ABSTRACT
Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.
Subject(s)
DNA Helicases/metabolism , DNA, Single-Stranded/metabolism , Deoxyribonuclease I/metabolism , Nucleic Acid Conformation , Protein Conformation , Protein Engineering/methods , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Trans-Activators/metabolism , Viral Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Circoviridae/enzymology , Conserved Sequence , Crystallography, X-Ray , DNA Helicases/chemistry , DNA, Single-Stranded/chemistry , Deoxyribonuclease I/chemistry , Gene Library , Models, Molecular , Molecular Docking Simulation , Molecular Sequence Data , Plant Viruses/enzymology , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Replication Origin , Sequence Alignment , Sequence Homology, Amino Acid , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Substrate Specificity , Trans-Activators/chemistry , Viral Proteins/chemistryABSTRACT
Cross-linking of nucleic acids to proteins in combination with mass spectrometry permits the precise identification of interacting residues between nucleic acid-protein complexes. However, the mass spectrometric identification and characterization of cross-linked nucleic acid-protein heteroconjugates within a complex sample is challenging. Here we establish a novel enzymatic differential 16O/18O-labeling approach, which uniquely labels heteroconjugates. We have developed an automated data analysis workflow based on OpenMS for the identification of differentially isotopically labeled heteroconjugates against a complex background. We validated our method using synthetic model DNA oligonucleotide-peptide heteroconjugates, which were subjected to the labeling reaction and analyzed by high-resolution FTICR mass spectrometry.
Subject(s)
Fungal Proteins/chemistry , Mass Spectrometry/methods , Nucleoproteins/analysis , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Trypsin/chemistry , Data Analysis , Isotope Labeling , Nucleoproteins/chemistry , Oxygen/chemistry , Oxygen Isotopes/chemistry , Software , WorkflowABSTRACT
Replication of DNA in multicellular organisms initiates from origin of replication (ori) sequences, which significantly differ in length and complexity. One of the best characterized is hamster dihydrofolate reductase (DHFR), which contains the ori-ß sequence with several functionally relevant domains, such as an AT-rich region, dinucleotide repeat element (DNR), sequence-induced bend DNA (BEND) and a RIP60 protein-binding site (RIP60). Prior to initiation, ori sequences are recognized by origin recognition complex (ORC), which is a hetero hexamer complex that serves as the landing pad for proteins of the pre-replication complex. The function of each ORC subunit is still unclear. In this study, we analyze the function of subunit 4 of the human ORC complex (HsOrc4) in interaction with a plasmid bearing the ori-ß DHFR sequence. We show that the topologically closed DHFR ori-ß replicator contains a bubble-like structure within its AT-rich region and that it is reversibly modified in the interaction with HsOrc4. The non-canonical structure of the AT-rich region in the topologically closed ori sequence is recognized and changed by HsOrc4 using the energy of supercoiled DNA. These findings could help to further elucidate DNA replication and its possible association with human genetic diseases.
Subject(s)
Cell Cycle Proteins/metabolism , Nucleic Acid Conformation , Origin Recognition Complex/metabolism , Replication Origin , Tetrahydrofolate Dehydrogenase/genetics , AT Rich Sequence , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Binding Sites , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cricetulus , DNA Replication , DNA-Binding Proteins , Origin Recognition Complex/chemistry , Origin Recognition Complex/genetics , Plant Proteins/chemistry , Plant Proteins/metabolism , Plasmids/chemistry , Plasmids/genetics , RNA-Binding Proteins , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Single-Strand Specific DNA and RNA Endonucleases/metabolismABSTRACT
Dicer cleaves double-stranded RNAs (dsRNAs) or precursor microRNAs (pre-miRNAs) to yield ≈ 22-nt RNA duplexes. The pre-miRNA structure requirement for human Dicer activity is incompletely understood. By large-scale in vitro dicing assays and mutagenesis studies, we showed that human Dicer cleaves most, although not all, of the 161 tested human pre-miRNAs efficiently. The stable association of RNAs with Dicer, as examined by gel shift assays, appears important but is not sufficient for cleavage. Human Dicer tolerates remarkable structural variation in its pre-miRNA substrates, although the dsRNA feature in the stem region and the 2-nt 3'-overhang structure in a pre-miRNA contribute to its binding and cleavage by Dicer, and a large terminal loop further enhances pre-miRNA cleavage. Dicer binding protects the terminal loop from digestion by S1 nuclease, suggesting that Dicer interacts directly with the terminal loop region.
Subject(s)
DEAD-box RNA Helicases/chemistry , MicroRNAs/chemistry , RNA Cleavage , RNA Precursors/chemistry , Ribonuclease III/chemistry , Base Sequence , Electrophoretic Mobility Shift Assay , Fungal Proteins/chemistry , HEK293 Cells , Humans , Inverted Repeat Sequences , Kinetics , MicroRNAs/genetics , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Protein Binding , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Substrate SpecificityABSTRACT
Global analyses of DNA methylation contribute important insights into biology and the wide-ranging role of DNA methylation. We describe the use of online solid-phase extraction and isotope-dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the simultaneous measurement of 5-methyl-2'-deoxycytidine (5-medC) and 2'-deoxycytidine (dC) in DNA. With the incorporation of isotope internal standards and online enrichment techniques, the detection limit of this method was estimated to be as low as 0.065 pg which enables human global DNA methylation detection using only picogram amounts of DNA. This method was applied to assess the optimal amounts of enzymes required for DNA digestion regarding an accurate global DNA methylation determination and completeness of digestion and to determine global methylation in human tumor adjacent lung tissue of 79 lung cancer patients. We further determined methylated (N7-methylguanine (N7-meG), O (6)-methylguanine (O (6)-meG), and N3-methyladenine (N3-meA)) and oxidized DNA lesions (8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG)) in lung cancer patients by LC-MS/MS. Optimization experiments revealed that dC was liberated from DNA much more readily than 5-medC by nuclease P1 and alkaline phosphatase (AP) in DNA, which could lead to an error in the global DNA methylation measurement following digestion with insufficient enzymes. Nuclease P1 showed more differential activity for 5-medC and dC than AP. Global DNA methylation levels in adenocarcinoma and squamous cell carcinoma patients were similar in the range of 3.16-4.01 %. Global DNA methylation levels were not affected by smoking and gender and were not correlated with N7-meG or 8-oxodG in lung cancer patients. Levels of O (6)-meG and N3-meA were however found to be undetectable in all lung tissue samples.
Subject(s)
Adenocarcinoma/chemistry , Carcinoma, Squamous Cell/chemistry , DNA, Neoplasm/metabolism , Lung Neoplasms/chemistry , Adenine/analogs & derivatives , Adenine/isolation & purification , Adenocarcinoma/diagnosis , Adenocarcinoma/metabolism , Aged , Carcinoma, Squamous Cell/diagnosis , Carcinoma, Squamous Cell/metabolism , Chromatography, Liquid , DNA Methylation , DNA, Neoplasm/isolation & purification , Deoxycytidine/analogs & derivatives , Deoxycytidine/isolation & purification , Female , Fungal Proteins/chemistry , Guanine/analogs & derivatives , Guanine/isolation & purification , Humans , Indicator Dilution Techniques , Limit of Detection , Lung Neoplasms/diagnosis , Lung Neoplasms/metabolism , Male , Middle Aged , Reproducibility of Results , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Solid Phase Extraction , Tandem Mass Spectrometry , Tumor MicroenvironmentABSTRACT
Restriction endonucleases (REases) are highly specific DNA scissors that have facilitated the development of modern molecular biology. Intensive studies of double strand (ds) cleavage activity of Type IIP REases, which recognize 4-8 bp palindromic sequences, have revealed a variety of mechanisms of molecular recognition and catalysis. Less well-studied are REases which cleave only one of the strands of dsDNA, creating a nick instead of a ds break. Naturally occurring nicking endonucleases (NEases) range from frequent cutters such as Nt.CviPII (^CCD; ^ denotes the cleavage site) to rare-cutting homing endonucleases (HEases) such as I-HmuI. In addition to these bona fida NEases, individual subunits of some heterodimeric Type IIS REases have recently been shown to be natural NEases. The discovery and characterization of more REases that recognize asymmetric sequences, particularly Types IIS and IIA REases, has revealed recognition and cleavage mechanisms drastically different from the canonical Type IIP mechanisms, and has allowed researchers to engineer highly strand-specific NEases. Monomeric LAGLIDADG HEases use two separate catalytic sites for cleavage. Exploitation of this characteristic has also resulted in useful nicking HEases. This review aims at providing an overview of the cleavage mechanisms of Types IIS and IIA REases and LAGLIDADG HEases, the engineering of their nicking variants, and the applications of NEases and nicking HEases.
Subject(s)
DNA Cleavage , Deoxyribonucleases, Type II Site-Specific/chemistry , Endodeoxyribonucleases/chemistry , Deoxyribonucleases, Type II Site-Specific/classification , Deoxyribonucleases, Type II Site-Specific/metabolism , Protein Engineering , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Substrate SpecificityABSTRACT
The hyperthermophilic Sulfolobus islandicus rod-shaped virus 2 (SIRV2) encodes a 25-kDa protein (SIRV2gp19) annotated as a hypothetical protein with sequence homology to the RecB nuclease superfamily. Even though SIRV2gp19 homologs are conserved throughout the rudivirus family and presumably play a role in the viral life cycle, SIRV2gp19 has not been functionally characterized. To define the minimal requirements for activity, SIRV2gp19 was purified and tested under varying conditions. SIRV2gp19 is a single-strand specific endonuclease that requires Mg(2+) for activity and is inactive on double-stranded DNA. A conserved aspartic acid in RecB nuclease superfamily Motif II (D89) is also essential for SIRV2gp19 activity and mutation to alanine (D89A) abolishes activity. Therefore, the SIRV2gp19 cleavage mechanism is similar to previously described RecB nucleases. Finally, SIRV2gp19 single-stranded DNA endonuclease activity could play a role in host chromosome degradation during SIRV2 lytic infection.
Subject(s)
Rudiviridae/enzymology , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Viral Proteins/metabolism , DNA, Archaeal/genetics , DNA, Archaeal/metabolism , Rudiviridae/genetics , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Single-Strand Specific DNA and RNA Endonucleases/genetics , Single-Strand Specific DNA and RNA Endonucleases/isolation & purification , Sulfolobus/enzymology , Sulfolobus/genetics , Sulfolobus/virology , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/isolation & purificationABSTRACT
Nuclease P1 (NP1) can hydrolyze nucleic acids into four 5'-mononucleotides, which are widely used in the pharmaceutical and food industries. In this paper, an aqueous two-phase system (ATPS) was developed to purify NP1 from Penicillium citrinum. Polyethylene glycol (PEG) and nucleotides salts were studied to form ATPSs, among which PEG3000/disodium guanosine monophosphate (GMPNa2) was researched, including the phase composition and pH. Using 14% (w/w) PEG3000 and 20% (w/w) GMPNa2 ATPS at pH 5.0, the best recovery and purification factor, 82.4% and 3.59, were obtained. The recovery of NP1 was 98.3% by the separation of ultrafiltration from the PEG-rich phase. The recycling use of GMPNa2 was also studied, and 95.1% of GMPNa2 in the salt-rich phase was obtained with the addition of ethanol as the solvent. These results showed that the ATPS was effective for purification of NP1.
Subject(s)
Fungal Proteins , Guanosine Monophosphate/chemistry , Penicillium/enzymology , Polyethylene Glycols/chemistry , Single-Strand Specific DNA and RNA Endonucleases , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Single-Strand Specific DNA and RNA Endonucleases/isolation & purificationABSTRACT
We report a label-free fluorescence turn-on approach for the selective sensing of potassium. A properly selected G-rich oligonucleotide (oligo-Y) folded into stable quadruplex structure when mixed with potassium in an aqueous solution. Single-stranded nucleic acid specific nuclease was subsequently added. Since an oligonucleotide in quadruplex structure is markedly more resistant to nuclease digestion than in its random coil conformation, oligo-Y digestion by nuclease was considerably slow. On the other hand, oligo-Y mixed with other common mono- or divalent ions was completely digested in 5 min under our experimental conditions because no quadruplex or less stable quadruplex was formed. Oligo-Y in potassium was subsequently mixed with a positively charged pyrene probe. Electrostatic interactions between oligo-Y (a polyanion) and the probe induced aggregation of the probe, which in turn induced strong pyrene excimer emission. The intensity of the induced excimer emission was directly proportional to the amount of potassium added. Our method shows good sensitivity, and good selectivity against other common interference ions.
Subject(s)
Fluorescence , G-Quadruplexes , Nucleic Acids/chemistry , Potassium/analysis , Molecular Structure , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Single-Strand Specific DNA and RNA Endonucleases/metabolismABSTRACT
Nuclease P1 is a trinuclear zinc enzyme that catalyzes the hydrolysis of single-stranded DNA and RNA. Density functional calculations are used to elucidate the reaction mechanism of this enzyme with a model of the active site designed on the basis of the X-ray crystal structure. 2-Tetrahydrofuranyl phosphate and methyl 2-tetrahydrofuranyl phosphate substrates are used to explore the phosphomonoesterase and phosphodiesterase activities of this enzyme, respectively. The calculations reveal that for both activities, a bridging hydroxide performs an in-line attack on the phosphorus center, resulting in inversion of the configuration. Simultaneously, the P-O bond is cleaved, and Zn2 stabilizes the negative charge of the leaving alkoxide anion and assists its departure. All three zinc ions, together with Arg48, provide electrostatic stabilization to the penta-coordinated transition state, thereby lowering the reaction barrier.
Subject(s)
Fungal Proteins/metabolism , Quantum Theory , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Zinc , Catalytic Domain , Esters , Fungal Proteins/chemistry , Hydrolysis , Hydroxides/metabolism , Models, Molecular , Phosphates/metabolism , Single-Strand Specific DNA and RNA Endonucleases/chemistryABSTRACT
By combining a pair of pseudo-complementary peptide nucleic acids (pcPNAs) with S1 nuclease, a novel tool to cut DNA at a predetermined site can be obtained. Complementary pcPNAs invade the DNA duplex and base pair to each strand of a target site, creating single-stranded regions that are cleaved by S1 nuclease. The scission site can be freely modulated by the design of pcPNAs. This method can be used to cleave a single site in the human genome. This protocol presents experimental details for site-selective scission using this versatile new tool. © 2019 by John Wiley & Sons, Inc.
Subject(s)
DNA Cleavage , DNA, Single-Stranded/chemistry , Fungal Proteins/chemistry , Gene Editing/methods , Genome, Human , Peptide Nucleic Acids/chemistry , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Base Pairing , Humans , HydrolysisABSTRACT
Herein, a simple and selective electrochemical method was developed for sulfadimethoxine detection based on the triggered cleavage activity of nuclease P1 by the formation of aptamer and sulfadimethoxine conjugate. After probe DNA was immobilized on gold electrode surface, aptamer DNA labeled with biotin at its 5'-terminal was then captured on electrode surface through the hybridization reaction between probe DNA and aptamer DNA. The formed double-stranded DNA (dsDNA) can block the digestion activity of Nuclease P1 towards the single-stranded probe DNA. Then, the anti-dsDNA antibody was further modified on electrode surface based on the specific interaction between dsDNA and antibody. Due to the electrostatic repulsion effect and steric-hindrance effect, a weak electrochemical signal was obtained at this electrode. However, in the presence of sulfadimethoxine, it can interact with aptamer DNA, and then the formation of dsDNA can be blocked. As a result, the probe DNA at its single-strand state can be digested by Nuclease P1, which leads to the failure of the immobilization of anti-dsDNA antibody. At this state, a strong electrochemical signal was obtained. Based on the change of the electrochemical signal, sulfadimethoxine can be detected with linear range of 0.1-500â¯nmol/L. The detection limit was 0.038â¯nmol/L. The developed method possesses high detection selectivity and sensitivity. The applicability of this method was also proved by detecting sulfadimethoxine in veterinary drug and milk with satisfactory results.
Subject(s)
Anti-Bacterial Agents/analysis , Biosensing Techniques/methods , Electrochemical Techniques/methods , Sulfadimethoxine/analysis , Anti-Bacterial Agents/chemistry , Antibodies/immunology , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/genetics , DNA/chemistry , DNA/genetics , DNA/immunology , DNA Probes/chemistry , DNA Probes/genetics , Electrochemical Techniques/instrumentation , Electrodes , Ferricyanides/chemistry , Fungal Proteins/chemistry , Limit of Detection , Nucleic Acid Hybridization , Penicillium/enzymology , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Sulfadimethoxine/chemistryABSTRACT
Some members of lactic acid bacteria are known to synthesize glutathione (GSH) or to import it from growth medium, whereas others are not. Analysis of the genome sequences of several Leuconostoc spp. indicate the presence of the gene gshA that encodes gamma-glutamylcysteine synthetase, but not the gene gshB encoding glutathione synthetase. We report here that, in cells of Leuconostoc kimchii and Leuconostoc mesenteroides, gamma-glutamylcysteine (gamma-GC) is present in large amount, whereas GSH is not detectable. The level of gamma-GC was higher at the stationary phase than at the exponential phase. Expression of the gshA gene in Leuconostoc spp. analyzed by S1 mapping showed the increased mRNA level upon hydrogen peroxide treatment. From high-resolution S1 mapping, the transcriptional start site was mapped and the putative promoter elements were suggested. This work suggests that gamma-GC has a significant role in Leuconostoc spp. as the major low-molecular-weight thiol.
Subject(s)
Bacterial Proteins/genetics , Dipeptides/biosynthesis , Glutamate-Cysteine Ligase/genetics , Leuconostoc/enzymology , Sulfhydryl Compounds/metabolism , Dipeptides/analysis , Dipeptides/genetics , Gene Expression , Genes, Bacterial , Hydrogen Peroxide/pharmacology , Lactic Acid/metabolism , Leuconostoc/drug effects , Leuconostoc/genetics , Molecular Weight , Oxidative Stress , Promoter Regions, Genetic , RNA, Messenger/metabolism , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Transcription Initiation SiteABSTRACT
3'-nucleases/nucleotidases of the S1-P1 family (EC 3.1.30.1) are single-strand-specific or non-specific zinc-dependent phosphoesterases present in plants, fungi, protozoan parasites, and in some bacteria. They participate in a wide variety of biological processes and their current biotechnological applications rely on their single-strand preference, nucleotide non-specificity, a broad range of catalytic conditions and high stability. We summarize the present and potential utilization of these enzymes in biotechnology and medicine in the context of their biochemical and structure-function properties. Explanation of unanswered questions for bacterial and trypanosomatid representatives could facilitate development of emerging applications in medicine.
Subject(s)
Antineoplastic Agents/pharmacology , Biotechnology/methods , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Single-Strand Specific DNA and RNA Endonucleases/metabolism , DNA Mutational Analysis/methods , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Host-Pathogen Interactions , Humans , Molecular Targeted Therapy/methods , Nucleotidases/metabolism , Single-Strand Specific DNA and RNA Endonucleases/genetics , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Structure-Activity Relationship , Substrate SpecificityABSTRACT
To probe the effect of nucleotide on the formation of ionic contacts between actin and the 567-578 residue loop of the heavy chain of rabbit skeletal muscle myosin subfragment 1 (S1), the complexes between F-actin and proteolytic derivatives of S1 were submitted to chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. We have shown that in the absence of nucleotide both 45 kDa and 5 kDa tryptic derivatives of the central 50 kDa heavy chain fragment of S1 can be cross-linked to actin, whereas in the presence of MgADP.AlF4, only the 5 kDa fragment is involved in cross-linking reaction. By the identification of the N-terminal sequence of the 5-kDa fragment, we have found that trypsin splits the 50 kDa heavy chain fragment between Lys-572 and Gly-573, the residues located within the 567-578 loop. Using S1 preparations cleaved with elastase, we could show that the residue of 567-578 loop that can be cross-linked to actin in the presence of MgADP.AlF4 is Lys-574. The observed nucleotide-dependent changes of the actin-subfragment 1 interface indicate that the 567-578 residue loop of skeletal muscle myosin participates in the communication between the nucleotide and actin binding sites.
Subject(s)
Actins/chemistry , Adenosine Diphosphate/chemistry , Myosin Heavy Chains/chemistry , Myosin Subfragments/chemistry , Protein Interaction Mapping , Amino Acid Sequence , Animals , Cross-Linking Reagents/chemistry , Ethyldimethylaminopropyl Carbodiimide/chemistry , Peptide Fragments/chemistry , Rabbits , Single-Strand Specific DNA and RNA Endonucleases/chemistryABSTRACT
DNA-directed synthesis represents a powerful new tool for molecular discovery. Its ultimate utility, however, hinges upon the diversity of chemical reactions that can be executed in the presence of unprotected DNA. We present a solid-phase reaction format that makes possible the use of standard organic reaction conditions and common reagents to facilitate chemical transformations on unprotected DNA supports. We demonstrate the feasibility of this strategy by comprehensively adapting solid-phase 9-fluorenylmethyoxycarbonyl-based peptide synthesis to be DNA-compatible, and we describe a set of tools for the adaptation of other chemistries. Efficient peptide coupling to DNA was observed for all 33 amino acids tested, and polypeptides as long as 12 amino acids were synthesized on DNA supports. Beyond the direct implications for synthesis of peptide-DNA conjugates, the methods described offer a general strategy for organic synthesis on unprotected DNA. Their employment can facilitate the generation of chemically diverse DNA-encoded molecular populations amenable to in vitro evolution and genetic manipulation.
Subject(s)
DNA/analysis , Genetic Techniques , Chromatography, High Pressure Liquid , Combinatorial Chemistry Techniques , DNA/chemistry , DNA Primers/chemistry , DNA, Single-Stranded/chemistry , Esters/chemistry , Fluorenes/chemistry , Genetic Engineering , Models, Chemical , Oligodeoxyribonucleotides/chemistry , Peptide Library , Peptides/chemistry , Protein Engineering , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Succinimides/chemistry , TemperatureABSTRACT
Single-walled carbon nanotubes (SWNTs) binding to human telomeric i-motif DNA can significantly accelerate S1 nuclease cleavage rate by increasing the enzyme turnover number.
Subject(s)
DNA/chemistry , Nanotubes, Carbon/chemistry , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Telomere/chemistry , Binding Sites , Humans , KineticsABSTRACT
A high affinity of apolipoprotein A-I for DNA and synthetic oligonucleotides was found using the affinity chromatography, affinity modification, and enzyme analysis. The competitive inhibition and Southern hybridization allowed disclosing the specificity of the interaction of the tetrahydrocortisol-apolipoprotein A-I complex (THC-ApoA-I) with high molecular weight DNA in regions contained GCC/CGG-sequences. The S1 nuclease sensitivity of the duplex CC(GCC)3 x GG(CGG)3 was found to occur under the action of THC-ApoA-I complex. The role of the interaction sites of eukaryotic DNA with steroid (THC, androsterone)-ApoA-I complexes in the initiation of the copy reaction in vitro was revealed.
Subject(s)
Apolipoprotein A-I/chemistry , DNA/chemistry , Nucleic Acid Conformation , Tetrahydrocortisol/chemistry , Animals , Base Sequence , Binding Sites , Blotting, Southern , Chromatography, Affinity , Oligonucleotides/chemistry , Rats , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Steroids/chemistryABSTRACT
By combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with S1 nuclease, a tool for site-selective and dual-strand scission of DNA/RNA hybrids has been developed. Both of the DNA and the RNA strands in the hybrids are hydrolyzed at desired sites to provide unique sticky ends. The scission fragments are directly ligated with other DNA/RNA hybrids by using T4 DNA ligase, resulting in the formation of desired recombinant DNA/RNA hybrids.
Subject(s)
Peptide Nucleic Acids/chemistry , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Base Sequence , DNA Cleavage , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , Humans , K562 Cells , RNA/chemistry , RNA/geneticsABSTRACT
Peroxynitrite (ONOO(-)) is a strong and potent oxidizing and nitrating agent, formed by rapid reaction of two highly reactive, nitric oxide and superoxide anion. The action of peroxynitrite generated by synergistic action of diethylamine NONOate (a nitric oxide donor) and 1,4-hydroquinone (a superoxide donor), on human placental DNA was monitored by ultraviolet and fluorescence spectroscopy, melting temperature studies, S1 nuclease digestibility and alkaline agarose electrophoresis. The peroxynitrite modified human DNA (ONOO(-)-DNA) was found to be highly immunogenic in rabbits inducing high titre immunogen specific antibodies. However, the induced antibodies exhibited appreciable cross-reactivity with various polynucleotides and nucleic acids. The data demonstrate that the antibodies, though cross-reactive, preferentially bind ONOO(-)-modified epitopes on DNA. Visual detection of immune complex formation with native and ONOO(-)-DNA reiterated preferential binding with modified human DNA. DNA modified by ONOO(-) presents unique epitopes which may be one of the factors for the induction of autoantibodies in cancer patients.