior reports of such associations, and underscore the need for more extensive replication of proposed links between stress and telomere biology in larger cohorts with appropriate phenotypic data.

  4. Telomere in Aging and Age-Related Diseases

    Directory of Open Access Journals (Sweden)

    Anna Meiliana

    2017-12-01

    Full Text Available BACKGROUND: The number of elderly population in the world keep increasing. In their advanced ages, many elderly face years of disability because of multiple chronic diseases, frailty, making them lost their independence. Consequently, this could have impacts on social and economic stability. A huge challenge has been sent for biomedical researchers to compress or at least eliminate this period of disability and increase the health span. CONTENT: Over the past decades, many studies of telomere biology have demonstrated that telomeres and telomere-associated proteins are implicated in human diseases. Accelerated telomere erosion was clearly correlated with a pack of metabolic and inflammatory diseases. Critically short telomeres or the unprotected end, are likely to form telomeric fusion, generating genomic instability, the cornerstone for carcinogenesis. Enlightening how telomeres involved in the mechanisms underlying the diseases’ pathogenesis was expected to uncover new molecular targets for any important diagnosis or therapeutic implications. SUMMARY: Telomere shortening was foreseen as an imporant mechanism to supress tumor by limiting cellular proliferative capacity by regulating senescence check point activation. Many human diseases and carcinogenesis are causally related to defective telomeres, asserting the importance of telomeres sustainment. Thus, telomere length assessment might serve as an important tool for clinical prognostic, diagnostic, monitoring and management. KEYWORDS: telomerase, cellular senescence, aging, cancer

  5. Telomere Biology—Insights into an Intriguing Phenomenon

    Directory of Open Access Journals (Sweden)

    Shriram Venkatesan

    2017-06-01

    Full Text Available Bacteria and viruses possess circular DNA, whereas eukaryotes with typically very large DNA molecules have had to evolve into linear chromosomes to circumvent the problem of supercoiling circular DNA of that size. Consequently, such organisms possess telomeres to cap chromosome ends. Telomeres are essentially tandem repeats of any DNA sequence that are present at the ends of chromosomes. Their biology has been an enigmatic one, involving various molecules interacting dynamically in an evolutionarily well-trimmed fashion. Telomeres range from canonical hexameric repeats in most eukaryotes to unimaginably random retrotransposons, which attach to chromosome ends and reverse-transcribe to DNA in some plants and insects. Telomeres invariably associate with specialised protein complexes that envelop it, also regulating access of the ends to legitimate enzymes involved in telomere metabolism. They also transcribe into repetitive RNA which also seems to be playing significant roles in telomere maintenance. Telomeres thus form the intersection of DNA, protein, and RNA molecules acting in concert to maintain chromosome integrity. Telomere biology is emerging to appear ever more complex than previously envisaged, with the continual discovery of more molecules and interplays at the telomeres. This review also includes a section dedicated to the history of telomere biology, and intends to target the scientific audience new to the field by rendering an understanding of the phenomenon of chromosome end protection at large, with more emphasis on the biology of human telomeres. The review provides an update on the field and mentions the questions that need to be addressed.

  6. Telomere tracking from birth to adulthood and residential traffic exposure.

    Science.gov (United States)

    Bijnens, Esmée M; Zeegers, Maurice P; Derom, Catherine; Martens, Dries S; Gielen, Marij; Hageman, Geja J; Plusquin, Michelle; Thiery, Evert; Vlietinck, Robert; Nawrot, Tim S

    2017-11-21

    Telomere attrition is extremely rapid during the first years of life, while lifestyle during adulthood exerts a minor impact. This suggests that early life is an important period in the determination of telomere length. We investigated the importance of the early-life environment on both telomere tracking and adult telomere length. Among 184 twins of the East Flanders Prospective Twin Survey, telomere length in placental tissue and in buccal cells in young adulthood was measured. Residential addresses at birth and in young adulthood were geocoded and residential traffic and greenness exposure was determined. We investigated individual telomere tracking from birth over a 20 year period (mean age (SD), 22.6 (3.1) years) in association with residential exposure to traffic and greenness. Telomere length in placental tissue and in buccal cells in young adulthood correlated positively (r = 0.31, P adulthood was negatively and significantly associated with residential traffic exposure at the birth address, while traffic exposure at the residential address at adult age was not associated with telomere length. Longitudinal evidence of telomere length tracking from birth to adulthood shows inverse associations of residential traffic exposure in association with telomere length at birth as well as accelerated telomere shortening in the first two decades of life.

  7. TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe.

    Science.gov (United States)

    Moravec, Martin; Wischnewski, Harry; Bah, Amadou; Hu, Yan; Liu, Na; Lafranchi, Lorenzo; King, Megan C; Azzalin, Claus M

    2016-07-01

    Telomerase-mediated telomere elongation provides cell populations with the ability to proliferate indefinitely. Telomerase is capable of recognizing and extending the shortest telomeres in cells; nevertheless, how this mechanism is executed remains unclear. Here, we show that, in the fission yeast Schizosaccharomyces pombe, shortened telomeres are highly transcribed into the evolutionarily conserved long noncoding RNA TERRA A fraction of TERRA produced upon telomere shortening is polyadenylated and largely devoid of telomeric repeats, and furthermore, telomerase physically interacts with this polyadenylated TERRA in vivo We also show that experimentally enhanced transcription of a manipulated telomere promotes its association with telomerase and concomitant elongation. Our data represent the first direct evidence that TERRA stimulates telomerase recruitment and activity at chromosome ends in an organism with human-like telomeres. © 2016 The Authors.

  8. Homeostasis of telomere length rather than telomere shortening after allogeneic peripheral blood stem cell transplantation

    NARCIS (Netherlands)

    Roelofs, Helene; de Pauw, Elmar S. D.; Zwinderman, Aeilko H.; Opdam, Sonja M.; Willemze, Roel; Tanke, Hans J.; Fibbe, Willem E.

    2003-01-01

    Hematopoietic reconstitution after stem cell transplantation requires excessive replicative activity because of the limited number of stem cells that are used for transplantation. Telomere shortening has been detected in hematopoietic cells after bone marrow transplantation. This has been thought to

  9. Repair of UV-induced DNA lesions in natural Saccharomyces cerevisiae telomeres is moderated by Sir2 and Sir3, and inhibited by yKu–Sir4 interaction

    Science.gov (United States)

    Guintini, Laetitia; Tremblay, Maxime; Toussaint, Martin; D’Amours, Annie; Wellinger, Ralf E.

    2017-01-01

    Abstract Ultraviolet light (UV) causes DNA damage that is removed by nucleotide excision repair (NER). UV-induced DNA lesions must be recognized and repaired in nucleosomal DNA, higher order structures of chromatin and within different nuclear sub-compartments. Telomeric DNA is made of short tandem repeats located at the ends of chromosomes and their maintenance is critical to prevent genome instability. In Saccharomyces cerevisiae the chromatin structure of natural telomeres is distinctive and contingent to telomeric DNA sequences. Namely, nucleosomes and Sir proteins form the heterochromatin like structure of X-type telomeres, whereas a more open conformation is present at Y’-type telomeres. It is proposed that there are no nucleosomes on the most distal telomeric repeat DNA, which is bound by a complex of proteins and folded into higher order structure. How these structures affect NER is poorly understood. Our data indicate that the X-type, but not the Y’-type, sub-telomeric chromatin modulates NER, a consequence of Sir protein-dependent nucleosome stability. The telomere terminal complex also prevents NER, however, this effect is largely dependent on the yKu–Sir4 interaction, but Sir2 and Sir3 independent. PMID:28334768

  10. Repair of UV-induced DNA lesions in natural Saccharomyces cerevisiae telomeres is moderated by Sir2 and Sir3, and inhibited by yKu-Sir4 interaction.

    Science.gov (United States)

    Guintini, Laetitia; Tremblay, Maxime; Toussaint, Martin; D'Amours, Annie; Wellinger, Ralf E; Wellinger, Raymund J; Conconi, Antonio

    2017-05-05

    Ultraviolet light (UV) causes DNA damage that is removed by nucleotide excision repair (NER). UV-induced DNA lesions must be recognized and repaired in nucleosomal DNA, higher order structures of chromatin and within different nuclear sub-compartments. Telomeric DNA is made of short tandem repeats located at the ends of chromosomes and their maintenance is critical to prevent genome instability. In Saccharomyces cerevisiae the chromatin structure of natural telomeres is distinctive and contingent to telomeric DNA sequences. Namely, nucleosomes and Sir proteins form the heterochromatin like structure of X-type telomeres, whereas a more open conformation is present at Y'-type telomeres. It is proposed that there are no nucleosomes on the most distal telomeric repeat DNA, which is bound by a complex of proteins and folded into higher order structure. How these structures affect NER is poorly understood. Our data indicate that the X-type, but not the Y'-type, sub-telomeric chromatin modulates NER, a consequence of Sir protein-dependent nucleosome stability. The telomere terminal complex also prevents NER, however, this effect is largely dependent on the yKu-Sir4 interaction, but Sir2 and Sir3 independent. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Telomeric expression sites are highly conserved in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Christiane Hertz-Fowler

    Full Text Available Subtelomeric regions are often under-represented in genome sequences of eukaryotes. One of the best known examples of the use of telomere proximity for adaptive purposes are the bloodstream expression sites (BESs of the African trypanosome Trypanosoma brucei. To enhance our understanding of BES structure and function in host adaptation and immune evasion, the BES repertoire from the Lister 427 strain of T. brucei were independently tagged and sequenced. BESs are polymorphic in size and structure but reveal a surprisingly conserved architecture in the context of extensive recombination. Very small BESs do exist and many functioning BESs do not contain the full complement of expression site associated genes (ESAGs. The consequences of duplicated or missing ESAGs, including ESAG9, a newly named ESAG12, and additional variant surface glycoprotein genes (VSGs were evaluated by functional assays after BESs were tagged with a drug-resistance gene. Phylogenetic analysis of constituent ESAG families suggests that BESs are sequence mosaics and that extensive recombination has shaped the evolution of the BES repertoire. This work opens important perspectives in understanding the molecular mechanisms of antigenic variation, a widely used strategy for immune evasion in pathogens, and telomere biology.

  12. Telomeric protein TRF2 protects Holliday junctions with telomeric arms from displacement by the Werner syndrome helicase.

    Science.gov (United States)

    Nora, Gerald J; Buncher, Noah A; Opresko, Patricia L

    2010-07-01

    WRN protein loss causes Werner syndrome (WS), which is characterized by premature aging as well as genomic and telomeric instability. WRN prevents telomere loss, but the telomeric protein complex must regulate WRN activities to prevent aberrant telomere processing. Telomere-binding TRF2 protein inhibits telomere t-loop deletion by blocking Holliday junction (HJ) resolvase cleavage activity, but whether TRF2 also modulates HJ displacement at t-loops is unknown. In this study, we used multiplex fluorophore imaging to track the fate of individual strands of HJ substrates. We report the novel finding that TRF2 inhibits WRN helicase strand displacement of HJs with telomeric repeats in duplex arms, but unwinding of HJs with a telomeric center or lacking telomeric sequence is unaffected. These data, together with results using TRF2 fragments and TRF2 HJ binding assays, indicate that both the TRF2 B- and Myb domains are required to inhibit WRN HJ activity. We propose a novel model whereby simultaneous binding of the TRF2 B-domain to the HJ core and the Myb domain to telomeric arms promote and stabilize HJs in a stacked arm conformation that is unfavorable for unwinding. Our biochemical study provides a mechanistic basis for the cellular findings that TRF2 regulates WRN activity at telomeres.

  13. Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions.

    Science.gov (United States)

    Nera, Bernadette; Huang, Hui-Shun; Lai, Thao; Xu, Lifeng

    2015-12-07

    The shelterin protein TRF2 is essential for chromosome-end protection. Depletion of TRF2 causes chromosome end-to-end fusions, initiating genomic instability that can be cancer promoting. Paradoxically, significant increased levels of TRF2 are observed in a subset of human cancers. Experimental overexpression of TRF2 has also been shown to induce telomere shortening, through an unknown mechanism. Here we report that TRF2 overexpression results in replication stalling in duplex telomeric repeat tracts and the subsequent formation of telomeric ultrafine anaphase bridges (UFBs), ultimately leading to stochastic loss of telomeric sequences. These TRF2 overexpression-induced telomere deletions generate chromosome fusions resembling those detected in human cancers and in mammalian cells containing critically shortened telomeres. Therefore, our findings have uncovered a second pathway by which altered TRF2 protein levels can induce end-to-end fusions. The observations also provide mechanistic insight into the molecular basis of genomic instability in tumour cells containing significantly increased TRF2 levels.

  14. Paternal age and telomere length in twins

    DEFF Research Database (Denmark)

    Hjelmborg, Jacob B; Dalgård, Christine; Mangino, Massimo

    2015-01-01

    . Based on two independent (discovery and replication) twin studies, comprising 889 twin pairs, we show an increase in the resemblance of leukocyte telomere length between dizygotic twins of older fathers, which is not seen in monozygotic twins. This phenomenon might result from a paternal age...

  15. Twin correlations of telomere length metrics

    DEFF Research Database (Denmark)

    Hjelmborg, Jacob B; Dalgård, Christine; Möller, Sören

    2015-01-01

    BACKGROUND: Leucocyte telomere length (LTL) is a complex trait associated with ageing and longevity. LTL dynamics are defined by LTL and its age-dependent attrition. Strong, but indirect evidence suggests that LTL at birth and its attrition during childhood largely explains interindividual LTL va...

  16. Molecular architecture of classical cytological landmarks: Centromeres and telomeres

    Energy Technology Data Exchange (ETDEWEB)

    Meyne, J.

    1994-11-01

    Both the human telomere repeat and the pericentromeric repeat sequence (GGAAT)n were isolated based on evolutionary conservation. Their isolation was based on the premise that chromosomal features as structurally and functionally important as telomeres and centromeres should be highly conserved. Both sequences were isolated by high stringency screening of a human repetitive DNA library with rodent repetitive DNA. The pHuR library (plasmid Human Repeat) used for this project was enriched for repetitive DNA by using a modification of the standard DNA library preparation method. Usually DNA for a library is cut with restriction enzymes, packaged, infected, and the library is screened. A problem with this approach is that many tandem repeats don`t have any (or many) common restriction sites. Therefore, many of the repeat sequences will not be represented in the library because they are not restricted to a viable length for the vector used. To prepare the pHuR library, human DNA was mechanically sheared to a small size. These relatively short DNA fragments were denatured and then renatured to C{sub o}t 50. Theoretically only repetitive DNA sequences should renature under C{sub o}t 50 conditions. The single-stranded regions were digested using S1 nuclease, leaving the double-stranded, renatured repeat sequences.

  17. Exploring possible DNA structures in real-time polymerase kinetics using Pacific Biosciences sequencer data.

    Science.gov (United States)

    Sawaya, Sterling; Boocock, James; Black, Michael A; Gemmell, Neil J

    2015-01-28

    Pausing of DNA polymerase can indicate the presence of a DNA structure that differs from the canonical double-helix. Here we detail a method to investigate how polymerase pausing in the Pacific Biosciences sequencer reads can be related to DNA sequences. The Pacific Biosciences sequencer uses optics to view a polymerase and its interaction with a single DNA molecule in real-time, offering a unique way to detect potential alternative DNA structures. We have developed a new way to examine polymerase kinetics data and relate it to the DNA sequence by using a wavelet transform of read information from the sequencer. We use this method to examine how polymerase kinetics are related to nucleotide base composition. We then examine tandem repeat sequences known for their ability to form different DNA structures: (CGG)n and (CG)n repeats which can, respectively, form G-quadruplex DNA and Z-DNA. We find pausing around the (CGG)n repeat that may indicate the presence of G-quadruplexes in some of the sequencer reads. The (CG)n repeat does not appear to cause polymerase pausing, but its kinetics signature nevertheless suggests the possibility that alternative nucleotide conformations may sometimes be present. We discuss the implications of using our method to discover DNA sequences capable of forming alternative structures. The analyses presented here can be reproduced on any Pacific Biosciences kinetics data for any DNA pattern of interest using an R package that we have made publicly available.

  18. Human Rap1 interacts directly with telomeric DNA and regulates TRF2 localization at the telomere.

    Science.gov (United States)

    Arat, N Özlem; Griffith, Jack D

    2012-12-07

    The TRF2-Rap1 complex suppresses non-homologous end joining and interacts with DNAPK-C to prevent end joining. We previously demonstrated that hTRF2 is a double strand telomere binding protein that forms t-loops in vitro and recognizes three- and four-way junctions independent of DNA sequence. How the DNA binding characteristics of hTRF2 to DNA is altered in the presence of hRap1 however is not known. Here we utilized EM and quantitative gel retardation to characterize the DNA binding properties of hRap1 and the TRF2-Rap1 complex. Both gel filtration chromatography and mass analysis from two-dimensional projections showed that the TRF2-Rap1 complex exists in solution and binds to DNA as a complex consisting of four monomers each of hRap1 and hTRF2. EM revealed for the first time that hRap1 binds to DNA templates in the absence of hTRF2 with a preference for double strand-single strand junctions in a sequence independent manner. When hTRF2 and hRap1 are in a complex, its affinity for ds telomeric sequences is 2-fold higher than TRF2 alone and more than 10-fold higher for telomeric 3' ends. This suggests that as hTRF2 recruits hRap1 to telomeric sequences, hRap1 alters the affinity of hTRF2 and its binding preference on telomeric DNA. Moreover, the TRF2-Rap1 complex has higher ability to re-model telomeric DNA than either component alone. This finding underlies the importance of complex formation between hRap1 and hTRF2 for telomere function and end protection.

  19. Human Rap1 Interacts Directly with Telomeric DNA and Regulates TRF2 Localization at the Telomere*

    Science.gov (United States)

    Arat, N. Özlem; Griffith, Jack D.

    2012-01-01

    The TRF2-Rap1 complex suppresses non-homologous end joining and interacts with DNAPK-C to prevent end joining. We previously demonstrated that hTRF2 is a double strand telomere binding protein that forms t-loops in vitro and recognizes three- and four-way junctions independent of DNA sequence. How the DNA binding characteristics of hTRF2 to DNA is altered in the presence of hRap1 however is not known. Here we utilized EM and quantitative gel retardation to characterize the DNA binding properties of hRap1 and the TRF2-Rap1 complex. Both gel filtration chromatography and mass analysis from two-dimensional projections showed that the TRF2-Rap1 complex exists in solution and binds to DNA as a complex consisting of four monomers each of hRap1 and hTRF2. EM revealed for the first time that hRap1 binds to DNA templates in the absence of hTRF2 with a preference for double strand-single strand junctions in a sequence independent manner. When hTRF2 and hRap1 are in a complex, its affinity for ds telomeric sequences is 2-fold higher than TRF2 alone and more than 10-fold higher for telomeric 3′ ends. This suggests that as hTRF2 recruits hRap1 to telomeric sequences, hRap1 alters the affinity of hTRF2 and its binding preference on telomeric DNA. Moreover, the TRF2-Rap1 complex has higher ability to re-model telomeric DNA than either component alone. This finding underlies the importance of complex formation between hRap1 and hTRF2 for telomere function and end protection. PMID:23086976

  20. Short telomeres in hatchling snakes: erythrocyte telomere dynamics and longevity in tropical pythons.

    Directory of Open Access Journals (Sweden)

    Beata Ujvari

    Full Text Available BACKGROUND: Telomere length (TL has been found to be associated with life span in birds and humans. However, other studies have demonstrated that TL does not affect survival among old humans. Furthermore, replicative senescence has been shown to be induced by changes in the protected status of the telomeres rather than the loss of TL. In the present study we explore whether age- and sex-specific telomere dynamics affect life span in a long-lived snake, the water python (Liasis fuscus. METHODOLOGY/PRINCIPAL FINDINGS: Erythrocyte TL was measured using the Telo TAGGG TL Assay Kit (Roche. In contrast to other vertebrates, TL of hatchling pythons was significantly shorter than that of older snakes. However, during their first year of life hatchling TL increased substantially. While TL of older snakes decreased w