The pyrimidine/purine-biased region of the epidermal growth factor receptor gene is sensitive to S1 nuclease and may form an intramolecular triplex.
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ABSTRACT:
SUBMITTER: Kato M
PROVIDER: S-EPMC1131408 | biostudies-other | 1990 May
REPOSITORIES: biostudies-other
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Project description:In vivo, left-handed DNA duplex (usually refers to Z-DNA) is mainly formed in the region of DNA with alternating purine pyrimidine (APP) sequence and plays significant biological roles. It is well known that d(CG)n sequence can form Z-DNA most easily under negative supercoil conditions, but its essence has not been well clarified. The study on sequence dependence of Z-DNA stability is very difficult without modification or inducers. Here, by the strong topological constraint caused by hybridization of two complementary short circular ssDNAs, left-handed duplex part was generated for various sequences, and their characteristics were investigated by using gel-shift after binding to specific proteins, CD and Tm analysis, and restriction enzyme cleavage. Under the strong topological constraint, non-APP sequences can also form left-handed DNA duplex as stable as that of APP sequences. As compared with non-APP sequences, the thermal stability difference for APP sequences between Z-form and B-form is smaller, which may be the reason that Z-DNA forms preferentially for APP ones. This result can help us to understand why nature selected APP sequences to regulate gene expression by transient Z-DNA formation, as well as why polymer with chirality can usually form both duplexes with left- or right-handed helix.
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Project description:We demonstrate that S1 nuclease converts supercoiled plasmid DNA to unit-length, linear dsDNA through the creation of a single, double-stranded break in a plasmid molecule. These double-stranded breaks occur not only in the origin of replication near inverted repeats but also at a wide variety of locations throughout the plasmid. S1 nuclease exhibits this activity under conditions typically employed for the nuclease's single-stranded nuclease activity. Thus, S1 nuclease digestion of plasmid DNA, unlike analogous digestion with DNaseI, effectively halts after the first double-stranded break. This property makes easier the construction of large domain insertion libraries in which the goal is to insert linear DNA at a variety of locations throughout a plasmid. We used this property to create a library in which a circularly permuted TEM1 ?-lactamase gene was inserted throughout a plasmid containing the gene encoding Escherichia coli ribose binding protein. Gene fusions that encode allosteric switch proteins in which ribose modulates ?-lactamase catalytic activity were isolated from this library using a combination of a genetic selection and a screen.
| S-EPMC3181043 | biostudies-literature
Project description:Triplex-forming oligonucleotides (TFOs) bind DNA in a sequence-specific manner at polypurine/polypyrimidine sites and mediate targeted genome modification. Triplexes are formed by either pyrimidine TFOs, which bind parallel to the purine strand of the duplex (pyrimidine, parallel motif), or purine TFOs, which bind in an anti-parallel orientation (purine, anti-parallel motif). Both purine and pyrimidine TFOs, when linked to psoralen, have been shown to direct psoralen adduct formation in cells, leading to mutagenesis or recombination. However, only purine TFOs have been shown to mediate genome modification without the need for a targeted DNA-adduct. In this work, we report the ability of a series of pyrimidine TFOs, with selected chemical modifications, to induce repair and recombination in two distinct episomal targets in mammalian cells in the absence of any DNA-reactive conjugate. We find that TFOs containing N3'-->P5' phosphoramidate (amidate), 5-(1-propynyl)-2'-deoxyuridine (pdU), 2'-O-methyl-ribose (2'-O-Me), 2'-O-(2-aminoethyl)-ribose, or 2'-O, 4'-C-methylene bridged or locked nucleic acid (LNA)-modified nucleotides show substantially increased formation of non-covalent triplexes under physiological conditions compared with unmodified DNA TFOs. However, of these modified TFOs, only the amidate and pdU-modified TFOs mediate induced recombination in cells and stimulate repair in cell extracts, at levels comparable to those seen with purine TFOs in similar assays. These results show that amidate and pdU-modified TFOs can be used as reagents to stimulate site-specific gene targeting without the need for conjugation to DNA-reactive molecules. By demonstrating the potential for induced repair and recombination with appropriately modified pyrimidine TFOs, this work expands the options available for triplex-mediated gene targeting.
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Project description:RNA duplex regions are often involved in tertiary interactions and protein binding and thus there is great potential in developing ligands that sequence-specifically bind to RNA duplexes. We have developed a convenient synthesis method for a modified peptide nucleic acid (PNA) monomer with a guanidine-modified 5-methyl cytosine base. We demonstrated by gel electrophoresis, fluorescence and thermal melting experiments that short PNAs incorporating the modified residue show high binding affinity and sequence specificity in the recognition of an RNA duplex containing an internal inverted Watson-Crick C-G base pair. Remarkably, the relatively short PNAs show no appreciable binding to DNA duplexes or single-stranded RNAs. The attached guanidine group stabilizes the base triple through hydrogen bonding with the G base in a C-G pair. Selective binding towards an RNA duplex over a single-stranded RNA can be rationalized by the fact that alkylation of the amine of a 5-methyl C base blocks the Watson-Crick edge. PNAs incorporating multiple guanidine-modified cytosine residues are able to enter HeLa cells without any transfection agent.
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Project description:The "RNA first" model for the origin of life holds that RNA emerged spontaneously on early Earth and developed into life through its dual capabilities for genetics and catalysis. The model's central weakness is the difficulty of making its building blocks, in particular, the glycosidic bond joining nucleobases to ribose. Thus, the focus of much of the modern literature on the topic is directed toward solving this difficulty and includes elegant, though indirect, methods for making this bond. Here, we report that the glycosidic bond in canonical pyrimidine and purine ribonucleotides can be formed by direct coupling of cyclic carbohydrate phosphates with free nucleobases, all reported to be available by experimentally supported pathways that might have operated on early Earth.
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Project description:Triplex-forming oligonucleotides (TFOs) are short, single-stranded oligomers that hybridise to a specific sequence of duplex DNA. TFOs can block transcription and thereby inhibit protein production, making them highly appealing in the field of antigene therapeutics. In this work, a primer extension protocol was developed to enzymatically prepare chemical nuclease TFO hybrid constructs, with gene-silencing applications. Click chemistry was employed to generate novel artificial metallo-nuclease (AMN)-dNTPs, which were selectively incorporated into the TFO strand by a DNA polymerase. This purely enzymatic protocol was then extended to facilitate the construction of 5-methylcytosine (5mC) modified TFOs that displayed increased thermal stability. The utility of the enzymatically synthesised di-(2-picolyl)amine (DPA)-TFOs was assessed and compared to a specifically prepared solid-phase synthesis counterpart through gel electrophoresis, quantitative PCR, and Sanger sequencing, which revealed similar recognition and damage properties to target genes. The specificity was then enhanced through coordinated designer intercalators-DPQ and DPPZ-and high-precision DNA cleavage was achieved. To our knowledge, this is the first example of the enzymatic production of an AMN-TFO hybrid and is the largest base modification incorporated using this method. These results indicate how chemical nuclease-TFOs may overcome limitations associated with non-molecularly targeted metallodrugs and open new avenues for artificial gene-editing technology.
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Project description:The sequence of the chicken alpha 2(I) collagen promoter from -712 to -85, relative to exon 1, has been shown to be important for transcriptional activity. Within this region a pyrimidine/purine asymmetrical element at -200 bp forms an in vitro S1 nuclease-sensitive site. The pyrimidine-rich strand of this element interacts specifically with single-stranded DNA-binding proteins present in fibroblast nuclear extracts [Bayarsaihan and Lukens (1996) Biochem. J. 314, 293-296]. To identify these proteins we performed expression screening of a chick embryo fibroblast cDNA library using a single-stranded polypyrimidine sequence derived from this element. One of the isolated clones was found to encode a member of the cold-shock gene family, either chicken YB-1 or a highly homologous protein. This protein and a known chicken Y-box protein were both found to bind sequence-specifically to the pyrimidine-rich strand of the pyrimidine/purine asymmetrical element in the chicken alpha 2(I) collagen promoter. The binding mechanism of these proteins could be based on the formation of a non-canonical triplex DNA structure (H-DNA). Although members of this widespread and conserved protein family have been reported to modulate the expression of a number of genes, the findings reported here provide the first evidence for a possible role of cold-shock proteins in the regulation of type I collagen genes.
| S-EPMC1217756 | biostudies-other
Project description:This paper describes the placement of a crosslinking agent (dibromobimane) between two thiols (Cys-522 and Cys-707) of a fragment, "S1," of the motor protein, myosin. It turns out that fastening the first anchor of the crosslinker is easy and rapid, but fastening the second anchor (Cys-522) is very temperature dependent, taking 30 min at room temperature but about a week on ice. Moreover, crystallography taken at 4 degrees C would seem to predict that the linkage is impossible, because the span of the crosslinking agent is much less than the interthiol distance. The simplest resolution of this seeming paradox is that structural fluctuations of the protein render the linkage increasingly likely as the temperature increases. Also, measurements of the affinity of MgADP for the protein, as well as the magnetic resonance of the P-atoms of the ADP once emplaced, suggest that binding the first reagent anchor to Cys-707 initiates an influence that travels to the rather distant ADP-binding site, and it is speculated what this "path of influence" might be.
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Project description:The nature of the first genetic polymer is the subject of major debate1. Although the 'RNA world' theory suggests that RNA was the first replicable information carrier of the prebiotic era-that is, prior to the dawn of life2,3-other evidence implies that life may have started with a heterogeneous nucleic acid genetic system that included both RNA and DNA4. Such a theory streamlines the eventual 'genetic takeover' of homogeneous DNA from RNA as the principal information-storage molecule, but requires a selective abiotic synthesis of both RNA and DNA building blocks in the same local primordial geochemical scenario. Here we demonstrate a high-yielding, completely stereo-, regio- and furanosyl-selective prebiotic synthesis of the purine deoxyribonucleosides: deoxyadenosine and deoxyinosine. Our synthesis uses key intermediates in the prebiotic synthesis of the canonical pyrimidine ribonucleosides (cytidine and uridine), and we show that, once generated, the pyrimidines persist throughout the synthesis of the purine deoxyribonucleosides, leading to a mixture of deoxyadenosine, deoxyinosine, cytidine and uridine. These results support the notion that purine deoxyribonucleosides and pyrimidine ribonucleosides may have coexisted before the emergence of life5.
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Project description:Understanding prebiotic nucleotide synthesis is a long standing challenge thought to be essential to elucidating the origins of life on Earth. Recently, remarkable progress has been made, but to date all proposed syntheses account separately for the pyrimidine and purine ribonucleotides; no divergent synthesis from common precursors has been proposed. Moreover, the prebiotic syntheses of pyrimidine and purine nucleotides that have been demonstrated operate under mutually incompatible conditions. Here, we tackle this mutual incompatibility by recognizing that the 8-oxo-purines share an underlying generational parity with the pyrimidine nucleotides. We present a divergent synthesis of pyrimidine and 8-oxo-purine nucleotides starting from a common prebiotic precursor that yields the β-ribo-stereochemistry found in the sugar phosphate backbone of biological nucleic acids. The generational relationship between pyrimidine and 8-oxo-purine nucleotides suggests that 8-oxo-purine ribonucleotides may have played a key role in primordial nucleic acids prior to the emergence of the canonical nucleotides of biology.
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