Estructuras no canónicas de ácidos nucleicos en telómeros y centrómeros

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 18-02-2015Los telómeros y los centrómeros son regiones del cromosoma que se caracterizan por la naturaleza repetitiva de las secuencias de DNA que las forman. En esta tesis se presenta una hipótesis sobre el origen y evolución de los telómeros y centrómeros. La hipótesis defiende que los primeros telómeros surgieron como consecuencia de la proliferación de retrotransposones en tándem en los finales de los primeros cromosomas lineales. El contenido en residuos de guanina de estos elementos móviles permitiría eventualmente la protección del final del cromosoma mediante la formación de estructuras no canónicas de DNA. Estos telómeros originales deberían poder cumplir una función doble que implica, tanto la protección del final del cromosoma, como la segregación del mismo durante la división celular. Más tarde, los centrómeros surgirían a partir de secuencias de las regiones subteloméricas. Estudios estructurales de secuencias centroméricas y teloméricas, como los abordados en esta tesis, son clave para la verificación de esta hipótesis. El RNA telomérico (o TERRA) es un RNA no codificante que forma parte de la heterocromatina telomérica. Los datos acerca de su estructura son escasos y se limitan a moléculas formadas por un número reducido de repeticiones. En este trabajo se ha llevado a cabo el estudio estructural de secuencias de TERRA compuestas por un número de repeticiones teloméricas cercano al que se encuentra en las moléculas de RNA telomérico presentes en la célula. La caracterización estructural de estas moléculas se ha realizado utilizando técnicas biofísicas como la RMN y el dicroísmo circular, y técnicas de molécula individual como las pinzas ópticas. Así ha sido posible determinar que las moléculas de TERRA estudiadas se pliegan formando G-quadruplexes consecutivos que interaccionan entre sí. Por otro lado, debido al interés de TERRA como diana terapéutica, se han utilizado oligonucleótidos de TERRA para su aplicación en la búsqueda de pequeños compuestos fluorados capaces de unir RNA telomérico. En esta tesis se presenta la metodología, basada en 19F-RMN, utilizada para la búsqueda de compuestos que unen TERRA así como las diferentes estrategias usadas para evaluar la afinidad y selectividad de los ligandos encontrados. Las secuencias de DNA centroméricas son el resultado de la repetición de secuencias simples o complejas denominadas DNA satélite. En esta tesis se ha abordado el estudio estructural de secuencias pertenecientes a satélites complejos, como es el caso del satélite alfoide humano, y de secuencias derivadas de satélites simples, como el satélite dodeca de Drosophila melanogaster. En el presente trabajo se muestra la estructura de alta resolución de una versión truncada de la secuencia A-box perteneciente al satélite alfoide humano. Además se muestran datos estructurales que indican que las versiones no truncadas adoptan también estructuras diméricas tipo i-motif a pH ácido. Por último, en esta tesis se presenta la estructura del centrómero del cromosoma 3 de D. melanogaster. El análisis estructural de secuencias derivadas de la hebra rica en citosinas del satélite dodeca muestra que estas secuencias son también capaces de formar i-motifs. Estos resultados sugieren un posible papel de la estructura i-motif en la formación de la cromatina centromérica y plantean una posible explicación a la paradoja del centrómero. Esta memoria se ha realizado de acuerdo a las normas de extensión y formato vigentes en el departamento de Biología Molecular de la Universidad Autónoma de Madrid en diciembre de 2014.Telomeres and centromeres are chromosomal regions characterised by the repetitive nature of their DNA sequences. In this thesis, we present a hypothesis on the origin and evolution of telomeres and centromeres. Our hypothesis argues that the first telomeres emerged as a consequence of the proliferation of tandemly arranged retrotransposons at the end of the nascent linear chromosomes. The guanine residue content of these mobile elements would eventually lead to protection or “capping” by means of formation of non-canonical DNA structures. These primal telomeres should be able to fulfil a dual function of protecting the end of the chromosome and allowing the correct segregation of the DNA during cell division. Later on, proper centromeres evolved at subtelomeric regions. Structural studies of telomeric and centromeric sequences, as those addressed in this thesis, are key elements to confirm the veracity of this hypothesis. Telomeric RNA or TERRRA is a non-coding RNA that acts as a scaffold for the formation of telomeric heterochromatin. The information about the structure of TERRA is scarce and limited to molecules with a small number of repeats. In this work, we have carried out the structural study of TERRA sequences composed by a number of telomeric repeats that approaches the size of the nuclear endogenous telomeric RNA. The structural characterization of these molecules has been performed by using biophysical techniques as NMR and circular dichroism, and single molecule techniques as the optical tweezers. Thus, it has been possible to determine that the studied TERRA molecules fold into consecutive G-quadruplexes that interact with each other. Furthermore, given the importance of these molecules as therapeutic targets, we have used TERRA oligonucleotides for the screening of small fluorinated fragments that interact with telomeric RNA. Here, we present a 19F-NMR based methodology for the screening of compounds that bind TERRA. In addition, we show the different strategies employed to analyse the affinity and selectivity of the hits identified in the screening. Centromeric DNA is the result of the recurrence of simple or complex repeated sequences known as satellite DNA. In this thesis, we have carried out the structural study of both sequences belonging to complex satellites, as the human alfa-satellite, and sequences from simple centromeric satellites, as the dodeca satellite of Drosophila melanogaster. In the present work, we present the high-resolution structure of a truncated version of the A-box sequence, a sequence that appears in the human alfa-satellite. Besides, we show structural evidences indicating that complete versions of the A-box also fold into dimeric i-motif structures at acidic pH. Finally, we show the structure of the centromere of the chromosome 3 of D. melanogaster. The structural analysis of sequences derived from the C-rich strand of dodeca satellite shows that these sequences are also able to form i-motif structures. These results suggest a potential role of the imotif in the formation of centromeric chromatin and also pose a possible explanation of the centromere paradox

Understanding the effect of the nature of the nucleobase in the loops on the stability of the i-motif structure

The nature and length of loops connecting cytosine tracts in i-motif structures may affect their stability. In this work, the influence of the nature of the nucleobases located in two of the loops of an intramolecular i-motif is studied using spectroscopy, separation techniques, and multivariate data analysis. Insertion of bases other than thymine induces an additional acid-base equilibrium with pKa~4.5. The presence of two guanine bases in the loops, placed opposite to each other, decreases the thermal stability of the structure. In contrast, thymine and cytosine bases in these positions stabilize the structur

The structure of an endogenous Drosophila centromere reveals the prevalence of tandemly repeated sequences able to form i-motifs

Centromeres are the chromosomal loci at which spindle microtubules attach to mediate chromosome segregation during mitosis and meiosis. In most eukaryotes, centromeres are made up of highly repetitive DNA sequences (satellite DNA) interspersed with middle repetitive DNA sequences (transposable elements). Despite the efforts to establish complete genomic sequences of eukaryotic organisms, the so-called 'finished' genomes are not actually complete because the centromeres have not been assembled due to the intrinsic difficulties in constructing both physical maps and complete sequence assemblies of long stretches of tandemly repetitive DNA. Here we show the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs. The finding of i-motif structures in simple and complex centromeric satellite DNAs leads us to suggest that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures.Peer Reviewe

Understanding the effect of the nature of the nucleobase in the loops on the stability of the i-motif structure

The nature and the length of loops connecting cytosine tracts in i-motif structures may affect their stability. In this work, the influence of the nature of the nucleobases located in two of the loops of an intramolecular i-motif is studied using spectroscopy, separation techniques, and multivariate data analysis. The insertion of bases other than thymine induces an additional acid-base equilibrium with pKa ∼ 4.5. The presence of two guanine bases in the loops, placed opposite to each other, decreases the thermal stability of the structure. In contrast, thymine and cytosine bases in these positions stabilize the structure. © the Owner Societies 2016.Anna Sadurnı´ (University of Barcelona) is acknowledged for carrying out some experiments. We acknowledge funding from the Spanish government (BFU2014-52864-R, CTQ2012-38616- C02-02 and CTQ2014-52588-R). This research has been recognized by the regional Catalan authorities (2014 SGR 1106).Peer reviewe

Detection of a G-Quadruplex as a Regulatory Element in Thymidylate synthase for Gene Silencing Using Polypurine Reverse Hoogsteen Hairpins

Thymidylate synthase (TYMS) enzyme is an anti-cancer target given its role in DNA biosynthesis. TYMS inhibitors (e.g., 5-Fluorouracil) can lead to drug resistance through an autoregulatory mechanism of TYMS that causes its overexpression. Since G-quadruplexes (G4) can modulate gene expression, we searched for putative G4 forming sequences (G4FS) in the TYMS gene that could be targeted using polypurine reverse Hoogsteen hairpins (PPRH). G4 structures in the TYMS gene were detected using the quadruplex forming G-rich sequences mapper and confirmed through spectroscopic approaches such as circular dichroism and NMR using synthetic oligonucleotides. Interactions between G4FS and TYMS protein or G4FS and a PPRH targeting this sequence (HpTYMS-G4-T) were studied by EMSA and thioflavin T staining. We identified a G4FS in the 5'UTR of the TYMS gene in both DNA and RNA capable of interacting with TYMS protein. The PPRH binds to its corresponding target dsDNA, promoting G4 formation. In cancer cells, HpTYMG-G4-T decreased TYMS mRNA and protein levels, leading to cell death, and showed a synergic effect when combined with 5-fluorouracil. These results reveal the presence of a G4 motif in the TYMS gene, probably involved in the autoregulation of TYMS expression, and the therapeutic potential of a PPRH targeted to the G4FS

Mechanical unfolding of long human telomeric RNA (TERRA)

[EN] We report the first single molecule investigation of TERRA molecules. By using optical-tweezers and other biophysical techniques, we have found that long RNA constructions of up to 25 GGGUUA repeats form higher order structures comprised of single parallel G-quadruplex blocks, which unfold at lower forces than their DNA counterparts.This work was supported by grants from the Spanish Ministry of Science and Innovation (grants RYC2007-01765 to JRA-G, BFU2011-30295-C02-01 to AV, and CTQ2010-21567-C02-02 to CG). MG was supported by the FPI fellowship BES-2009-027909. RB and EH-G were supported by Comunidad de Madrid, grant CAM-S2009MAT-1507. AV acknowledges an institutional grant from the Fundacion Ramon Areces to the CBMSO. JRA-G wants to thank Prof. J. L. Carrascosa and Prof. J. M. Valpuesta (CNB-CSIC) for their continuous support and encouragement in this research. We also acknowledge the excellent technical assistance of Beatriz de Pablos (CBMSO).Garavís, M.; Bocanegra, R.; Herrero-Galán, E.; González, C.; Villasante, A.; Arias-Gonzalez, JR. (2013). Mechanical unfolding of long human telomeric RNA (TERRA). Chemical Communications. 49(57):6397-6399. https://doi.org/10.1039/c3cc42981dS639763994957De Lange, T. (2005). Shelterin: the protein complex that shapes and safeguards human telomeres. Genes & Development, 19(18), 2100-2110. doi:10.1101/gad.1346005Blackburn, E. H. (1991). Structure and function of telomeres. Nature, 350(6319), 569-573. doi:10.1038/350569a0Biffi, G., Tannahill, D., McCafferty, J., & Balasubramanian, S. (2013). Quantitative visualization of DNA G-quadruplex structures in human cells. Nature Chemistry, 5(3), 182-186. doi:10.1038/nchem.1548Paeschke, K., Simonsson, T., Postberg, J., Rhodes, D., & Lipps, H. J. (2005). Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo. 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On the origin of the eukaryotic chromosome: the role of noncanonical DNA structures in telomere evolution.

The transition of an ancestral circular genome to multiple linear chromosomes was crucial for eukaryogenesis because it allowed rapid adaptive evolution through aneuploidy. Here, we propose that the ends of nascent linear chromosomes should have had a dual function in chromosome end protection (capping) and chromosome segregation to give rise to the >proto-telomeres.> Later on, proper centromeres evolved at subtelomeric regions. We also propose that both noncanonical structures based on guanine-guanine interactions and the end-protection proteins recruited by the emergent telomeric heterochromatin have been required for telomere maintenance through evolution. We further suggest that the origin of Drosophila telomeres may be reminiscent of how the first telomeres arose.Ministerio de Ciencia e Innovación; Ministerio de Economía y Competitividad; Fundación Ramón ArecesPeer Reviewe

Rpb1-CTD phosphorylation is differentially modulated by Rpb4/7

Póster presentado a la EMBO Conference: "Gene Transcrition in Yeast: From Chromatin to RNA and back", celebrada en Sant Feliu de Guixols (España) del 11 al 16 de junio de 2016.The Rpb4 and Rpb7 subunits of eukaryotic RNA polymerase II (RNAPII) participate in a variety of processes from transcription, DNA repair, mRNA export and decay, to translation regulation and stress response. In addition, we have recently shown that the Rpb4/7 heterodimer in S. cerevisiae plays a key role in controlling phosphorylation of the carboxy terminal domain (CTD) of the Rpb1 subunit of RNAPII. Deletion of RPB4, and mutations that disrupt the integrity of Rpb4/7 or its recruitment to the RNAPII complex, increased phosphorylation of Ser2, Ser5, Ser7. We showed that Rpb4 is important for Ssu72 and Fcp1 phosphatases association, recruitment and/or accessibility to the CTD, and that this correlates strongly with Ser5P and Ser2P levels, respectively. Here we show that, in addition, rpb4D cells display increased Thr4P and Tyr1P. Our data suggest that Fcp1 is the Thr4P phosphatase in yeast, as in vertebrate. Moreover, we present evidences that Rpb4 may be also linked to the function of the CTD phosphatase Rtr1, which has been involved in Ser5P and Tyr1P dephosporylation. On the other hand, Rpb4 also influences the recruitment of the CTD-Ser2 kinase Ctk1 during transcription elongation. We proposed a model where Rpb1-CTD phosphorylation levels are differentially modulated by Rpb4/7. Thus, increased Ser2P phoshorylation levels in the rpb4D mutants are due to altered Fcp1 and Ctk1 functions, while increased Ser5 phosphorylation is the result of changes in Ssu72. Our data and others, and the close localization of Rpb4/7 to the CTD, suggest that Rpb4/7 might modulate the access of the CTD modifying enzymes to their substrate during the whole transcription cycle.This work was funded by the MINECO (BFU2013-48374-P)Peer Reviewe

The Effects of FANA Modifications on Non-canonical Nucleic Acid Structures

37 pags., 24 figs., 6 tabs. -- Commemorates the 70th anniversary of the DNA double helix discoveryInitially explored for its antiviral applications, the 2′-deoxy-2′-fluoroarabino nucleoside is now widely incorporated into oligonucleotides for applications in structure elucidation, synthetic genetics, and therapeutics, among others. This chapter explores the use of 2′-fluoroarabino nucleic acids (FANA) in the context of noncanonical nucleic acid structures, namely triple helices, G-quadruplexes, and i-motifs. For the last three decades, FANA has been utilized to understand biophysical properties associated with these structures and to stabilize certain conformers or even manipulate their topology. Owing to its DNA-like character and the flexibility of its sugar pucker, FANA has also been explored in biochemical contexts, including its interactions with thrombin and human telomerase. Despite years of research, novel properties of FANA are still being uncovered today; indeed, FANA promises to consistently serve as an exemplary tool for chemists and molecular biologists investigating nucleic acid phenomena.The authors would like to acknowledge the following funding agencies: the European Union Marie Sklodowska Curie Action (799693, to M.G.), the Canadian Natural Sciences and Engineering Research Council of Canada (Discovery Grant to M.J.D.), and the Fonds de Recherche du Québec – Nature et Technologies Doctoral Scholarship (DE, to R.E.K)

i-Motif DNA: Structural Features and Significance to Cell Biology

19 pags., 7 figs.The i-motif represents a paradigmatic example of the wide structural versatility of nucleic acids. In remarkable contrast to duplex DNA, i-motifs are four-stranded DNA structures held together by hemi- protonated and intercalated cytosine base pairs (C:C+). First observed 25 years ago, and considered by many as a mere structural oddity, interest in and discussion on the biological role of i-motifs have grown dramatically in recent years. In this review we focus on structural aspects of i-motif formation, the factors leading to its stabilization and recent studies describing the possible role of i-motifs in fundamental biological processes.Natural Sciences and Engineering Research Council of Canada (NSERCC); MINECO grant [BFU2017-89707-P]; Juan de la Cierva postdoctoral Fellowship [FJCI-2016-28474 to M.G.]. Funding for open access charge: NSERCC Discovery grant ( to M.J.D.); MINECO grant [BFU2017-89707-P to C.G.].Peer Reviewe