More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1-TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1-TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex.

• MeSH
• chlorid sodný farmakologie   MeSH
• DNA chemie   genetika   metabolismus   MeSH
• fluorescenční polarizace MeSH
• fluorescenční spektrometrie MeSH
• kinetika MeSH
• kompetitivní vazba účinky léků   MeSH
• lidé MeSH
• povrchová plasmonová rezonance MeSH
• protein TRF2 chemie   genetika   metabolismus   MeSH
• proteiny vázající telomery chemie   genetika   metabolismus   MeSH
• statická elektřina MeSH
• telomery genetika   metabolismus   MeSH
• vazba proteinů účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

There are two basic mechanisms that are associated with the maintenance of the telomere length, which endows cancer cells with unlimited proliferative potential. One mechanism, referred to as alternative lengthening of telomeres (ALT), accounts for approximately 10-15% of all human cancers. Tumours engaged in the ALT pathway are characterised by the presence of the single stranded 5'-C-rich telomeric overhang (C-overhang). This recently identified hallmark of ALT cancers distinguishes them from healthy tissues and renders the C-overhang as a clear target for anticancer therapy. We analysed structures of the 5'-C-rich and 3'-G-rich telomeric overhangs from human and Caenorhabditis elegans, the recently established multicellular in vivo model of ALT tumours. We show that the telomeric DNA from C. elegans and humans forms fundamentally different secondary structures. The unique structural characteristics of C. elegans telomeric DNA that are distinct not only from those of humans but also from those of other multicellular eukaryotes allowed us to identify evolutionarily conserved properties of telomeric DNA. Differences in structural organisation of the telomeric DNA between the C. elegans and human impose limitations on the use of the C. elegans as an ALT tumour model.

• MeSH
• Caenorhabditis elegans genetika   MeSH
• DNA chemie   MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• molekulární evoluce * MeSH
• telomery chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

DNA concentration has been recently suggested to be the reason why different arrangements are revealed for K(+)-stabilized human telomere quadruplexes by experimental methods requiring DNA concentrations differing by orders of magnitude. As Raman spectroscopy can be applied to DNA samples ranging from those accessible by absorption and CD spectroscopies up to extremely concentrated solutions, gels and even crystals; it has been used here to clarify polymorphism of a core human telomeric sequence G(3)(TTAG(3))(3) in the presence of K(+) and Na(+) ions throughout wide range of DNA concentrations. We demonstrate that the K(+)-structure of G(3)(TTAG(3))(3) at low DNA concentration is close to the antiparallel fold of Na(+)-stabilized quadruplex. On the increase of G(3)(TTAG(3))(3) concentration, a gradual transition from antiparallel to intramolecular parallel arrangement was observed, but only for thermodynamically equilibrated K(+)-stabilized samples. The transition is synergically supported by increased K(+) concentration. However, even for extremely high G(3)(TTAG(3))(3) and K(+) concentrations, an intramolecular antiparallel quadruplex is spontaneously formed from desalted non-quadruplex single-strand after addition of K(+) ions. Thermal destabilization or long dwell time are necessary to induce interquadruplex transition. On the contrary, Na(+)-stabilized G(3)(TTAG(3))(3) retains its antiparallel folding regardless of the extremely high DNA and/or Na(+) concentrations, thermal destabilization or annealing.

• MeSH
• DNA chemie   MeSH
• draslík chemie   MeSH
• G-kvadruplexy * MeSH
• lidé MeSH
• Ramanova spektroskopie MeSH
• telomery chemie   MeSH
• teplota MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• DNA chemie   ultrastruktura   MeSH
• kationty dvojmocné MeSH
• soli MeSH
• telomery genetika   chemie   MeSH

Telomere homeostasis is regulated at multiple levels, including the local chromatin structure of telomeres and subtelomeres. Recent reports demonstrated that a decrease in repressive chromatin marks, such as levels of cytosine methylation in subtelomeric regions, results in telomere elongation in mouse cells. Here we show that a considerable fraction of cytosines is methylated not only in subtelomeric, but also in telomeric DNA of tobacco BY-2 cells. Drug-induced hypomethylation (demonstrated at subtelomeric, telomeric, and global DNA levels) results in activation of telomerase. However, in contrast to mouse cells, the decrease in 5-methylcytosine levels and upregulation of telomerase do not result in any changes of telomere lengths. These results demonstrate the involvement of epigenetic mechanisms in the multilevel process of regulation of telomerase activity in plant cells and, at the same time, they indicate that changes in telomerase activity can be overridden by other factors governing telomere length stability.

• MeSH
• adenin analogy a deriváty   farmakologie   MeSH
• aktivace enzymů účinky léků   MeSH
• cytidin analogy a deriváty   farmakologie   MeSH
• DNA rostlinná chemie   účinky léků   MeSH
• epigeneze genetická MeSH
• genetická transkripce účinky léků   MeSH
• kultivované buňky MeSH
• metylace DNA účinky léků   MeSH
• nukleozomy účinky léků   fyziologie   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• tabák cytologie   účinky léků   genetika   metabolismus   MeSH
• telomerasa metabolismus   MeSH
• telomery chemie   účinky léků   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The i-motif is a tetrameric DNA structure based on the formation of hemiprotonated cytosine-cytosine (C+.C) base pairs. i-motifs are widely used in nanotechnology. In biological systems, i-motifs are involved in gene regulation and in control of genome integrity. In vivo, the i-motif forming sequences are subjects of epigenetic modifications, particularly 5-cytosine methylation. In plants, natively occurring methylation patterns lead to a complex network of C+.C, 5mC+.C and 5mC+.5mC base-pairs in the i-motif stem. The impact of complex methylation patterns (CMPs) on i-motif formation propensity is currently unknown. METHODS: We employed CD and UV-absorption spectroscopies, native PAGE, thermal denaturation and quantum-chemical calculations to analyse the effects of native, native-like, and non-native CMPs in the i-motif stem on the i-motif stability and pKa. RESULTS: CMPs have strong influence on i-motif stability and pKa and influence these parameters in sequence-specific manner. In contrast to a general belief, i) CMPs do not invariably stabilize the i-motif, and ii) when the CMPs do stabilize the i-motif, the extent of the stabilization depends (in a complex manner) on the number and pattern of symmetric 5mC+.5mC or asymmetric 5mC+.C base pairs in the i-motif stem. CONCLUSIONS: CMPs can be effectively used to fine-tune i-motif properties. Our data support the notion of epigenetic modifications as a plausible control mechanism of i-motif formation in vivo. GENERAL SIGNIFICANCE: Our results have implications in epigenetic regulation of telomeric DNA in plants and highlight the potential and limitations of engineered patterning of cytosine methylations on the i-motif scaffold in nanotechnological applications.

• MeSH
• cytosin metabolismus   MeSH
• DNA rostlinná chemie   genetika   MeSH
• epigeneze genetická * MeSH
• metylace DNA * MeSH
• molekulární modely MeSH
• nanotechnologie * MeSH
• nukleotidové motivy genetika   MeSH
• sekvence nukleotidů MeSH
• telomery genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The alkaloid berberine presents many biological activities related to its potential to bind DNA structures, such as duplex or G-quadruplex. Recently, it has been proposed that berberine may interact with i-motif structures formed from the folding of cytosine-rich sequences. In the present work, the interaction of this alkaloid with the i-motif formed by the human telomere cytosine-rich sequence, as well as with several positive and negative controls, has been studied. Molecular fluorescence and circular dichroism spectroscopies, as well as nuclear magnetic resonance spectrometry and competitive dialysis, have been used with this purpose. The results shown here reveal that the interaction of berberine with this i-motif is weak, mostly electrostatics in nature and takes place with bases not involved in C·C+ base pairs. Moreover, this ligand is not selective for i-motif structures, as binds equally to both, folded structure, and unfolded strand, without producing any stabilization of the i-motif. As a conclusion, the development of analytical methods based on the interaction of fluorescent ligands, such as berberine, with i-motif structures should consider the thermodynamic aspects related with the interaction, as well as the selectivity of the proposed ligands with different DNA structures, including unfolded strands.

• MeSH
• alkaloidy * MeSH
• berberin * MeSH
• cirkulární dichroismus MeSH
• dialýza ledvin MeSH
• DNA MeSH
• G-kvadruplexy * MeSH
• lidé MeSH
• telomery MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Although the impact of telomeres on physiology stands well established, a question remains: how do telomeres impact cellular functions at a molecular level? This is because current understanding limits the influence of telomeres to adjacent subtelomeric regions despite the wide-ranging impact of telomeres. Emerging work in two distinct aspects offers opportunities to bridge this gap. First, telomere-binding factors were found with non-telomeric functions. Second, locally induced DNA secondary structures called G-quadruplexes are notably abundant in telomeres, and gene regulatory regions genome wide. Many telomeric factors bind to G-quadruplexes for non-telomeric functions. Here we discuss a more general model of how telomeres impact the non-telomeric genome - through factors that associate at telomeres and genome wide - and influence cell-intrinsic functions, particularly aging, cancer, and pluripotency.

• MeSH
• DNA metabolismus   MeSH
• G-kvadruplexy * MeSH
• heterochromatin MeSH
• telomery * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

• MeSH
• buněčné jádro ultrastruktura   MeSH
• DNA ultrastruktura   MeSH
• finanční podpora výzkumu jako téma MeSH
• hybridizace in situ MeSH
• Lepidoptera genetika   MeSH
• meióza MeSH
• počítačové zpracování obrazu MeSH
• telomery ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

The ends of eukaryotic chromosomes typically contain a 3' ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing-based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.

• MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA metabolismus   MeSH
• G-kvadruplexy MeSH
• homeostáza telomer fyziologie   MeSH
• jednovláknová DNA metabolismus   MeSH
• kinetika MeSH
• konformace nukleové kyseliny MeSH
• oligonukleotidy genetika   MeSH
• proteiny vázající telomery metabolismus   MeSH
• retardační test MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• telomerasa genetika   MeSH
• telomery metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH