G-quadruplex structure of the C. elegans telomeric repeat: a two tetrads basket type conformation stabilized by a non-canonical C-T base-pair.
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ABSTRACT:
SUBMITTER: Marquevielle J
PROVIDER: S-EPMC9262591 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Human telomeres contain numerous copies of the (TTAGGG)(n).(AATCCC)(n) repeated sequence with multiple TTAGGG repeats in 3' single-stranded overhangs. Single-stranded oligonucleotides consisting of four TTAGGG repeats can fold into various intramolecular quadruplex structures stabilized by quartets of guanines. The quadruplex structures are believed to play a role in telomere functions and considered as targets for anticancer drug design. In an effort to create a more realistic model of telomeric DNA, we designed oligonucleotides containing a duplex region at the 5' end and four telomeric repeats in the 3' overhang. We applied CD spectroscopy and (125)I radioprobing to determine the conformation of the quadruplexes formed in the 3' overhangs. We found that in the presence of NaCl the conformation of the quadruplex changes with formation of the 5' duplex and depends on the position of the interface between the duplex and the 3' telomeric sequence. When the duplex region extended to the first T of the first TTAGGG repeat, both CD and radioprobing data are consistent with the parallel propeller conformation of the overhang. In the presence of KCl, formation of the duplex at the 5' end of DNA molecules did not change the fold of the quadruplex in the overhang which was interpreted as a mixture of two isomers of 3+1 conformation regardless of the duplex-overhang interface position. Our results demonstrate that the interface between the duplex and single-stranded overhang can affect the conformation of the telomeric quadruplex.
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Project description:Telomeres consisting of tandem guanine-rich repeats can form secondary DNA structures called G-quadruplexes that represent potential targets for DNA repair enzymes. While G-quadruplexes interfere with DNA synthesis in vitro, the impact of G-quadruplex formation on telomeric repeat replication in human cells is not clear. We investigated the mutagenicity of telomeric repeats as a function of G-quadruplex folding opportunity and thermal stability using a shuttle vector mutagenesis assay. Since single-stranded DNA during lagging strand replication increases the opportunity for G-quadruplex folding, we tested vectors with G-rich sequences on the lagging versus the leading strand. Contrary to our prediction, vectors containing human [TTAGGG]?? repeats with a G-rich lagging strand were significantly less mutagenic than vectors with a G-rich leading strand, after replication in normal human cells. We show by UV melting experiments that G-quadruplexes from ciliates [TTGGGG]? and [TTTTGGGG]? are thermally more stable compared to human [TTAGGG]?. Consistent with this, replication of vectors with ciliate [TTGGGG]?? repeats yielded a 3-fold higher mutant rate compared to the human [TTAGGG]?? vectors. Furthermore, we observed significantly more mutagenic events in the ciliate repeats compared to the human repeats. Our data demonstrate that increased G-quadruplex opportunity (repeat orientation) in human telomeric repeats decreased mutagenicity, while increased thermal stability of telomeric G-quadruplexes was associated with increased mutagenicity.
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Project description:Prediction of RNA tertiary structure from sequence is an important problem, but generating accurate structure models for even short sequences remains difficult. Predictions of RNA tertiary structure tend to be least accurate in loop regions, where non-canonical pairs are important for determining the details of structure. Non-canonical pairs can be predicted using a knowledge-based model of structure that scores nucleotide cyclic motifs, or NCMs. In this work, a partition function algorithm is introduced that allows the estimation of base pairing probabilities for both canonical and non-canonical interactions. Pairs that are predicted to be probable are more likely to be found in the true structure than pairs of lower probability. Pair probability estimates can be further improved by predicting the structure conserved across multiple homologous sequences using the TurboFold algorithm. These pairing probabilities, used in concert with prior knowledge of the canonical secondary structure, allow accurate inference of non-canonical pairs, an important step towards accurate prediction of the full tertiary structure. Software to predict non-canonical base pairs and pairing probabilities is now provided as part of the RNAstructure software package.
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Project description:MOTIVATION:Quadruplexes attract the attention of researchers from many fields of bio-science. Due to a specific structure, these tertiary motifs are involved in various biological processes. They are also promising therapeutic targets in many strategies of drug development, including anticancer and neurological disease treatment. The uniqueness and diversity of their forms cause that quadruplexes show great potential in novel biological applications. The existing approaches for quadruplex analysis are based on sequence or 3D structure features and address canonical motifs only. RESULTS:In our study, we analyzed tetrads and quadruplexes contained in nucleic acid molecules deposited in Protein Data Bank. Focusing on their secondary structure topology, we adjusted its graphical diagram and proposed new dot-bracket and arc representations. We defined the novel classification of these motifs. It can handle both canonical and non-canonical cases. Based on this new taxonomy, we implemented a method that automatically recognizes the types of tetrads and quadruplexes occurring as unimolecular structures. Finally, we conducted a statistical analysis of these motifs found in experimentally determined nucleic acid structures in relation to the new classification. AVAILABILITY AND IMPLEMENTATION:https://github.com/tzok/eltetrado/. SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.
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Project description:Telomeric repeat-containing RNA (TERRA) is important for telomere regulation, but the structural basis for how TERRA localizes to chromosome ends is unknown. Here we report on studies exploring whether the TERRA G-quadruplex structure is critical for binding to telomeres. We demonstrate that the telomeric protein TRF2 binds TERRA via interactions that necessitate the formation of a G-quadruplex structure rather than the TERRA sequence per se. We also show that TRF2 simultaneously binds TERRA and telomeric duplex or G-quadruplex DNA. These observations suggest that the TERRA G-quadruplex is a key feature of telomere organization.
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Project description:A nucleoside analogue comprising the ligand 1H-imidazo[4,5-f][1,10]phenanthroline (P) was applied to develop a molecular beacon capable of discriminating the canonical nucleobases cytosine and thymine. The beacon is based on the formation of a stable Ag+-mediated base pair between P and cytosine, whereas the presence of Ag+ strongly destabilizes nucleic acids comprising an artificial base pair between P and thymine. Metal-mediated base pair formation was investigated by temperature-dependent UV spectroscopy and CD spectroscopy and complemented by extensive DFT calculations. The molecular beacon significantly extends the application spectrum of nucleic acids with metal-mediated base pairs. It is of potential use in the detection of single-nucleotide polymorphisms.
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Project description:The flipping-out of a DNA base from the double helical structure is a key step of many cellular processes, such as DNA replication, modification and repair. Base pair opening is the first step of base flipping and the exact mechanism is still not well understood. We investigate sequence effects on base pair opening using extensive classical molecular dynamics simulations targeting the opening of 11 different canonical base pairs in two DNA sequences. Two popular biomolecular force fields are applied. To enhance sampling and calculate free energies, we bias the simulation along a simple distance coordinate using a newly developed adaptive sampling algorithm. The simulation is guided back and forth along the coordinate, allowing for multiple opening pathways. We compare the calculated free energies with those from an NMR study and check assumptions of the model used for interpreting the NMR data. Our results further show that the neighboring sequence is an important factor for the opening free energy, but also indicates that other sequence effects may play a role. All base pairs are observed to have a propensity for opening toward the major groove. The preferred opening base is cytosine for GC base pairs, while for AT there is sequence dependent competition between the two bases. For AT opening, we identify two non-canonical base pair interactions contributing to a local minimum in the free energy profile. For both AT and CG we observe long-lived interactions with water and with sodium ions at specific sites on the open base pair.
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Project description:Guanine-rich DNA sequences can form G-quadruplexes. These four-stranded structures are known to form in several genomic regions and to influence certain biological activities. Sometimes, the instability of G-quadruplexes causes the abnormal biological processes. Mutation is a culprit for the destabilization of G-quadruplexes, but the details of mutated G-quadruplexes are poorly understood. In this article, we investigated the conformational dynamics of single-base mutated human telomeric G-quadruplexes in the presence of K(+) with single-molecule FRET spectroscopy. We observed that the replacement of single guanine by thymine in a G-track induces various folded structures, i.e. structural polymorphism. Moreover, direct observation of their dynamics revealed that a single-base mutation causes fast unfolding of folded states under physiological conditions. Furthermore, we found that the degree of destabilization varies according to mutation positions. When the central guanine of a G-track is replaced, the G-quadruplexes unfold quickly at any K(+) concentrations and temperature. Meanwhile, outer-quartet mutated G-quadruplexes have heterogeneous dynamics at intermediate K(+) concentrations and longstanding folded states at high K(+) concentrations. Several factors such as base-stacking interaction and K(+) coordination are responsible for the different dynamics according to the mutation position.
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Project description:Telomeres are hot spots for mutagenic oxidative and methylation base damage due to their high guanine content. We used single-molecule fluorescence resonance energy transfer detection and biochemical assays to determine how different positions and types of guanine damage and mutations alter telomeric G-quadruplex structure and telomerase activity. We compared 15 modifications, including 8-oxoguanine (8oxoG), O-6-methylguanine (O6mG), and all three possible point mutations (G to A, T, and C) at the 3' three terminal guanine positions of a telomeric G-quadruplex, which is the critical access point for telomerase. We found that G-quadruplex structural instability was induced in the order C < T < A ≤ 8oxoG < O6mG, with the perturbation caused by O6mG far exceeding the perturbation caused by other base alterations. For all base modifications, the central G position was the most destabilizing among the three terminal guanines. While the structural disruption by 8oxoG and O6mG led to concomitant increases in telomerase binding and extension activity, the structural perturbation by point mutations (A, T, and C) did not, due to disrupted annealing between the telomeric overhang and the telomerase RNA template. Repositioning the same mutations away from the terminal guanines caused both G-quadruplex structural instability and elevated telomerase activity. Our findings demonstrate how a single-base modification drives structural alterations and telomere lengthening in a position-dependent manner. Furthermore, our results suggest a long-term and inheritable effect of telomeric DNA damage that can lead to telomere lengthening, which potentially contributes to oncogenesis.
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Project description:G-quadruplexes are noncanonical secondary structures formed in DNA sequences containing consecutive runs of guanines. It has been shown that the 3' G-rich single-stranded overhangs of human telomeres can form G-quadruplex structures, and the human telomeric DNA G-quadruplexes are considered attractive targets for anticancer drugs. G-quadruplex-interactive compounds have been shown to inhibit telomerase access as well as telomere capping. Nuclear magnetic resonance (NMR) spectroscopy is a powerful method in determining the G-quadruplex structures under physiologically relevant conditions. We present the NMR and biophysical methodology used in our research group for the study of G-quadruplex structures in physiologically relevant solution and their interactions with small-molecule compounds.
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