RESUMEN
The symmetry of the responses of the human DNA (cytosine-5)methyltransferase to alternative placements of 5-methylcytosine in model oligodeoxynucleotide duplexes containing unusual structures has been examined. The results of these experiments more clearly define the DNA recognition specificity of the enzyme. A simple three-nucleotide recognition motif within the CG dinucleotide pair can be identified in each enzymatically methylated duplex. The data can be summarized by numbering the four nucleotides in the dinucleotide pair thus: 1 4/2 3. With reference to this numbering scheme, position 1 can be occupied by cytosine or 5-methylcytosine; position 2 can be occupied by guanosine or inosine; position 3, the site of enzymatic methylation, can be occupied only by cytosine; and position 4 can be occupied by guanosine, inosine, O6-methylguanosine, cytosine, adenosine, an abasic site, or the 3' hydroxyl group at the end of a gapped molecule. Replacing the guanosine normally found at position 4 with any of the moieties introduces unusual (non-Watson-Crick) pairing at position 3 and generally enhances methylation of the cytosine at that site. The exceptional facility of the enzyme in actively methylating unusual DNA structures suggests that the evolution of the DNA methyltransferase, and perhaps DNA methylation itself, may be linked to the biological occurrence of unusual DNA structures.
Asunto(s)
ADN-Citosina Metilasas/metabolismo , ADN/metabolismo , 5-Metilcitosina , Secuencia de Bases , Citosina/análogos & derivados , Citosina/metabolismo , ADN/química , Fosfatos de Dinucleósidos/metabolismo , Femenino , Humanos , Metilación , Modelos Moleculares , Datos de Secuencia Molecular , Ácidos Nucleicos HeterodúplexRESUMEN
An abasic site in DNA creates a strong block to DNA polymerase and is a mutagenic base lesion. In this study, we present structural and dynamic properties of duplex oligodeoxynucleotides containing G, C and T opposite a model abasic site studied by one and two-dimensional nuclear magnetic resonance spectroscopy. We have demonstrated that A opposite the abasic site was positioned within the helix as if paired with T, and that the A residue melted co-operatively with the surrounding helix. We report here that G opposite the abasic site is also observed to be predominantly intrahelical in a normal anti conformation at low temperature. With increasing temperature, the mobility of the G residue increases rapidly and apparently is in a "melted state" well before denaturation of the helix. At low temperature, two species are found for T opposite the abasic site; one, intrahelical, one extrahelical. These species are in slow exchange with one another on a proton nuclear magnetic resonance time-scale. The two species then move into fast exchange with increasing temperature and the proportion of the extra-helical form increases. When C is positioned opposite the abasic site, both the C residue and the abasic sugar are extrahelical, the helix collapses, and the adjacent G.C base-pairs stack over one another. On the basis of these observations, we propose a model that explains why the abasic site acts to block DNA replication. Further, we suggest an explanation for the observed polymerase preference for base selection at abasic sites.
Asunto(s)
Citosina , ADN Polimerasa Dirigida por ADN , Guanina , Oligodesoxirribonucleótidos , Timina , Secuencia de Bases , ADN , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación de Ácido NucleicoRESUMEN
The binding of human immunodeficiency virus type 1 (HIV-1) Rev protein to its viral RNA target, stem-loop IIB (SLIIB) within the Rev Response element (RRE), mediates the export of singly-spliced and unspliced viral mRNA from the nucleus to the cytoplasm of infected cells; this Rev-mediated transport of viral RNA is absolutely required for the replication of infectious virus. To identify important features that influence the binding affinity and specificity of this Rev-RRE interaction, we have characterized the arginine side-chain dynamics of the Rev arginine-rich motif (ARM) while bound to a 34 nt RNA oligomer that corresponds to SLIIB. As the specificity of the Rev-RRE interaction varies with salt concentration, arginine side-chain dynamics were characterized at two different salt conditions. Following NMR measurements of (15)N spin relaxation parameters for the arginine (15)N(epsilon) nuclei, the dynamics of the corresponding N(epsilon)-H(epsilon) bond vectors were interpreted in terms of Lipari-Szabo model-free parameters using anisotropic expressions for the spectral density functions. Results from these analyses indicate that a number of arginine side-chains display a surprising degree of conformational freedom when bound to RNA, and that arginine residues having known importance for specific RRE recognition show striking differences in side-chain mobility. The (15)N relaxation measurements at different salt conditions suggest that the previously reported increase in Rev-RRE specificity at elevated salt concentrations is likely due to reduced affinity of non-specific Rev-RNA interactions. The observed dynamical behavior of the arginine side-chains at this protein-RNA interface likely plays an important role in the specificity and affinity of Rev-SLIIB complex formation.
Asunto(s)
Arginina/metabolismo , Productos del Gen rev/metabolismo , VIH-1/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anisotropía , Secuencia de Bases , Sitios de Unión , Transporte Biológico , Difusión , Productos del Gen rev/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligorribonucleótidos/química , Oligorribonucleótidos/genética , Oligorribonucleótidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Conformación Proteica , ARN Viral/química , ARN Viral/genética , Proteínas de Unión al ARN/química , Sales (Química)/farmacología , Especificidad por Sustrato , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
Structure-function studies of the insulin molecule indicate that an insulin B chain domain comprising residues 22-26 is involved both in binding to the insulin receptor (INSR) and in insulin dimer formation, suggesting that this domain might also interact with a structure resembling the insulin dimer interface in the INSR. Expression of a mutant INSR cDNA with a deletion of the region corresponding to exon 2 of the INSR gene produces a protein devoid of insulin-binding activity, although the mutant protein is processed appropriately to alpha- and beta-subunits, suggesting that the insulin-binding domain is encoded at least in part by exon 2. Within this region of the INSR molecule, the sequence 83-103 fulfills the structural criteria for a dimer interface. Studies of mutant INSRs with substitutions for phenylalanine 88 or 89 show that the presence of phenylalanine at position 89 is essential for full binding affinity.
Asunto(s)
Genes/genética , Ligandos , Receptor de Insulina/genética , Secuencia de Aminoácidos , Células Cultivadas , ADN/genética , Exones , Humanos , Insulina/metabolismo , Datos de Secuencia Molecular , Mutación , Fenilalanina/análisis , Receptor de Insulina/metabolismo , Receptor de Insulina/fisiología , Transcripción Genética , TransfecciónRESUMEN
We have synthesized and studied by proton NMR a duplex heptaoligonucleotide containing a 5-bromouracil (brU)-adenine base pair. This represents the first structural characterization of a B-form DNA containing brU. The brU.A base pair is Watson-Crick rather than Hoogsteen as seen for the monomers in the crystalline state. From analysis of the NOESY sepctra at very short mixing times evidence is presented that substitution of brU for T induces significant conformational changes from that of a normal B DNA. The helix twist between brU4.A11 and G3.C12 is ca. 15 degrees and for both brU4 and G3 the glycosyl torsion angles are significantly changed. The imino proton of the bru.A base pair shows a pH insensitive line with which shows that the pK of brU in this base pair is very much higher than that of the monomer.
Asunto(s)
Adenina , Bromouracilo , ADN , Composición de Base , ADN/análisis , Espectroscopía de Resonancia Magnética , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/análisisAsunto(s)
Respuesta Galvánica de la Piel , Adolescente , Adulto , Emociones , Retroalimentación , Femenino , Humanos , Imaginación , Masculino , Pruebas PsicológicasAsunto(s)
Psicología Experimental/instrumentación , Goma , Transductores , Humanos , Métodos , RespiraciónAsunto(s)
Conducta , Volición , Peso Corporal , Femenino , Humanos , Masculino , Motivación , AutoimagenRESUMEN
This paper deals with the crisis of cancer and the communication loss faced by the laryngectomized patient. It considers the particular issues for the patient and his family and how the use of crisis intervention can reinforce the coping patterns of the patient and his family.
Asunto(s)
Neoplasias de Cabeza y Cuello/psicología , Adaptación Psicológica , Adulto , Intervención en la Crisis (Psiquiatría) , Mecanismos de Defensa , Femenino , Humanos , Masculino , Ajuste Social , Asistencia Social en Psiquiatría , Estrés Psicológico/psicologíaRESUMEN
One- and two-dimensional nuclear Overhauser effect experiments demonstrate that a single hydrogen bond between a T imino proton and purine N3 is sufficient to hold the base pair dPu.dT in d(CGPuAATTTCG) by a Watson-Crick fashion rather than a Hoogsteen type. In addition, the dPu.dT base pair is well stacked with neighboring base pairs. The spin-lattice relaxation measurements at 30 and 35 degrees C of two decamers, d(CGPuAATTTCG) and d(CGAAATTTCG), reveal that the elimination of two single hydrogen bonds of dA.dT base pairs (due to the substitution of adenine for purine) in the sequence results in an increase in the overall imino proton exchange rate from 7 to 36 s-1 at the site of mismatch.
Asunto(s)
Composición de Base , Oligonucleótidos , Timina , Secuencia de Bases , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética/métodos , Conformación de Ácido NucleicoRESUMEN
The use of a reverse-phase polystyrene resin for ion-pair HPLC purification of large amounts of synthetic chimeric DNA-RNA oligomers that is faster and more reliable than previously used techniques has been developed. The preparation of synthetic oligomers containing RNA requires the use of tetrabutylammonium fluoride in the final step, the cleavage of the tert-butyl-dimethyl silyl protecting group from the ribonucleotides. Cleavage is accompanied by the serendipitous formation of ion pairs between tetrabutylammonium cations and the oligomer phosphates. The formation of these ion pairs retards the elution of the oligomer during HPLC, which allows rapid removal of excess tetrabutylammonium fluoride and the concomitant purification of chimeric ribozymes. This technique is based on a correlation between the length of ion-paired oligomers and their retardation during HPLC. The advantages of reverse-phase ion-pair HPLC on polystyrene resin for the fast purification of oligoribonucleotides are discussed and illustrated through the examples of synthesized chimeric ribozymes.
Asunto(s)
ARN Catalítico/análisis , Proteínas Recombinantes de Fusión/análisis , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Datos de Secuencia Molecular , PoliestirenosRESUMEN
In order to specify the recognition requirements of the human DNA (cytosine-5-)-methyltransferase, two isomeric 48mers were synthesized so as to link a long block of DNA with a shorter complementary block of DNA through a tether consisting of five thymidine residues. These isomeric foldback molecules, differing only in the location of the 5-methyldeoxycytosine, were shown to be unimolecular, to contain a region of duplex DNA, and to contain a region of single-stranded DNA. When used as substrates for the DNA methyltransferase, only one of the isomers was methylated. A comparison of the structures of the two isomers allows us to begin to define the potential sites of interaction between the enzyme and the three nucleotides forming a structural motif consisting of 5-methyldeoxycytosine, its base-paired deoxyguanosine, and a deoxycytosine 5' to the paired deoxyguanosine.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN/química , Secuencia de Bases , ADN/metabolismo , Femenino , Humanos , Cinética , Metilación , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Placenta/enzimología , Embarazo , Especificidad por SustratoRESUMEN
Metallothionein is a naturally occurring metal-binding protein with high cysteine content. Oligopeptides containing three cysteinyl residues and having amino acid sequences analogous to portions of this protein were synthesized by the solid-phase method. Strong affinity of the synthetic peptides to Cd2+ and Zn2+ was observed, and the dissociation constants of the peptide-metal complexes were 2-4 orders of magnitude lower than those of cysteine-metal and dithioerythritol-metal complexes. Effectiveness of detoxification of the peptides against Cd toxicity was demonstrated by the higher survival rates of mice treated with the peptides and by the neutralization of Cd toxicity by the peptides in tissue cultures.
Asunto(s)
Cationes Bivalentes , Metaloproteínas , Metalotioneína , Oligopéptidos/metabolismo , Cationes Bivalentes/antagonistas & inhibidores , Cationes Bivalentes/metabolismo , Cationes Bivalentes/toxicidad , Células Cultivadas/efectos de los fármacos , Cisteína , Metaloproteínas/metabolismo , Metalotioneína/análogos & derivados , Metalotioneína/metabolismo , Metales , Relación Estructura-ActividadRESUMEN
An N-acetylgalactosaminyltransferase, which converts blood group O red blood cells to A cells, was purified to homogeneity from plasma of blood group A1 subjects. The enzyme was adsorbed on Sepharose 4B, and after washing out the impurities, the enzyme was eluted with UDP. This procedure resulted in a 70,000- to 100,000-fold increase in specific activity with recovery of about 80%. Further purification of the enzyme was achieved by Bio-Gel P treatment. The final enzyme preparation showed a single protein band, which coincided with enzyme activity, on acrylamide gel electrophoresis, and revealed a single protein band on sodium dodecyl sulfate-gel electrophoresis. Judging from the molecular weight (90,000 to 100,000), which was estimated by Sephadex gel filtration, and the subunit size estimated by sodium dodecyl sulfate-gel electrophoresis, the enzyme is presumably in a dimeric form. The enzyme required Mn2+ and had optimum activity at pH 6.5 to 7.0.
Asunto(s)
Sistema del Grupo Sanguíneo ABO , Galactosiltransferasas/sangre , Acetilgalactosamina , Galactosiltransferasas/aislamiento & purificación , Humanos , Cinética , Peso MolecularRESUMEN
An oligodeoxynucleotide duplex containing the chemotherapeutic agent 5-fluorouracil (FU) has been constructed by solid phase phosphotriester synthesis and has been studied in solution by proton NMR. In this study, we provide the first structural characterization of a DNA complex containing a FU.A base pair. It has been determined that the 7-mer duplex containing a central FU.A base pair adopts a normal right-handed configuration and the A residue in the FU.A pair is oriented in the normal anticonfiguration giving a Watson-Crick base pair. The significant difference between T.A and FU.A base pairs is dynamic, not structural: the FU.A base pair opens faster than normal base pairs in the oligonucleotide studied. We provide evidence that the FU.A base pair has a significantly enhanced opening rate resulting form decreased stacking of the 5-fluorouracil residue and not from the enhanced acidity of the 5'-fluorouracil imino proton.
Asunto(s)
Adenina , ADN , Fluorouracilo , Espectroscopía de Resonancia Magnética , Concentración de Iones de Hidrógeno , Conformación de Ácido NucleicoRESUMEN
A heteroduplex containing the mismatch 2-aminopurine (AP)-adenine has been synthesized and studied by proton NMR. The mismatch was incorporated into the sequence d[CGG(AP)GGC].d-(GCCACCG). One-dimensional nuclear Overhauser effect measurements in H2O and two-dimensional nuclear Overhauser effect spectra in D2O show AP.A base pairs in a wobble structure in which both bases are in the anti conformation. The adenine is stacked well in the helix, but the helix twist between the adenine and neighboring cytosine in the 3' direction is unusually small. As a result, the aminopurine on the opposite strand is somewhat pushed out of the helix. From the measurements of the imino proton line widths, the two adjacent G.C base pairs are not found to be significantly destabilized by the presence of the purine-purine wobble pair.
Asunto(s)
2-Aminopurina , Adenina , Composición de Base , Oligodesoxirribonucleótidos , Adenina/análogos & derivados , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética/métodos , Conformación de Ácido NucleicoRESUMEN
Oligonucleotides containing the nonbase residues 1,3-propanediol or 1,4-anhydro-2-deoxy-D-ribitol were synthesized and used as primers for the polymerase chain reaction (PCR). Since these residues cannot be replicated by a DNA polymerase, the resulting PCR products have protruding 5' ends. Primers were designed with three regions, a 3' region complementary to the desired template, a 5' region complementary to a preselected nucleotide sequence, and a nonreplicable element interposed between these two containing 1-3 of the nonbase residues. The primers were used in a PCR and the products hybridized without denaturation to a solid support containing an immobilized preselected nucleotide sequence. Studies are reported showing the effects of the nonreplicable elements in primer extension reactions and the application to the capture of PCR products.
Asunto(s)
Cartilla de ADN , Oligonucleótidos/síntesis química , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , ADN/biosíntesis , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribosa/análogos & derivados , Datos de Secuencia Molecular , Glicoles de PropilenoRESUMEN
A gel assay is introduced to measure DNA polymerase insertion kinetics at single sites along a DNA template strand. The assay is used to analyze the kinetics of inserting deoxynucleotides opposite a synthetic abasic (apurinic/apyrimidinic) lesions using Drosophila DNA polymerase alpha. The location of the abasic lesion next to different nearest-neighbor bases allows the effects of base stacking on the specificity of insertion to be evaluated. The specificity of nucleotide insertion, Vmax/Km, is 6-11 times greater for A over G and about 20-50 times greater for A over C and T. The insertion specificity at the abasic lesion appears to depend more on differences in Vmax than Km. Apparent Michaelis constants for inserting A and G deoxynucleotides are similar to within about a factor of 2. The insertion of A or G occurs most efficiently at the abasic lesion when T is the 5'-nearest neighbor on the primer strand and least efficiently when G is the 5'-nearest neighbor. The presence of different base stacking partners adjacent to the site of insertion has up to a 4-fold effect on specificity.
Asunto(s)
Ácido Apurínico , Daño del ADN , ADN Polimerasa II/metabolismo , Polinucleótidos , Animales , Secuencia de Bases , Drosophila/enzimología , Cinética , Matemática , Modelos Genéticos , Oligodesoxirribonucleótidos/síntesis química , Moldes GenéticosRESUMEN
2-Aminopurine (AP), a potent mutagenic base analogue, most frequently pairs with thymine. In the AP X T base pair, both bases adopt normal tautomeric forms. The mechanism for the mutagenic activity arises from its observed pairing with cytosine, which has been ascribed to an enhanced tendency to adopt the rare imino tautomeric form. NMR studies in H2O on all the exchangeable protons in an oligonucleotide duplex containing an AP X T base pair show Watson-Crick hydrogen bonding. When the thymine is replaced by cytosine in the duplex, we observe an AP X C base pair. Both amino protons of AP are seen excluding the rare tautomeric form. Although several alternative structures are possible, it is shown that the second hydrogen bond is formed by protonation of the AP X C base pair and that this is the dominant species under physiological conditions.