RESUMEN
Using a combination of nuclear magnetic resonance (NMR) spectroscopy experiments and molecular dynamics, we have analyzed the structure and dynamics of a complex between the bisnaphthalimide drug LU-79553 and the DNA duplex d(ATGCAT)(2). LU-79553 is a DNA-binding topoisomerase II inhibitor that is particularly effective against human solid tumors that are refractory to other drugs. We have found that the two naphthalimide chromophores of the drug bisintercalate at the TpG and CpA steps of the DNA hexanucleotide, stacking mainly with the purine G and A bases from opposite strands. The 3, 7-diazanonylene linker lies in the major groove of the DNA molecule, with its two amino groups hydrogen-bonded to the symmetry-related guanine bases. Unexpectedly, we have detected an unprecedented exchange process between two equivalent and intercalated states of the naphthalimide rings in the drug-DNA complex. The interconversion process takes place by rotational ring flipping, has an activation energy of 22 kcal mol(-)(1) for the two rings, and does not affect the aminoalkyl linker region of the drug. The exchange rate is intermediate to fast on the chemical shift time scale at 36 degrees C (1800 s(-)(1)) but slow at 2 degrees C (20 s(-)(1)). We have also observed limited flexibility for the drug linker on the picosecond time scale on the basis of NMR data and a time-averaged restrained molecular dynamics simulation. The implications of the structural and dynamic features of the DNA-LU-79553 complex on the binding specificity and on the antitumor activity of bisnaphthalimide agents are discussed.
Asunto(s)
Amidas/química , Antineoplásicos/química , ADN/química , Sustancias Intercalantes/química , Isoquinolinas/química , Ácidos Nucleicos Heterodúplex/química , Secuencia de Bases , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodos , Oligonucleótidos/química , Conformación Proteica , Soluciones , TermodinámicaRESUMEN
We have carried out a physicochemical and computational analysis on the stability of the intercalated structures formed by cytosine-rich DNA strands. In the computational study, the electrostatic energy components have been calculated using a Poisson-Boltzmann model, and the non-polar energy components have been computed with a van der Waals function and/or a term dependent on the solvent-accessible surface area of the molecules. The results have been compared with those obtained for Watson-Crick duplexes and with thermodynamic data derived from UV experiments. We have found that intercalated DNA is mainly stabilized by very favorable electrostatic interactions between hydrogen-bonded protonated and neutral cytosines, and by non-polar forces including the hydrophobic effect and enhanced van der Waals contacts. Cytosine protonation electrostatically promotes the association of DNA strands into a tetrameric structure. The electrostatic interactions between stacked C.C+ pairs are strongly attenuated by the reaction field of the solvent, and are modulated by a complex interplay of geometric and protonation factors. The forces stabilizing intercalated DNA must offset an entropic penalty due to the uptake of protons for cytosine protonation, at neutral pH, and also the electrostatic contribution to the solvation free energy. The latter energy component is less favorable for protonated DNA due to the partial neutralization of the negative charge of the molecule, and probably affects other protonated DNA and RNA structures such as C+-containing triplexes.
Asunto(s)
Centrómero/química , ADN/química , Conformación de Ácido Nucleico , Simulación por Computador , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Desnaturalización de Ácido Nucleico , Electricidad Estática , Temperatura , TermodinámicaRESUMEN
Eukaryotic RNases H from Saccharomyces cerevisiae , Schizosaccharomyces pombe and Crithidia fasciculata , unlike the related Escherichia coli RNase HI, contain a non-RNase H domain with a common motif. Previously we showed that S.cerevisiae RNase H1 binds to duplex RNAs (either RNA-DNA hybrids or double-stranded RNA) through a region related to the double-stranded RNA binding motif. A very similar amino acid sequence is present in caulimovirus ORF VI proteins. The hallmark of the RNase H/caulimovirus nucleic acid binding motif is a stretch of 40 amino acids with 11 highly conserved residues, seven of which are aromatic. Point mutations, insertions and deletions indicated that integrity of the motif is important for binding. However, additional amino acids are required because a minimal peptide containing the motif was disordered in solution and failed to bind to duplex RNAs, whereas a longer protein bound well. Schizosaccharomyces pombe RNase H1 also bound to duplex RNAs, as did proteins in which the S.cerevisiae RNase H1 binding motif was replaced by either the C.fasciculata or by the cauliflower mosaic virus ORF VI sequence. The similarity between the RNase H and the caulimovirus domain suggest a common interaction with duplex RNAs of these two different groups of proteins.
Asunto(s)
Caulimovirus/metabolismo , ARN Bicatenario/metabolismo , Ribonucleasa H/química , Ribonucleasa H/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Caulimovirus/genética , Pollos , Secuencia de Consenso , Secuencia Conservada , Crithidia fasciculata/enzimología , Escherichia coli/enzimología , Datos de Secuencia Molecular , ARN Bicatenario/química , Ribonucleasa H/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , Schizosaccharomyces/enzimología , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The three-dimensional solution structure of the hybrid duplex r(gaggacug):d(CAGTCCTC) has been determined by two-dimensional NMR, distance geometry (DG), restrained molecular dynamics (rMD) and NOE back-calculation methods. This hybrid, consisting of a purine-rich RNA strand and a pyrimidine-rich DNA strand, is related to the polypurine (+)-strand primer formed after (-)-strand DNA synthesis and RNase H degradation of the viral RNA strand and contains the site of a specific cleavage by reverse transcription (RT) RNase H at the end of the HIV-1 polypurine tract. This polypurine primer is an important intermediate in the formation of virally encoded double-stranded DNA prior to HIV-1 retrovirus integration. The correct processing of this primer is vital in the life cycle of the human immunodeficiency virus type (HIV-1) retrovirus. The structure of the r(gaggacug):d(CAGTCCTC) hybrid, as determined in solution by NMR, is intermediate between canonical A-type and B-type double helices, and has mixed structural characteristics. It is quantitatively different from the previously determined solution structures of other RNA-DNA hybrids, particularly in the width and shape of the major groove, which is wider than the major groove of other hybrids and is close to the dimension of the major groove of B-type DNA duplexes. The structure of this hybrid duplex contains a prominent bend in the double helix with a magnitude and direction similar to the bend in Okazaki fragments. The structural features of the present duplex may explain the unique interactions of this sequence with HIV-1 RT during both (-)-strand and (+)-strand DNA synthesis.
Asunto(s)
ADN/química , Modelos Genéticos , Ácidos Nucleicos Heterodúplex/química , Oligonucleótidos/química , ARN/química , Adenosina/genética , Cartilla de ADN , Replicación del ADN , ADN Viral/biosíntesis , Genes gag , Guanosina/genética , VIH-1/genética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Movimiento (Física) , Oligodesoxirribonucleótidos/química , Oligorribonucleótidos/química , ADN Polimerasa Dirigida por ARN/metabolismo , Soluciones , Transcripción GenéticaRESUMEN
The Watson-Crick G x C and A x T base-paired DNA duplex has been the single most important milestone in modem molecular biology. However, it is possible that other types of stable DNA structures besides the double helix might exist, since only about 5% of the human chromosome is transcribed and expressed. Stable, four-stranded G-tetraplex DNA structures occur in the extensive tandem repeated sequences at the telomeres of chromosome. Formation of stable triplexes of the Py x Pu x Py or Pu x Pu x Py type have been implicated at the control regions of certain human genes. We review and discuss the various types of DNA duplex structures containing stable sheared base-pairs and compare their structural characteristics with that of B-DNA. Pu x Pu structural motifs are found in the highly conserved sequences at the replication origins of several single-stranded DNA viruses and in the peri-centromeric regions of human chromosomes, and may be involved in important biological functions, such as viral DNA replication and centromere formation.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Nucleótidos de Purina/química , Secuencia de Bases , Centrómero/química , Replicación del ADN , ADN de Cadena Simple/química , ADN Viral/química , Humanos , Modelos Moleculares , Datos de Secuencia MolecularRESUMEN
The solution structures of the oligodeoxynucleotides d(CCCGTTTCC) and d(TCCCGTTTCCA) have been determined by two-dimensional NMR spectroscopy. These oligomers are part of a DNA box in human centromeric alpha satellite targeted by the centromere protein B (CENP-B). Both CENP-B and its recognition box in alphoid DNA are conserved in mammals, suggesting an important biological role. At acidic pH, d(CCCGTTTCC), d(TCCCGTTTCCA) and the full d(TCCCGTTTCCAACGAAG) CENP-B box strand all fold and dimerize in solution forming a stable bimolecular structure containing two GTTT hairpin loops that interact through a novel T : G : G : T tetrad. The stem region of the dimer is a four-stranded intercalated motif in which the hairpin monomers are parallel and held together by C : C+ hydrogen-bonding and intercalation. The loops are at the same end of the dimer and lie across the narrow grooves of the tetraplex. They are remarkably structured and stabilized by base-base cross-stacking, sugar-base stacking, and parallel G:G and antiparallel G:T pairing. In the d(TCCCGTTTCCA)2 structure, the intercalated motif is continued at the other end of the dimer with unpaired but stacked adenine and thymine bases. The possible biological implications of these structures are discussed.
Asunto(s)
Autoantígenos , Proteínas de Unión al ADN , ADN/química , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Centrómero/química , Proteína B del Centrómero , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Simulación por Computador , Dimerización , Humanos , Espectroscopía de Resonancia Magnética , Modelos Químicos , Modelos Moleculares , Protones , SolucionesRESUMEN
The DNA undecamers GTACAAAGTAC (AAA 11-mer) and GTACGAGGTAC (GAG 11-mer) have been studied in solution by high-resolution NMR spectroscopy. Both duplexes form stable hairpins containing single deoxyadenosine loops and stems containing five base-pairs that are closed at the loop end by sheared AxA and GxC pairs, respectively. These molecules thus contain new AAA and GAG loop turn motifs. All protons, including the chiral H5'/H5" protons of the loop residues, were assigned using NOESY, DQF-COSY and heteronuclear 1H-31P COSY experiments. The backbone torsion angles were constrained using experimental data from NOE crosspeaks, three-bond 1H-1H coupling constants and four-bond 1H-31P coupling constants and four-bond 1H-31P coupling constants. The AAA and GAG 11-mers form similar structures in solution. The detailed structure of the AAA 11-mer was determined by the combined use of NMR, distance geometry and energy minimization methods. This structure exhibits good stacking of the loop adenosine base on the closing 5Ax7A sheared pair, with the 6A base stacking on the 5A base and the 6A deoxyribose stacking with the 7A base. All sugars in the AAA 11-mer hairpin adopt the typical DNA C2'-endo conformation and a sharp backbone turn occurs between residues 6A and 7A. This loop turn is brought about mainly by a change in the backbone phosphate torsion angles from zeta(g-) alpha(g-) to zeta(g+) alphat(g+) at the turn. The gamma torsion angle of residue 7A in the closing sheared pair also changes from gauche+ to trans. In Pu1NPu2 loop turns of the GCA, AAA and GAG types, the chemical shift of the H4' proton of the loop deoxyribose depends on the nature of Pu2; this reflects the stacking of the loop sugar on the Pu2 base and the different ring current effects of A or G in this position.
Asunto(s)
ADN/química , Oligodesoxirribonucleótidos/química , Adenina/química , Composición de Base , Secuencia de Bases , ADN/genética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/genética , Protones , SolucionesRESUMEN
Recently, we established that satellite III (TGGAA)n tandem repeats, which occur at the centromeres of human chromosomes, pair with themselves to form an unusual "self-complementary" antiparallel duplex containing (GGA)2 motifs in which two unpaired guanines from opposite strands intercalate between sheared G.A base pairs. In separate studies, we have also established that the GCA triplet does not form bimolecular (GCA)2 motifs but instead promotes the formation of hairpins containing a GCA-turn motif in which the loop contains a single cytidine closed by a sheared G.A pair. Since TGCAA is the most frequent variant of TGGAA found in satellite III repeats, we reasoned that the potential of this variant to form GCA-turn miniloop fold-back structures might be an important factor in modulating the local structure in natural (TGGAA)n repeats. We report here the NMR-derived solution structure of the heptadecadeoxynucleotide (G)TGGAATGCAATGGAA(C) in which a central TGCAA pentamer is flanked by two TGGAA pentamers. This 17-mer forms a rather unusual and very stable hairpin structure containing eight base pairs in the stem, only four of which are Watson-Crick pairs, and a loop consisting of a single cytidine residue. The stem contains a (GGA)2 motif with intercalative 14G/4G stacking between two sheared G.A base pairs; the loop end of the stem consists of a sheared 8G.10A closing pair with the cytosine base of the 9C loop stacked on 8G. The remarkable stability of this unusual hairpin structure (Tm = 63 degrees C) suggests that it probably plays an important role in modulating the folding of satellite III (TGGAA)n repeats at the centromere.
Asunto(s)
Centrómero/genética , Conformación de Ácido Nucleico , Secuencias Repetitivas de Ácidos Nucleicos , Centrómero/química , Citosina , Guanina , Humanos , Espectroscopía de Resonancia MagnéticaRESUMEN
The three-dimensional solution structure of the hybrid-chimeric duplex r(gcca)d(CTGC).d(GCAGTGGC) has been determined by two-dimensional NMR, restrained molecular dynamics (rMD), and NOE back-calculation methods. This chimera, consisting of a chimeric RNA-DNA strand and its complementary DNA strand, is formed after priming (-)-strand DNA synthesis by tRNA(Lys3) and subsequent (+)-strand DNA synthesis by reverse transcriptase and is an obligatory intermediate in the formation of double-stranded DNA prior to HIV-1 retrovirus integration. The duplex consists of two different types of double helix: a hybrid form (H-form) and a B-form structure connected by a junction. It is chemically similar to several other Okazaki fragments whose structures have been previously determined in our laboratory. However, some structural parameters are not the same and were found to be sequence dependent. In particular, the sugar conformations at the DNA base pair proximal to the hybrid segment vary from O4'-endo to C2'-endo depending on the base composition. The position of the transition from the relatively wide groove of H-form to the narrow groove of B-form is also sequence dependent, occurring either exactly at the RNA-DNA junction or within the purely DNA segment of the chimera-as is the case in the structure of the present HIV-1 (-)-strand primer. This structural change produces a kink at the DNA-DNA step adjacent to the RNA-DNA junction in the HIV-1 (-)-strand primer. The sequence dependence of structures of RNA-DNA chimeric duplexes may be responsible for the variable cleavage pattern of different Okazaki fragments by reverse transcriptase RNase H.
Asunto(s)
ADN Viral/química , ADN/química , VIH-1/genética , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/química , Composición de Base , Secuencia de Bases , ADN/metabolismo , ADN Viral/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , ARN Viral/química , ARN Viral/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Ribonucleasa H/metabolismoRESUMEN
We have determined the solution structure of the synthetic chimeric duplex r(ccca)d(AATGA).d(TCATTTGGG) by two-dimensional NMR, distance geometry, restrained molecular dynamics, and full relaxation matrix simulation of the two-dimensional nuclear Overhauser effect spectra at various mixing times. The chimeric strand of this duplex consists of the last four residues of the tRNA(Pro) primer for (-) strand DNA synthesis of Moloney murine leukemia virus and the first five residues of the (-) strand DNA produced by extending this primer; the complementary DNA strand corresponds to the (+) strand product from this template. The hybrid section of this chimeric duplex assumes a structure similar to that found for pure hybrid duplexes of mixed sequence, while the DNA section assumes a conformation closer to B-form DNA. There is significant distortion of the duplex at the hybrid-DNA junction which is manifested in marked changes in the helical parameters buckle, roll, and tip, changes in glycosidic torsion angles, and changes in the backbone torsion angles delta, epsilon, and zeta. The sugar conformations also undergo large changes, from heteromerous puckers in the hybrid section to a more B-form in the DNA section. Furthermore, the intrastrand phosphate separation in the chimeric strand is more typical of A-form duplexes in the RNA section but more like B-form duplexes in the DNA section. In the DNA section the minor groove width changes gradually from B-form at the periphery and approaches hybrid-like dimensions closer to the junction. The structural discontinuities act synergistically to produce a bend of 18 +/- 3 degrees at the junction. The global structure of this sequence is similar to that previously found in the chemically analogous Okazaki fragment r(gcg)d(TATACCC).d(GGGTATACGC) in solution. Such structure homology suggests a possible link between structure and function with respect to the recognition and cleavage of the junction RNA residues in both retroviral chimeras and Okazaki fragments during reverse transcription and normal DNA replication.
Asunto(s)
ADN Viral/química , ADN/química , Virus de la Leucemia Murina de Moloney/química , Conformación de Ácido Nucleico , ARN Viral/química , Secuencia de Bases , Simulación por Computador , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , ARN de Transferencia de Prolina/química , SolucionesRESUMEN
While tandem repeats of the human centromere DNA pentamer sequence TGRAA form stable "self-complementary" [TGRAATGRAA]2 duplexes (R = G or A) containing the GA-bracketed unpaired purine stack motif, their phase-shifted variants NAATGNAATG (N = A, G, C, T) were found to exist in solution as an equilibrium mixture of a duplex containing the GA-bracketed unpaired stack motif and a hairpin containing a single-residue loop closed by a sheared G x A pair. The stability of the hairpin form relative to duplex form of GNA triplets was found to be GCA>GAA/GTA>>GGA, with the CAATGCAATG sequence mostly in the hairpin form and the GAATGGAATG sequence mostly in the [GAATGGAATG]2 duplex form. The chemical shifts of the H1' and H4' protons of the central N residue in GNA triplets were found to differ markedly in the duplex and hairpin forms and are diagnostic indicators of which conformation the oligonucleotide adopts. Comparison between the structures of the G x A-closed C loop motif and the G x A-bracketed unpaired G-stack [GGA]2 motif reveals remarkably similar stacking by the loop C residue and the intercalated G residue on the adjacent sheared G x A pair. The anomalous upfield chemical shifts of the H1' and H4' protons in [GGA]2 motifs and the H4' proton in GCA loops, and the different sugar conformations in these two motifs, can be explained by interstrand versus intrastrand stacking of the central (G or C) deoxyribose with the adenine base. Based on these studies, a DNA sequence GTGGAATGGAATGGAAC was designed and shown to form a duplex containing three [GGA]2 motifs, while its 9G-->9C analog GTGGAATGCAATGGAAC was found to adopt a stable hairpin containing a (GGA)2 motif in the stem and a G x A-closed single C-loop.
Asunto(s)
Centrómero/química , Conformación de Ácido Nucleico , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Bases , Centrómero/genética , Humanos , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Datos de Secuencia MolecularRESUMEN
At TpA steps in DNA, the adenine base experiences exceptionally large amplitude (20 degrees-50 degrees) and slow (10 ms-1 microsecond) motion [Kennedy et al. (1993) Biochemistry 32, 8022-8035] which has been correlated with transitions between multiple conformational states [Lefevre et al. (1985) FEBS Lett. 190, 37-40]. The base dynamics can be detected in one-dimensional 1H NMR spectra as excess line width of the aromatic proton resonances. The magnitude of the excess line width is temperature dependent and reaches a maximum at some temperature. In order to better understand the origin of the dynamics, we have studied the effect of N6-methylation of the TpA adenine on both the line widths and its local structure. Here, solution-state 500 and 750 MHz 1H NMR data collected on [d(CGAGGTTTAAACCTCG)]2 show that the excess line width of the TpA adenine-H2 is diminished when the TpA adenine is N6-methylated and that the line width no longer experiences a maximum as the temperature is varied. The resonances sharpen upon methylation because the amplitude of base motion is restricted due to steric effects and due to other structural changes at the TpA site. Additionally, both the TpA adenine-H8 and the exchangeable imino resonance of thymine at the TpA step were also found to have excess line width that is diminished upon N6-methylation. In order to elucidate the structural features responsible for TpA base dynamics, solution-state NMR structures of [d(CGAGGTTTAAACCTCG)]2 and its A9 N6-methylated derivative were determined at 750 MHz. Comparison of the structures shows that poor base stacking at the TpA step may contribute to, or be the origin of, its base dynamics.
Asunto(s)
Adenina/metabolismo , ADN/química , Secuencia de Bases , Espectroscopía de Resonancia Magnética , Metilación , Modelos Estructurales , Datos de Secuencia Molecular , Conformación de Ácido NucleicoRESUMEN
The solution structure of the DNA duplex [GTGGAATGGAAC]2 containing a tandem repeat of the human centromere (TGGAA)n unit has been determined by two-dimensional nuclear magnetic resonance (2D-NMR), distance geometry (DG) and molecular dynamics/energy minimization (MD/EM) methods. This remarkably stable "self-complementary" antiparallel duplex contains a tandem repeated motif in which unpaired guanine residues from opposite strands intercalate and costack between sheared G.A pairs. Twelve independent refined structures were determined from the NMR data and found to converge to a single family of closely related structures with pair-wise r.m.s.d. values of 0.55 +/- 0.25 Angstrum. All sugar residues are in the normal C2'-endo conformation except for the unpaired guanosines, which are in the unusual C3'-endo conformation. The guanosine residues of the bracketing G.A pairs have high-antiglycosidic torsion angles and zeta backbone torsion angles close to the trans domain. The structure exhibits many unusual interstrand interactions, including base-sugar stacking, base-phosphate hydrogen bonding and cross-strand base stacking. The [GGA]2 unit contains a stack of four contiguous guanine residues, all of which have their hydrogen-bonding surface (N2H-N1H-O6-N7) exposed to solvent and available for interaction with other bases or ligands. This unexpected property may explain the unique morphology and function of the human centromere in mitosis.
Asunto(s)
Centrómero/química , Centrómero/genética , Cromosomas Humanos , Mitosis , Conformación de Ácido Nucleico , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Bases , Humanos , Espectroscopía de Resonancia Magnética/métodos , Repeticiones de Minisatélite , Modelos Moleculares , Datos de Secuencia Molecular , ProtonesRESUMEN
The widely used antitumor drug cis-diamminedichloroplatinum(II) (cisplatin or cis-DDP) reacts with DNA, cross-linking two purine residues through the N7 atoms, which reside in the major groove in B-form DNA. The solution structure of the short duplex [d(CAT-AGCTATG)]2 cross-linked at the GC:GC site was determined by nuclear magnetic resonance (NMR). The deoxyguanosine-bridging cis-diammineplatinum(II) lies in the minor groove, and the complementary deoxycytidines are extrahelical. The double helix is locally reversed to a left-handed form, and the helix is unwound and bent toward the minor groove. These findings were independently confirmed by results from a phase-sensitive gel electrophoresis bending assay. The NMR structure differs markedly from previously proposed models but accounts for the chemical reactivity, the unwinding, and the bending of cis-DDP interstrand cross-linked DNA and may be important in the formation and repair of these cross-links in chromatin.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , ADN/química , Conformación de Ácido Nucleico , Secuencia de Bases , ADN/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , SolucionesRESUMEN
In certain contexts the DNA triplet GGA, when juxtaposed on opposite strands of a DNA duplex, shows the unusual property of pairing with itself in an antiparallel orientation to form the (GGA)2 motif. In this motif the central guanines do not pair but intercalate and stack between sheared G.A pairs. Similar studies with GCA triplets reveal that they do not form analogous paired (GCA)2 motifs but instead strongly promote formation of a hairpin, the structure of which is now reported here. The GCA hairpin loop consists of a single cytidine residue closed by a sheared G.A pair and this structure is discussed in the context of triplet expansions in triplet-repeat diseases.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Secuencia de Bases , Simulación por Computador , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/etiología , Enfermedades del Sistema Nervioso/congénito , Enfermedades del Sistema Nervioso/etiología , Repeticiones de TrinucleótidosRESUMEN
An improved 2D NOESY simulation program, BIRDER (back-calculation considering incomplete recovery and differential external relaxation), has been developed. BIRDER has the following features: (1) It allows for differential external relaxation rates for protons with different chemical environments, (2) it automatically accounts for incomplete recovery of z magnetization between scans, and (3) it incorporates anisotropic tumbling motional modes for nonspherical molecules. It was found that a lower R factor is obtained if differential external relaxation rates are used in the NOESY spectrum simulation. The method for experimentally determining these external relaxation rates is discussed. In practice, the ability to account for incomplete recovery of z magnetization is very useful in the quantitative analysis of 2D NOESY spectra of spin systems with long longitudinal relaxation times (T1). For example, the T1's of some protons in RNA duplexes are approximately 4-6 s. Thus, a more than 15 s relaxation delay is needed to allow the z magnetization to return to thermal equilibrium between scans, requiring more than a week to obtain a single NOESY spectrum. Unless incomplete recovery of z magnetization is explicitly considered, distance errors may arise when shorter relaxation delays are used. The situation is further complicated in studies of DNA:RNA hybrid or DNA-RNA chimeric duplexes in which the DNA protons relax much faster than the RNA protons. It is shown that BIRDER successfully corrects for the incomplete recovery of z magnetization and makes it possible to save spectrometer time by using shorter relaxation delays without introducing serious errors. The nonsymmetrical nature of NOESY spectra when the spin system is not fully relaxed is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
ADN/análisis , Espectroscopía de Resonancia Magnética/métodos , ARN/análisis , Secuencia de Bases , Simulación por Computador , Matemática , Datos de Secuencia MolecularRESUMEN
Hybrid duplexes and chimeric duplexes containing hybrid segments linked to pure DNA (or pure RNA) segments are involved in transcription and replication, as well as reverse transcription. A complete understanding of the mechanism of these processes requires detailed information on such duplexes and the junctions between duplexes of differing structure. Using two-dimensional NMR, restrained molecular dynamics and mechanics, and back-calculation refinement against the nuclear Overhauser effect spectra at various mixing times, we have determined the solution structure of the chimeric duplex [r(cgcg)d-(TATACGCG)]2 containing a pure DNA segment in the center of a hybrid duplex. The solution structure differs from the previously determined X-ray structure of the analogous duplex [r(gcg)d(TATACGC)]2, which was found to be A-form throughout [Wang, A.H.-J., et al. (1982) Nature 299, 601-604]. The basic features of the solution structure are (a) the RNA residues are all A-form with C3'-endo sugar conformations, (b) the central DNA segment is B-form, (c) the transition from A-form RNA sugar conformations to B-form DNA sugar conformations involves only the dT5 base step, and (d) although the sugar conformations of the DNA residues A6-G12 are closer to B-form, the basic helical properties of the peripheral RNA.DNA hybrid segments are closer to typical A-form than to B-form.
Asunto(s)
Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligonucleótidos/química , Secuencia de Bases , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , SolucionesRESUMEN
An effective correlation time that accounts for position dependence of the combined local angular motions and collective twisting and bending deformations, as well as for anisotropic uniform rotation, is defined in terms of the magnetization-transfer rate and expressed in terms of molecular parameters. Application to measured magnetization-transfer rates from H6 to H5 of cytosine in cases where all other relevant data, including the uniform rotational diffusion coefficients, are known, suggests that the amplitude of local angular motion, as well as that due to collective deformations, is significantly greater for a penultimate base pair than for base pairs near the center of the molecule, and that such amplitudes might be approximately transferable from one molecule to another. Protocols are suggested for using estimated ratios of effective correlation times in the initial calibrations of internuclear distances and in the subsequent structure-refinement process.
Asunto(s)
Citosina/química , ADN/química , Algoritmos , Composición de Base , Calibración , Transferencia de Energía , Guanosina/química , Magnetismo , Modelos Químicos , Estructura Molecular , Rotación , Factores de TiempoRESUMEN
The centromere of human chromosomes contains multiple repeats of the DNA sequence d(TGGAA)n. This sequence has the interesting property of pairing with itself to form stable duplexes. We have determined the solution structure of the unusual DNA duplex 5'-TGGAATGGAA:TGGAATGGAA-3' at atomic resolution. The duplex contains unpaired staggered guanosine residues, which co-stack by intercalation between sheared G.A and A.G base-pairs to form an interesting new structural motif, the GA-bracketed G-stack. The TGGAA repeat unit contains six "steps", four of which are not Watson-Crick base-pairs.
Asunto(s)
Centrómero/química , ADN/química , Conformación de Ácido Nucleico , Secuencias Repetitivas de Ácidos Nucleicos , Adenosina/química , Composición de Base , Secuencia de Bases , Centrómero/genética , Gráficos por Computador , Guanosina/química , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia MolecularRESUMEN
Solid-state 2H NMR spectroscopy has been used to investigate the base dynamics of a RNA oligonucleotide with a defined sequence, [r(CGCGAAUUCGCG)]2, which contains the RNA analogue of the EcoRI binding site. The C8 protons of all purines in the self-complementary dodecamer were exchanged for deuterons. The quadrupole-echo lineshapes and spin-lattice relaxation times as a function of hydration for the sample in the form of the Na salt have previously been reported. In that study the 2H NMR lineshapes and T1 values of [r(CG*CG*A*A*UUCG*CG*)]2 were compared with those of the analogously labeled DNA sequence, [(CG*CG*A*A*TTCG*CG*)]2 (Wang et al., J. Am. Chem. Soc. 114, 6583, 1992). It was concluded that the amplitudes of purine motion for DNA and RNA are similar at all hydration levels; however, the rate difference observed at low-hydration levels may or may not persist at high hydration. Here the internal motions of the purine bases in the RNA oligomer have been thoroughly investigated. Three models were used to simulate the motion: (1) two-site jump, (2) diffusion in a cone, and (3) restricted diffusion on the surface of a cone. The purine motion is best simulated by the restricted-diffusion on a cone model with an amplitude of +/- 9.5 degrees and a rate between 8.0 x 10(6) rad/s at 90% RH and 8.4 x 10(8) rad/s at 0% RH. This small amplitude and fast rate of purine motion for RNA are similar to previous results obtained for DNA purines.