RESUMO
This short review assembles the contributions of the author's laboratory to the structural aspects of DNA. DNA was modified by small ligands and/or substituents. There are three aspects to this work: a) Protonation of guanosine and DNA and the formation of triple- and quadruple-strands of guanosine, its nucleotides, their polymers and DNA. b) Substitution of the 2'-position of deoxyribose by the most polar atom, fluorine: studies on 2'-deoxy-2'-fluro-nucleosides, -nucleotides and their polymers, studied both by structural and biological methods. c) The effect of introducing the methyl group in the large groove of DNA: NMR studies of oligonucleotides containing N6-methylated adenine residues, and enzymatic and molecular biology work on Dam methylase are reported.
Assuntos
DNA/química , Ligantes , Conformação de Ácido Nucleico , Adenina/análogos & derivados , Sequência de Bases , DNA/efeitos dos fármacos , Desoxirribose , Nucleotídeos de Guanina , Guanosina , Espectroscopia de Ressonância Magnética , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Plasmídeos , Polidesoxirribonucleotídeos/química , S-Adenosilmetionina/farmacologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/farmacologiaRESUMO
DNA adenine methyltransferase (Dam methylase) has been crosslinked with its cofactor S-adenosyl methionine (AdoMet) by UV irradiation. About 3% of the enzyme was radioactively labelled after the crosslinking reaction performed either with (methyl-3H)-AdoMet or with (carboxy-14C)-AdoMet. Radiolabelled peptides were purified after trypsinolysis by high performance liquid chromatography in two steps. They could not be sequenced due to radiolysis. Therefore we performed the same experiment using non-radioactive AdoMet and were able to identify the peptide modified by the crosslinking reaction by comparison of the separation profiles obtained from two analytical control experiments performed with 3H-AdoMet and Dam methylase without crosslink, respectively. This approach was possible due to the high reproducibility of the chromatography profiles. In these three experiments only one radioactively labelled peptide was present in the tryptic digestions of the crosslinked enzyme. Its sequence was found to be XA-GGK, corresponding to amino acids 10-14 of Dam methylase. The non-identified amino acid in the first sequence cycle should be a tryptophan, which is presumably modified by the crosslinking reaction. The importance of this region near the N-terminus for the structure and function of the enzyme was also demonstrated by proteolysis and site-directed mutagenesis experiments.
Assuntos
Escherichia coli/enzimologia , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica) , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Reagentes de Ligações Cruzadas , Proteínas de Escherichia coli , Metiltransferases/química , Dados de Sequência Molecular , Peptídeos/química , Ligação ProteicaRESUMO
Double-stranded polynucleotides, which are composed of two complementary homopolyribonucleotides containing no genetic information, are synthetic molecules capable of mimicking the action of natural double-stranded RNA or viral RNA on cells. Double-stranded polyribonucleotides act as an alarm system alerting the cell to the presence of an external aggression, e.g. a viral attack. In addition, polyribonucleotides have a more active function in that they trigger cell defense processes through activation of a family of genes, of which some encode cytokines, activation of cytoplasmic enzymes involved in antiviral mechanisms or signal transduction, and activation of nonspecific immune responses. Double-stranded polyribonucleotides containing one mismatched base pair per helix have been found to be especially interesting. The best known example is poly(I).poly(C12U), also called ampligen. Poly(I).poly(C12U) is capable, in experimental models, of limiting the development of viruses (including HIV), reducing tumor growth, eliminating metastases, and, according to one report, preventing steady declines in T-cell counts in HIV-positive patients. Therapeutic doses used in the USA as an experimental drug induced little toxicity. In vitro, poly(I).poly(C12U) acts synergistically with interferon, interleukin 2 or AZT, suggesting that these latter drugs may be effective in lower, less toxic doses when used in combination with poly(I).poly(C12U). The therapeutic activity of poly(I).poly(C12U) holds promise. More extensive prospective studies of this agent are warranted.
Assuntos
Infecções por HIV/terapia , Polirribonucleotídeos/uso terapêutico , Infecções por Citomegalovirus/terapia , Sinergismo Farmacológico , Hepatite/terapia , Herpes Simples/terapia , Humanos , Interferons/uso terapêutico , Neoplasias/tratamento farmacológico , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Polirribonucleotídeos/biossíntese , Polirribonucleotídeos/química , Polirribonucleotídeos/genética , RNA de Cadeia Dupla/genéticaRESUMO
Adenine methylation of GATC sequences in DNA is carried out by the DNA adenine methyltransferase with the methyl group source being the cofactor S-adenosylmethionine. We report 3H NMR studies on the interaction of DNA adenine methyltransferase with S-adenosylmethionine and the reaction when the ternary complex is formed with an oligonucleotide containing a GATC site. The methylation reaction was also studied in the presence of a competitive inhibitor and this showed two successive stages involved in the methylation and two sites of binding for S-adenosylmethionine.
Assuntos
Escherichia coli/enzimologia , S-Adenosilmetionina/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Sítio Alostérico , Sítios de Ligação , Cinética , Espectroscopia de Ressonância Magnética/métodos , Oligonucleotídeos/metabolismo , TrítioRESUMO
The DNA duplex 5'-d(GCCACAAGCTC).d(GAGCTGGTGGC), which contains a central G.A mismatch has been studied by one and two-dimensional NMR techniques. The duplex corresponds to the sequence 29-39 of the K-ras gene. The mismatch position is that of the first base of the Gly12 codon, a hot spot for mutations. The observed NOEs of the nonexchangeable protons show that both of the bases of the mismatched pair are intrahelical over a wide range of pH. However, the structure of the G.A mispair and the conformation of the central part of the duplex change with pH. This structural change shows a pK of 6.0. At low pH, the G.A bases are base paired with hydrogen bonds between the keto group of the G residue and the amino group of the A residue and, secondly, between the N7 of the G and a proton on N1 of A. This causes the G residue to adopt a syn conformation. On raising the pH, the N1-H proton of the protonated A residue is removed, and the base pair rearranges. In the neutral G.A base pair both residues adopt an anti conformation, and the mismatch is stabilized by hydrogen bonds. Our results on the exchangeable and A(H2) protons of the mismatched pair indicate a shift from a classical face-to-face two hydrogen-bonded structure to a slipped structure stabilized by bifurcated hydrogen bonds. This may be a particular characteristics of this oncogenic sequence in which the G.A error is poorly repaired.
Assuntos
Reparo do DNA , Genes ras , Mutação , Composição de Bases , Sequência de Bases , DNA/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , SoluçõesRESUMO
Adenine residues in DNA are oxidized under the action of ionizing radiation at the C-8 position to give 7,8-dihydro-8-oxoadenine. The formation of this lesion can be considered a cause of mutations and carcinogenesis. Oligodeoxyribonucleotides 39 and 47 bases long containing a single 7,8-dihydro-8-oxoadenine (8-hydroxyadenine) residue were synthesized by using nucleoside phosphoramidites. They were used as templates to study the copies obtained in vitro by the Klenow fragment and the thermostable Taq DNA polymerase. 7,8-Dihydro-8-oxoadenine does not block the replication and thymine is incorporated opposite the damage. The modifications of the DNA duplex conformation provoked by 7,8-dihydro-8-oxoadenine are minor. 1H-NMR spectroscopy shows that the duplex is in a B form, the sugar in a normal position in the helix and the modified base in the anti position. NMR confirms that 7,8-dihydro-8-oxoadenine exists predominantly in the keto form.
Assuntos
Adenina/análogos & derivados , Replicação do DNA , DNA/química , Adenina/química , Adenina/metabolismo , Composição de Bases , Sequência de Bases , DNA/biossíntese , DNA/genética , Eletroforese em Gel de Poliacrilamida , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/genética , Oxirredução , Prótons , Moldes GenéticosRESUMO
Highly purified DNA-adenine methyltransferase was irradiated in the presence of different concentrations of radiolabelled S-adenosyl-methionine (AdoMet) with a conventional Mineralight UV-lamp from several minutes up to 1 h while incubating in ice. Incorporation of radioactivity was monitored by electrophoresis of the crosslink between S-adenosyl-methionine and Dam methylase on SDS-polyacrylamide gels followed by fluorography. Crosslinking reached a maximum in presence of 10 microM S-adenosyl-methionine; it was inhibited in the presence of substances which competitively inhibit methylation of DNA by Dam methylase, like sinefungin or S-adenosyl-homocysteine, but not in the presence of non-inhibitors like ATP or S-isobutyl-adenosine. The crosslink obtained was resistant against a wide range of even drastic conditions commonly used in protein and peptide chemistry. Proteins, which do not bind S-adenosyl-methionine, as well as heat inactivated Dam methylase were not photolabelled. After limited proteolysis the radioactive label appeared only in certain of the peptides obtained. From Western blots carried out with polyclonal antibodies produced against a synthetic peptide corresponding in its sequence to amino acids 92-106 of the Dam methylase, the crosslinking of AdoMet could be tentatively mapped at a position after amino acid 106.
Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica) , Ligação Competitiva , Endopeptidases/farmacologia , Ativação Enzimática , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos da radiação , Temperatura Alta , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/efeitos da radiação , Raios UltravioletaRESUMO
The turnover of DNA-adenine-methylase of E. coli strongly decreases when the temperature is lowered. This has allowed us to study the binding of Dam methylase on 14 bp DNA fragments at 0 degrees C by gel retardation in the presence of Ado-Met, but without methylation taking place. The enzyme can bind non-specific DNA with low affinity. Binding to the specific sequence occurs in the absence of S-adenosyl-methionine (Ado-Met), but is activated by the presence of the methyl donor. The two competitive inhibitors of Ado-Met, sinefungin and S-adenosyl-homocysteine, can neither activate this binding to DNA by themselves, nor inhibit this activation by Ado-Met. This suggests that Ado-Met could bind to Dam methylase in two different environments. In one of them, it could play the role of an allosteric effector which would reinforce the affinity of the enzyme for the GATC site. The analogues can not compete for such binding. In the other environment Ado-Met would be in the catalytic site and could be exchanged by its analogues. We have also visualized conformational changes in Dam methylase induced by the simultaneous binding of Ado-Met and the specific target sequence of the enzyme, by an anomaly of migration and partial resistance to proteolytic treatment of the ternary complex Ado-Met/Dam methylase/GATC.
Assuntos
Escherichia coli/enzimologia , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica) , Adenosina/análogos & derivados , Adenosina/farmacologia , Regulação Alostérica , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli , Cinética , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , S-Adenosil-Homocisteína/farmacologia , S-Adenosilmetionina/farmacologia , Cloreto de Sódio/farmacologia , Temperatura , TermodinâmicaRESUMO
Previous comparison of the amino acid sequences of the GATC-methylating Escherichia coli Dam methyltransferase (MTase) with those of other adenine MTases (M.EcoRV, M.DpnII and T4Dam) localized four conserved regions. Regions III and IV have similarities with many other MTases. The sequence DPPY (or NPPY) is always present in region IV. It was suggested to be the AdoMet binding site. Publication of the nucleotide and amino acid sequences of M.CviBIII, M.DpnA and MutH give further credence to this assignment: M.DpnA, which also methylates GATC, has strong similarities with regions III and IV; M.CviBIII, a cytosine methylase, has a characteristic NPPY sequence in region IV, and only limited resemblance in region III; MutH, the GATC-specific endonuclease in DNA mismatch repair, has significant similarities uniquely in region III. The presently available evidence suggests that region III is the GAT(C) binding site and region IV is the AdoMet binding site. This hypothesis is strengthened by recent genetic findings.
Assuntos
Proteínas de Bactérias/metabolismo , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA , Escherichia coli/enzimologia , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica) , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Metilases de Modificação do DNA/genética , Reparo do DNA , Endodesoxirribonucleases/genética , Proteínas de Escherichia coli , Metiltransferases/genética , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência do Ácido NucleicoRESUMO
We have determined the three-dimensional structure of a non-selfcomplementary nonanucleotide duplex which contains an abasic (apyrimidinic) site in the centre, i.e. a deoxyribose residue opposite an adenosine. The majority of the base and sugar proton resonances were assigned by NOESY, COSY and 2DQF spectra in D2O and H2O. We have measured the initial slope of buildup of NOEs in NOESY spectra at very short mixing times (25 to 50 ms), and from these were able to establish interproton distances for the central part of the duplex. We propose a different strategy for proton-proton distance determinations which takes into account the observed variations in correlation times for particular proton-proton vectors. A set of 31 measured interproton distances was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two structures were obtained which retain all aspects of a classical B DNA in which the unpaired adenine and the abasic deoxyribose lie inside the helix. We observe that the non-hydrogen bonded adenine is held well in the helix, the Tm of this base being the same as that of the A.T base pairs in the same duplex.
Assuntos
DNA , Algoritmos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
The amino acid sequence of EcoRV DNA methyltransferase which methylates the amino group of the 5'-adenine residue of the target sequence GATATC has been found to be closely related to that of three other adenine methyltransferases, DpnII, dam and damT4, the target sequence of which is GATC. Despite large differences on the DNA level, the four sequences show four blocks of homologies. One of these blocks has the sequence DVYXDPPY and is found with little modification in numerous other DNA methyltransferases. It is speculated that it could be the binding site of the methyl donor, S-adenosylmethionine. On the other hand, the identification of a DNA-binding region is more tenuous. As expected, no analogies with (dimeric) repressors and cro proteins which have the characteristic helix-turn-helix motif have been observed.
Assuntos
Sequência de Bases , DNA (Citosina-5-)-Metiltransferases/genética , Homologia de Sequência do Ácido Nucleico , Fagos T/genética , Sequência de Aminoácidos , Evolução Biológica , Proteínas de Ligação a DNA/genética , Metilação , Fagos T/enzimologiaRESUMO
We have recorded NOESY spectra of two non-selfcomplementary undecanucleotide duplexes. From the observed NOEs we do not detect any significant distortion of the helix when a G-C pair is replaced by a G-T pair and the normal interresidue connectivities can be followed through the mismatch site. We conclude that the 2D spectra of the non-exchangeable protons do not allow differentiation between a wobble or rare tautomer form for the mismatch. NOE measurements in H2O, however, clearly show that the mismatch adopts a wobble structure and give information on the hydration in the minor groove for the G-T base pair which is embedded between two A-T base pairs in the sequence.
Assuntos
Composição de Bases , Espectroscopia de Ressonância Magnética/métodos , Conformação de Ácido Nucleico , Adenosina , Sequência de Bases , Oligodesoxirribonucleotídeos , PrótonsRESUMO
The enzyme dam methylase which recognizes and methylates the adenine in the palindromic sequence GATC in DNA was isolated and the secondary structure was determined by CD spectroscopy and various predicting methods from the amino acid sequence. The interaction of dam methylase with S-adenosylmethionine was studied by CD spectroscopy indicating a decrease of the percentage of alpha-helix as the amount of S-adenosylmethionine bound to the enzyme was increased.
Assuntos
Escherichia coli/enzimologia , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , Dicroísmo Circular , Conformação Proteica , DNA Metiltransferases Sítio Específica (Adenina-Específica)RESUMO
The conformation of two hexanucleotides, d(GGATCC) and d(GGm6ATCC), has been studied by proton nuclear magnetic resonance. Nuclear Overhauser effect (NOE) measurements on d(GGATCC) are in agreement with a normal B form right-handed helical structure. The single- and double-strand resonances are in fast exchange on a proton NMR time scale. The exchange is observed to be slow for d(GGm6ATCC); up to the Tm, separate resonances are observed for each state, though above the Tm exchange becomes more rapid. The preferred orientation of the adenosine methylamino group (methyl cis to N1) hinders base-pair formation. At 0 degree C irradiation of the m6A-T imino proton gives an NOE to AH2, showing that base pairing is Watson-Crick. Intra- and interresidue NOEs show that the helix is right handed and in the B form. Comparing results on the two oligomers demonstrates that adenosine methylation induces little or no change in the conformation of the helix but reduces the Tm from 45 to 32 degrees C. All of the amino proton resonances, as well as the imino resonances, have been assigned. From NOE experiments on the unmethylated oligomer we have located the Watson-Crick and non-Watson-Crick adenosine amino protons. At 0 degree C these resonances show broadening due to rotation of the amino group, and their rotation is slightly slower than for the adjacent guanosine amino group, though both these amino groups have lifetimes of less than 10 ms at 0 degree C. The imino protons show normal behavior, disappearing from the spectra ca. 20 degrees C below the Tm.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos , Oligorribonucleotídeos , Sequência de Bases , Espectroscopia de Ressonância Magnética/métodos , Metilação , Relação Estrutura-AtividadeRESUMO
All exchangeable protons in a short DNA helix, d(CG)3 sodium salt, have been studied by proton nuclear magnetic resonance. The cytidine and guanosine amino protons have been assigned for the first time. As a function of temperature the cytidine amino protons and the imino protons behave very similarly, their relaxation is dominated by exchange with solvent above 30 degrees C. The guanosine amino protons, however, show that helix opening can only be described by a multistate model. The most rapid process observed is probably a twist about the helix axis which lengthens or breaks the guanosine amino hydrogen bond and allows rotation of the amino group. The second fastest process is a scissor opening into the major groove which gives rise to solvent exchange with the imino and cytidine amino protons. The slowest process observed is the complete base pair opening in which the guanosine amino protons also exchange with solvent. For the ammonium salt of the oligonucleotide, a specific ammonium ion complex is observed which at low temperature may catalyze exchange of the guanosine amino protons with the protons of the ammonium ion, but retards exchange with solvent. The complex appears to be specific for the sequence d(CpG).
Assuntos
Monofosfato de Citidina , Nucleotídeos de Citosina , DNA , Fosfatos de Dinucleosídeos , Guanosina/análogos & derivados , Conformação de Ácido Nucleico , Aminas , Fenômenos Químicos , Química , Monofosfato de Citidina/análogos & derivados , Iminas , Espectroscopia de Ressonância Magnética/métodos , TermodinâmicaRESUMO
Six polynucleotide duplexes containing polydeoxyadenylic acid, polyadenylic acid or poly-2'-deoxy-2'-fluoro-adenylic acid in one strand, and polydeoxyuridylic acid or polydeoxythymidylic acid in the other strand have been studied by circular dichroism, ionic strength dependence of melting temperatures and binding of the DNA specific antibiotic netropsin. Circular dichroism spectra of (dA)n . (dT)n and (dA)n . (dU)n indicated the presence of the B-form of DNA, while those of (dAfl)n . (dT)n and (rA)n . (dT)n (and the corresponding (dU)n hybrids) indicated the presence of the A-form. (dAfl)n . (dT)n and (dAfl)n . (dU)n bound netropsin only slightly less than the (dA)n containing duplexes, while replacement by (rA)n decreased netropsin binding to a large degree. Since netropsin requires B-DNA for binding, it is concluded that the A to B transition is facilitated in the case of fluorine substitution in the sugar moiety, while the 2'-OH group greatly limits this conformational change.
Assuntos
Adenosina , Floxuridina , Guanidinas , Netropsina , Conformação de Ácido Nucleico , Polidesoxirribonucleotídeos , Dicroísmo Circular , Relação Estrutura-AtividadeRESUMO
The four disulfide bonds of nine homologous short curare-like polypeptides are cleaved by reduced dithiothreitol. Air oxidation renaturations of the reduced compounds are followed by far-ultraviolet circular dichroism analysis, and the kinetics of refolding thus determined are compared. They indicate that three toxins refold 4--10 times more slowly than the six others. It is shown that a significant difference between the refolding kinetics still subsists when renaturations are made in the presence of various concentrations of thiol-disulfide exchange reagents or at various pH values. From an examination of the toxin sequences, it is proposed that a single additional amino acid insertion is responsible for the difference in the observed kinetics. This proposal is supported by temperature studies of renaturation kinetics.
Assuntos
Neurotoxinas , Venenos de Serpentes , Animais , Dicroísmo Circular , Venenos Elapídicos , Cinética , Oxirredução , Conformação Proteica , Desnaturação Proteica , Especificidade da Espécie , Termodinâmica , Difração de Raios XRESUMO
The proton and 13C NMR spectra of uridine, deoxyuridine and four 2' substituted uridines (dUn, dUz, dUcl and dUfl) are reported. A linear relationship between the electronegativity of the 2'-substituent and the carbon-13 chemical shift of C2' is observed. Taking into account the effect of electronegativity by using the correction proposed by Karplus or by Jankowski, the proton-proton coupling constants have been used to compute the conformational equilibria of the six uridines. It is shown that the contribution of the N form (3'-endo -2'-exo) increases with the electronegativity of the 2' substituent. Thus dUfl contains some 85% N form in solution. - Applying similar corrections to published data in the adenosine series, a similar correlation is observed. This observation, that the most polar substituent pulls the pucker to its side, holds also for 3'-substituted compounds, like cordycepin (3'dAdo) and 3'-deoxy-3'-amino-adenosine. It is suggested that the influence of the electronegativity could be the dominating effect of nucleoside conformations and would also hold for arabinosides and xylosides. This effect should therefore also be the principal force which determines the differences between DNA and RNA.