High-Resolution Three-Dimensional NMR Structure Of The KRAS Proto-Oncogene Promoter Reveals Key Features Of A G-Quadruplex Involved In Transcriptional Regulation

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

Quadruplexes In ‘Dicty’: Crystal Structure Of A Four-Quartet G-Quadruplex Formed By G-Rich Motif Found In The Dictyostelium Discoideum Genome

Guanine-rich DNA has the potential to fold into non-canonical G-quadruplex (G4) structures. Analysis of the genome of the social amoeba Dictyostelium discoideum indicates a low number of sequences with G4-forming potential (249–1055). Therefore, D. discoideum is a perfect model organism to investigate the relationship between the presence of G4s and their biological functions. As a first step in this investigation, we crystallized the dGGGGGAGGGGTACAGGGGTACAGGGG sequence from the putative promoter region of two divergent genes in D. discoideum. According to the crystal structure, this sequence folds into a four-quartet intramolecular antiparallel G4 with two lateral and one diagonal loops. The G-quadruplex core is further stabilized by a G-C Watson–Crick base pair and a A–T–A triad and displays high thermal stability (Tm \u3e 90°C at 100 mM KCl). Biophysical characterization of the native sequence and loop mutants suggests that the DNA adopts the same structure in solution and in crystalline form, and that loop interactions are important for the G4 stability but not for its folding. Four-tetrad G4 structures are sparse. Thus, our work advances understanding of the structural diversity of G-quadruplexes and yields coordinates for in silico drug screening programs and G4 predictive tools

Interaction Of Human Telomeric DNA With N-Methyl Mesoporphyrin IX

The remarkable selectivity of N-methyl mesoporphyrin IX (NMM) for G-quadruplexes (GQs) is long known, however its ability to stabilize and bind GQs has not been investigated in detail. Through the use of circular dichroism, UV-visible spectroscopy and fluorescence resonance energy transfer (FRET) melting assay we have shown that NMM stabilizes human telomeric DNA dAG(3)(TTAG(3))(3) (Tel22) and is selective for its parallel conformation to which it binds in 1:1 stoichiometry with a binding constant of similar to 1.0 x 10(5) M-1. NMM does not interact with an antiparallel conformation of Tel22 in sodium buffer and is the second example in the literature, after TOxaPy, of a ligand with an excellent selectivity for a specific GQ structure. NMM\u27s stabilizing ability toward predominantly parallel GQ conformation is universal: it stabilizes a variety of biologically relevant G-rich sequences including telomeres and oncogene promoters. The N-methyl group is integral for selectivity and stabilization, as the unmethylated analogue, mesoporphyrin IX, does not stabilize GQ DNA in FRET melting assays. Finally, NMM induces the isomerization of Tel22 into a structure with increased parallel component in K+ but not in Na+ buffer. The ability of NMM to cause structural rearrangement and efficient stabilization of Tel22 may bear biological significance

Thioflavin T As A Fluorescence Light-Up Probe For G4 Formation

Thioflavin T (ThT) becomes fluorescent in the presence of the G-quadruplex structure such as that formed by the human telomeric motif. In this report, we extend and generalize these observations and show that this dye may be used as a convenient and specific quadruplex probe. In the presence of most, but not all, G4-forming sequences, we observed a large increase in ThT fluorescence emission, whereas the presence of control duplexes and single strands had a more limited effect on emission. This differential behavior allowed us to design a high-throughput assay to detect G4 formation. Hundreds of different oligonucleotides may be tested in parallel for G4 formation with a simple fluorescence plate reader. We applied this technique to a family of aptamers not previously recognized as G4-forming sequences and demonstrated that ThT fluorescence signal may be used to predict G4 formation

Investigation Of The Interactions Between Pt(II) And Pd(II) Derivatives Of 5,10,15,20-Tetrakis (N-Methyl-4-Pyridyl) Porphyrin And G-Quadruplex DNA

G-quadruplexes are non-canonical DNA structures formed by guanine-rich DNA sequences that are implicated in cancer and aging. Understanding how small molecule ligands interact with quadruplexes is essential both to the development of novel anticancer therapeutics and to the design of new quadruplex-selective probes needed for elucidation of quadruplex biological functions. In this work, UV–visible, fluorescence, and circular dichroism spectroscopies, fluorescence resonance energy transfer (FRET) melting assays, and resonance light scattering were used to investigate how the Pt(II) and Pd(II) derivatives of the well-studied 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) interact with quadruplexes formed by the human telomeric DNA, Tel22, and by the G-rich sequences from oncogene promoters. Our results suggest that Pt- and PdTMPyP4 interact with Tel22 via efficient π–π stacking with a binding affinity of 106–107 M−1. Under porphyrin excess, PtTMPyP4 aggregates using Tel22 as a template; the aggregates reach maximum size at [PtTMPyP4]/[Tel22] ~8 and dissolve at [PtTMPyP4]/[Tel22] ≤ 2. FRET assays reveal that both porphyrins are excellent stabilizers of human telomeric DNA, with stabilization temperature of 30.7 ± 0.6 °C for PtTMPyP4 and 30.9 ± 0.4 °C for PdTMPyP4 at [PtTMPyP4]/[Tel22] = 2 in K+ buffer, values significantly higher as compared to those for TMPyP4. The porphyrins display modest selectivity for quadruplex vs. duplex DNA, with selectivity ratios of 150 and 330 for Pt- and PdTMPyP4, respectively. This selectivity was confirmed by observed ‘light switch’ effect: fluorescence of PtTMPyP4 increases significantly in the presence of a variety of DNA secondary structures, yet the strongest effect is produced by quadruplex DNA

Protocol for a national audit on self-reported confidence levels, training requirements and current practice among trainee doctors in the UK: The Trainees Own Perception of Delivery of Care in Diabetes (TOPDOC) Study

Background: As the incidence and prevalence of diabetes increases across the world, resource pressures require doctors without specialist training to provide care for people with diabetes. In the UK, national standards have been set to ensure quality diabetes care from diagnosis to the management of complications. In a multi-centre pilot study, we have demonstrated a lack of confidence among UK trainee doctors in managing diabetes. Suboptimal confidence was identified in a number of areas, including the management of diabetes emergencies. A national survey would clarify whether the results of our pilot study are representative and reproducible. Methods/Design: Target cohort: All postgraduate trainee doctors in the UK. Domains Studied: The self reported online survey questionnaire has 5 domains: (1) confidence levels in the diagnosis and management of diabetes, (2) working with diabetes specialists, (3) perceived adequacy of training in diabetes (4) current practice in optimising glycaemic control and (5) perceived barriers to seeking euglycaemia. Assessment tools: Self-reported confidence is assessed using the 'Confidence Rating' (CR) scale for trainee doctors developed by the Royal College of Physicians. This scale has four points - ('not confident' (CR1), 'satisfactory but lacking confidence' (CR2), 'confident in some cases (CR3) and 'fully confident in most cases' (CR4). Frequency of aspects of day-to-day practice is assessed using a six-point scale. Respondents have a choice of 'always' (100%), 'almost always' (80-99%), 'often' (50-79%), 'not very often' (20-49%) and 'rarely' (5-19%) or never (less than 5%). Discussion: It is anticipated that the results of this national study will clarify confidence levels and current practice among trainee doctors in the provision of care for people with diabetes. The responses will inform efforts to enhance postgraduate training in diabetes, potentially improving the quality of care for people with diabetes.</p

Rudimentary G-Quadruplex-Based Telomere Capping In Saccharomyces Cerevisiae

Telomere capping conceals chromosome ends from exonucleases and checkpoints, but the full range of capping mechanisms is not well defined. Telomeres have the potential to form G-quadruplex (G4) DNA, although evidence for telomere G4 DNA function in vivo is limited. In budding yeast, capping requires the Cdc13 protein and is lost at nonpermissive temperatures in cdc13-1 mutants. Here, we use several independent G4 DNA-stabilizing treatments to suppress cdc13-1 capping defects. These include overexpression of three different G4 DNA binding proteins, loss of the G4 DNA unwinding helicase Sgs1, or treatment with small molecule G4 DNA ligands. In vitro, we show that protein-bound G4 DNA at a 3\u27 overhang inhibits 5\u27-\u3e 3\u27 resection of a paired strand by exonuclease I. These findings demonstrate that, at least in the absence of full natural capping, G4 DNA can play a positive role at telomeres in vivo

The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences

A Conserved Stem Loop Motif in the 5′Untranslated Region Regulates Transforming Growth Factor-β1 Translation

Transforming growth factor-β1 (TGF-β1) regulates cellular proliferation, differentiation, migration, and survival. The human TGF-β1 transcript is inherently poorly translated, and translational activation has been documented in relation to several stimuli. In this paper, we have sought to identify in cis regulatory elements within the TGF-β1 5′Untranslated Region (5′UTR). In silico analysis predicted formation of stable secondary structure in a G/C-rich element between nucleotides +77 to +106, and demonstrated that this element is highly conserved across species. Circular dichroism spectroscopy confirmed the presence of secondary structure in this region. The proximal 5′UTR was inhibitory to translation in reporter gene experiments, and mutation of the secondary structure motif increased translational efficiency. Translational regulation of TGF-β1 mRNA is linked to altered binding of YB-1 protein to its 5′UTR. Immunoprecipitation-RT-qPCR demonstrated a high basal association of YB-1 with TGF-β1 mRNA. However, mutation of the secondary structure motif did not prevent interaction of YB-1 with the 5′UTR, suggesting that YB-1 binds to this region due to its G/C-rich composition, rather than a specific, sequence-dependent, binding site. These data identify a highly conserved element within the TGF-β1 5′UTR that forms stable secondary structure, and is responsible for the inherent low translation efficiency of this cytokine