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International Journal of Molecular... Nov 2019The tumor suppressor functions of p53 and its roles in regulating the cell cycle, apoptosis, senescence, and metabolism are accomplished mainly by its interactions with... (Review)
Review
The tumor suppressor functions of p53 and its roles in regulating the cell cycle, apoptosis, senescence, and metabolism are accomplished mainly by its interactions with DNA. p53 works as a transcription factor for a significant number of genes. Most p53 target genes contain so-called p53 response elements in their promoters, consisting of 20 bp long canonical consensus sequences. Compared to other transcription factors, which usually bind to one concrete and clearly defined DNA target, the p53 consensus sequence is not strict, but contains two repeats of a 5'RRRCWWGYYY3' sequence; therefore it varies remarkably among target genes. Moreover, p53 binds also to DNA fragments that at least partially and often completely lack this consensus sequence. p53 also binds with high affinity to a variety of non-B DNA structures including Holliday junctions, cruciform structures, quadruplex DNA, triplex DNA, DNA loops, bulged DNA, and hemicatenane DNA. In this review, we summarize information of the interactions of p53 with various DNA targets and discuss the functional consequences of the rich world of p53 DNA binding targets for its complex regulatory functions.
Topics: Amino Acid Sequence; Animals; Binding Sites; Consensus Sequence; DNA; Humans; Models, Molecular; Nucleic Acid Conformation; Protein Binding; Protein Conformation; Tumor Suppressor Protein p53
PubMed: 31717504
DOI: 10.3390/ijms20225605 -
Current Protocols in Molecular Biology May 2001Column chromatography has evolved to provide a rapid and effective alternative to more laborious methods for preparing high-quality DNA, such as CsCl-gradient...
Column chromatography has evolved to provide a rapid and effective alternative to more laborious methods for preparing high-quality DNA, such as CsCl-gradient centrifugation. This unit describes the use of a column made of a unique anion-exchange resin that selectively binds nucleic acids, allowing rapid separation of DNA from contaminating RNA, proteins, carbohydrates, and metabolites. The procedure employs columns supplied by QIAGEN; other preparation methods are available from other suppliers. A crude nucleic acid sample (usually a cleared cell lysate) is applied to the QIAGEN tip under conditions that favor binding. Contaminants in the sample are washed from the column with a moderate-salt buffer, and DNA is eluted using a high-salt buffer.
Topics: Anion Exchange Resins; Chromatography, Ion Exchange; DNA
PubMed: 18265181
DOI: 10.1002/0471142727.mb0201bs42 -
Methods in Molecular Biology (Clifton,... 2022R-loops are three-stranded RNA:DNA hybrid structures that frequently form during transcription. While R-loop misregulation is associated with genome instability, cells...
R-loops are three-stranded RNA:DNA hybrid structures that frequently form during transcription. While R-loop misregulation is associated with genome instability, cells also harness RNA-DNA hybrids in scheduled, "regulatory," R-loops to control gene expression. One regulatory role involves epigenetic gene regulation by the R-loop reader Growth Arrest and DNA Damage 45A (GADD45A). This small stress related protein promotes DNA demethylation by recruiting TET dioxygenase and Thymine DNA glycosylase to specific genomic loci. GADD45A requires adapters for its genomic localization. One such class of adapters are R-loops formed at certain CpG island promoters to which GADD45A binds directly, targets the demethylation machinery, and confers an open chromatin state. Here, we describe protocols for carrying out in vitro binding assays with GADD45A to RNA:DNA hybrids to biochemically study its direct binding to R-loops, specifically GADD45A pulldown and EMSA (electrophoretic mobility shift) assays.
Topics: Cell Cycle Proteins; DNA; DNA Damage; Genomic Instability; Humans; Nuclear Proteins; RNA
PubMed: 35704198
DOI: 10.1007/978-1-0716-2477-7_18 -
Acta Biochimica Et Biophysica Sinica Sep 2017FOXP3, a lineage-specific forkhead (FKH) transcription factor, plays essential roles in the development and function of regulatory T cells. However, the DNA-binding...
FOXP3, a lineage-specific forkhead (FKH) transcription factor, plays essential roles in the development and function of regulatory T cells. However, the DNA-binding properties of FOXP3 are not well understood. In this study, FOXP3 fragments containing different domains were purified, and their DNA-binding properties were investigated using electrophoretic mobility shift assay and isothermal titration calorimetry (ITC). Both the FKH and leucine-zipper domains were required for optimal DNA binding for FOXP3. FOXP3 protein not only binds with DNA sequences containing one FKH consensus sequence, but also binds with DNA sequences with two direct repeats of consensus sequences separated by three-nucleotides (DRE3). Our results shed lights on the mechanisms by which FOXP3 recognizes cognate DNA elements, and would facilitate further structural and functional studies of FOXP3.
Topics: Amino Acid Sequence; Base Sequence; Binding Sites; DNA; Electrophoretic Mobility Shift Assay; Forkhead Transcription Factors; Humans; Models, Molecular; Nucleic Acid Conformation; Protein Binding; Protein Domains
PubMed: 28910978
DOI: 10.1093/abbs/gmx079 -
The Journal of Physical Chemistry. B Jul 2022A pair of ruthenium(II) complex enantiomers, - and -[Ru(bpy)MBIP] (bpy = 2,2'-bipyridine, MBIP = 2-(3-bromophenyl)imidazo[5,6-]phenanthroline), were designed,...
A pair of ruthenium(II) complex enantiomers, - and -[Ru(bpy)MBIP] (bpy = 2,2'-bipyridine, MBIP = 2-(3-bromophenyl)imidazo[5,6-]phenanthroline), were designed, synthesized, and characterized. Comparative studies between the enantiomers on their binding behaviors to calf thymus DNA (CT-DNA) were conducted using UV-visible, fluorescence, and circular dichroism spectroscopies, viscosity measurements, isothermal titration calorimetry, a photocleavage experiment, and molecular simulation. The experimental results indicated that both the enantiomers spontaneously bound to CT-DNA through intercalation stabilized by the van der Waals force or the hydrogen bond and driven by enthalpy and that -[Ru(bpy)MBIP] intercalated into DNA more deeply than -[Ru(bpy)MBIP] did and exhibited a better DNA photocleavage ability. Molecular simulation further indicated that -[Ru(bpy)MBIP] more preferentially intercalated between the base pairs of CT-DNA to the major groove, and -[Ru(bpy)MBIP] more favorably intercalated to the minor groove. These research findings should be very helpful to the understanding of the stereoselectivity mechanism of DNA-bindings of metal complexes, and be useful for the design of novel metal-complex-based antitumor drugs with higher efficacy and lower toxicity.
Topics: DNA; Molecular Structure; Organometallic Compounds; Phenanthrolines; Ruthenium; Stereoisomerism
PubMed: 35731588
DOI: 10.1021/acs.jpcb.2c02104 -
Organic & Biomolecular Chemistry Mar 2023A disulfide-functionalized bis-benzo[]quinolizinium is presented that is transformed quantitatively into its cyclomers in a fast intramolecular [4 + 4]...
A disulfide-functionalized bis-benzo[]quinolizinium is presented that is transformed quantitatively into its cyclomers in a fast intramolecular [4 + 4] photocycloaddition. Both the bis-quinolizinium and the photocyclomers react with glutathione (GSH) or dithiothreitol (DTT) to give 9-(sulfanylmethyl)benzo[]quinolizinium as the only product. As all components of this reaction sequence have different DNA-binding properties, it enables the external control and switching of DNA association. Hence, the bis-benzo[]quinolizinium binds strongly to DNA and is deactivated upon photocycloaddition to the non-binding cyclomers. In turn, the subsequent cleavage of the cyclomers with DTT regains a DNA-intercalating benzoquinolizinium ligand. Notably, this sequence of controlled deactivation and recovery of DNA-binding properties can be performed directly in the presence of DNA.
Topics: Quinolizines; Ligands; Oxidation-Reduction; DNA
PubMed: 36762516
DOI: 10.1039/d3ob00013c -
International Journal of Molecular... Mar 2022The winged helix superfamily comprises a large number of structurally related nucleic acid-binding proteins. While these proteins are often shown to bind dsDNA, few are...
The winged helix superfamily comprises a large number of structurally related nucleic acid-binding proteins. While these proteins are often shown to bind dsDNA, few are known to bind ssDNA. Here, we report the identification and characterization of Sul7s, a novel winged-helix single-stranded DNA binding protein family highly conserved in . Sul7s from binds ssDNA with an affinity approximately 15-fold higher than that for dsDNA in vitro. It prefers binding oligo(dT) over oligo(dC) or a dG-rich 30-nt oligonucleotide, and barely binds oligo(dA). Further, binding by Sul7s inhibits DNA strand annealing, but shows little effect on the melting temperature of DNA duplexes. The solution structure of Sul7s determined by NMR shows a winged helix-turn-helix fold, consisting of three α-helices, three β-strands, and two short wings. It interacts with ssDNA via a large positively charged binding surface, presumably resulting in ssDNA deformation. Our results shed significant light on not only non-OB fold single-stranded DNA binding proteins in Archaea, but also the divergence of the winged-helix proteins in both function and structure during evolution.
Topics: Archaea; DNA; DNA, Single-Stranded; DNA-Binding Proteins; Sulfolobus
PubMed: 35408816
DOI: 10.3390/ijms23073455 -
Nucleosides, Nucleotides & Nucleic Acids 2021The interaction mechanism of guaifenesin drug; ()-3-(2-methoxyphenoxy)propane-1,2-diol; and calf thymus DNA was characterized by multiple spectroscopic and molecular...
The interaction mechanism of guaifenesin drug; ()-3-(2-methoxyphenoxy)propane-1,2-diol; and calf thymus DNA was characterized by multiple spectroscopic and molecular docking approaches. The changes in drug electronic absorption with increasing DNA concentration and also the observed significant quenching of guaifenesin emission in the presence of DNA proved the complex formation between guaifenesin and DNA during the interactions. Both the binding constant and thermodynamic parameters for the interaction have been calculated in 283, 298, and 310 K at pH 7.4. The results = 17.87 kJ/mol and = 143.31 J/mol.K confirmed the role of hydrophobic force in the guaifenesin-DNA interaction. Circular dichroism study showed that guaifenesin causes decrease in the negative band of CT-DNA and at the same time the positive band increases which indicated the transition of DNA conformation from B to A. KI quenching experiment specifies that guaifenesin binds to DNA via nonintercalative mode. The competitive studies based on known Hoechst 33258 and methylene blue probes proved the groove binding mode in guaifenesin-DNA adduct. Further, full agreement of molecular docking simulation with the experimental results of binding constant and interaction mode, support high accuracy of the results.
Topics: DNA; Guaifenesin; Molecular Docking Simulation; Nucleic Acid Conformation; Osmolar Concentration; Spectrum Analysis; Thermodynamics
PubMed: 33463400
DOI: 10.1080/15257770.2021.1872793 -
Organic & Biomolecular Chemistry Jul 2023A disulfide-functionalized photoactive DNA ligand is presented that enables the control of its DNA-binding properties by a combination of a photocycloaddition reaction...
A disulfide-functionalized photoactive DNA ligand is presented that enables the control of its DNA-binding properties by a combination of a photocycloaddition reaction and the redox reactivity of the sulfide/disulfide functionalities. In particular, the initially applied ligand binds to DNA by a combination of intercalation and groove-binding of separate benzo[]quinolizinium units. The association to DNA is interrupted by an intramolecular [4 + 4] photocycloaddition to the non-binding head-to-head cyclomers. In turn, the subsequent cleavage of these cyclomers with dithiothreitol (DTT) regains temporarily a DNA-intercalating benzoquinolizinium ligand that is eventually converted into a non-binding benzothiophene. As a special feature, this sequence of controlled deactivation, recovery and internal shut-off of DNA-binding properties can be performed directly in the presence of DNA.
Topics: Ligands; Intercalating Agents; Oxidation-Reduction; DNA
PubMed: 37401249
DOI: 10.1039/d3ob00879g -
Journal of Biomolecular Structure &... Nov 2022The interaction between antihistaminic drug oxatomide () and calf-thymus DNA (CT-DNA) has been investigated in a physiological buffer (pH 7.4) using UV-Vis,...
The interaction between antihistaminic drug oxatomide () and calf-thymus DNA (CT-DNA) has been investigated in a physiological buffer (pH 7.4) using UV-Vis, fluorescence, H NMR and circular dichroism spectral techniques coupled with viscosity measurements, KI quenching, voltammetry and molecular modeling studies. binds with CT-DNA in a concentration-dependent manner. At a lower [Drug]/[CT-DNA] molar ratio (0.6-0.1), intercalates into the base pairs of CT-DNA, while at a higher [Drug]/[CT-DNA] molar ratio (13-6), the drug binds in the minor grooves of CT-DNA. The binding constants for the interaction are found to be in the order of 10-10 M, and the groove binding mode of interaction exhibits a slightly higher binding constant than that of intercalative mode. Thermodynamic analysis of binding constants at three different temperatures suggests that both these modes of binding are mainly driven by hydrophobic interactions (Δ > 0 and Δ > 0). Voltammetric investigations indicate that the electro-reduction of is an adsorption controlled process and shifts in reduction peak potentials reiterate the concentration-dependent mode of binding of the drug with CT-DNA. The free energy landscape obtained at the all-atom level, using metadynamics simulation studies, revealed two major binding forces: partial intercalation and minor groove binding, which corroborate well with the experimental results.Communicated by Ramaswamy H. Sarma.
Topics: Circular Dichroism; DNA; Molecular Docking Simulation; Piperazines; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Thermodynamics
PubMed: 33896411
DOI: 10.1080/07391102.2021.1911860