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1.
Nucleic Acids Res ; 48(6): 3366-3378, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32052019

RESUMO

RNAs play major roles in the regulation of gene expression. Hence, designer RNA molecules are increasingly explored as regulatory switches in synthetic biology. Among these, the TetR-binding RNA aptamer was selected by its ability to compete with operator DNA for binding to the bacterial repressor TetR. A fortuitous finding was that induction of TetR by tetracycline abolishes both RNA aptamer and operator DNA binding in TetR. This enabled numerous applications exploiting both the specificity of the RNA aptamer and the efficient gene repressor properties of TetR. Here, we present the crystal structure of the TetR-RNA aptamer complex at 2.7 Å resolution together with a comprehensive characterization of the TetR-RNA aptamer versus TetR-operator DNA interaction using site-directed mutagenesis, size exclusion chromatography, electrophoretic mobility shift assays and isothermal titration calorimetry. The fold of the RNA aptamer bears no resemblance to regular B-DNA, and neither does the thermodynamic characterization of the complex formation reaction. Nevertheless, the functional aptamer-binding epitope of TetR is fully contained within its DNA-binding epitope. In the RNA aptamer complex, TetR adopts the well-characterized DNA-binding-competent conformation of TetR, thus revealing how the synthetic TetR-binding aptamer strikes the chords of the bimodal allosteric behaviour of TetR to function as a synthetic regulator.


Assuntos
Aptâmeros de Nucleotídeos/química , Proteínas de Ligação a DNA/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Conformação Proteica , Aptâmeros de Nucleotídeos/genética , Cristalografia por Raios X , DNA de Forma B/química , DNA de Forma B/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Regulação da Expressão Gênica/genética , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Modelos Moleculares , Ligação Proteica/genética , RNA/química , RNA/genética
2.
BMC Genomics ; 21(1): 60, 2020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31959108

RESUMO

BACKGROUND: Lactoferrampin (LFampin), Lactoferricin (LFcin), and LFchimera are three well-known antimicrobial peptides derived from Lactoferrin and proposed as alternatives for antibiotics. Although the intracellular activity of these peptides has been previously demonstrated, their mode of action is not yet fully understood. Here, we performed a molecular dynamics simulation study to understand the molecular interactions between camel Lactoferrin derived peptides, including CLFampin, CLFcin, and CLFchimera, and DNA as an important intracellular target. RESULTS: Our results indicate that all three peptides bind to DNA, albeit with different propensities, with CLFchimera showing the highest binding affinity. The secondary structures of the peptides, modeled on Lactoferrin, did not undergo significant changes during simulation, supporting their functional relevance. Main residues involved in the peptide-DNA interaction were identified based on binding free energy estimates calculated over 200 ns, which, as expected, confirmed strong electrostatic interactions between DNA phosphate groups and positively charged peptide side chains. Interaction between the different concentrations of CLFchimera and DNA revealed that after binding of four copies of CLFchimera to DNA, hydrogen bonds between the two strands of DNA start to break from one of the termini. CONCLUSIONS: Importantly, our results revealed that there is no DNA-sequence preference for peptide binding, in line with a broad antimicrobial activity. Moreover, the results showed that the strength of the interaction between DNA and CLFchimera is concentration dependent. The insight provided by these results can be used for the rational redesign of natural antimicrobial peptides targeting the bacterial DNA.


Assuntos
DNA de Forma B/química , Lactoferrina/química , Peptídeos/química , Ligação de Hidrogênio , Lactoferrina/genética , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Proteínas Recombinantes de Fusão/química
3.
Nucleic Acids Res ; 48(5): e29, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31956910

RESUMO

We present a new coarse grained method for the simulation of duplex DNA. The algorithm uses a generalized multi-harmonic model that can represent any multi-normal distribution of helical parameters, thus avoiding caveats of current mesoscopic models for DNA simulation and representing a breakthrough in the field. The method has been parameterized from accurate parmbsc1 atomistic molecular dynamics simulations of all unique tetranucleotide sequences of DNA embedded in long duplexes and takes advantage of the correlation between helical states and backbone configurations to derive atomistic representations of DNA. The algorithm, which is implemented in a simple web interface and in a standalone package reproduces with high computational efficiency the structural landscape of long segments of DNA untreatable by atomistic molecular dynamics simulations.


Assuntos
Algoritmos , DNA de Forma B/química , Simulação de Dinâmica Molecular/estatística & dados numéricos , Internet , Repetições de Microssatélites , Método de Monte Carlo , Software , Termodinâmica
4.
Nucleic Acids Res ; 47(21): 11090-11102, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31624840

RESUMO

We present a multi-laboratory effort to describe the structural and dynamical properties of duplex B-DNA under physiological conditions. By processing a large amount of atomistic molecular dynamics simulations, we determine the sequence-dependent structural properties of DNA as expressed in the equilibrium distribution of its stochastic dynamics. Our analysis includes a study of first and second moments of the equilibrium distribution, which can be accurately captured by a harmonic model, but with nonlocal sequence-dependence. We characterize the sequence-dependent choreography of backbone and base movements modulating the non-Gaussian or anharmonic effects manifested in the higher moments of the dynamics of the duplex when sampling the equilibrium distribution. Contrary to prior assumptions, such anharmonic deformations are not rare in DNA and can play a significant role in determining DNA conformation within complexes. Polymorphisms in helical geometries are particularly prevalent for certain tetranucleotide sequence contexts and are always coupled to a complex network of coordinated changes in the backbone. The analysis of our simulations, which contain instances of all tetranucleotide sequences, allow us to extend Calladine-Dickerson rules used for decades to interpret the average geometry of DNA, leading to a set of rules with quantitative predictive power that encompass nonlocal sequence-dependence and anharmonic fluctuations.


Assuntos
DNA de Forma B/química , DNA/química , Simulação de Dinâmica Molecular , Sequência de Bases
5.
Nat Commun ; 10(1): 4818, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645548

RESUMO

Metal-mediated base pairs expand the repertoire of nucleic acid structures and dynamics. Here we report solution structures and dynamics of duplex DNA containing two all-natural C-HgII-T metallo base pairs separated by six canonical base pairs. NMR experiments reveal a 3:1 ratio of well-resolved structures in dynamic equilibrium. The major species contains two (N3)T-HgII-(N3)C base pairs in a predominantly B-form helix. The minor species contains (N3)T-HgII-(N4)C base pairs and greater A-form characteristics. Ten-fold different 1J coupling constants (15N,199Hg) are observed for (N3)C-HgII (114 Hz) versus (N4)C-HgII (1052 Hz) connectivities, reflecting differences in cytosine ionization and metal-bonding strengths. Dynamic interconversion between the two types of C-HgII-T base pairs are coupled to a global conformational exchange between the helices. These observations inspired the design of a repetitive DNA sequence capable of undergoing a global B-to-A-form helical transition upon adding HgII, demonstrating that C-HgII-T has unique switching potential in DNA-based materials and devices.


Assuntos
DNA Forma A/ultraestrutura , DNA de Forma B/ultraestrutura , Mercúrio/química , Pareamento de Bases , Citosina , DNA/química , DNA/ultraestrutura , DNA Forma A/química , DNA de Forma B/química , Metais , Modelos Moleculares , Conformação de Ácido Nucleico , Espectroscopia de Prótons por Ressonância Magnética , Soluções , Timina
6.
Nat Struct Mol Biol ; 26(11): 1013-1022, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31659330

RESUMO

P element transposase catalyzes the mobility of P element DNA transposons within the Drosophila genome. P element transposase exhibits several unique properties, including the requirement for a guanosine triphosphate cofactor and the generation of long staggered DNA breaks during transposition. To gain insights into these features, we determined the atomic structure of the Drosophila P element transposase strand transfer complex using cryo-EM. The structure of this post-transposition nucleoprotein complex reveals that the terminal single-stranded transposon DNA adopts unusual A-form and distorted B-form helical geometries that are stabilized by extensive protein-DNA interactions. Additionally, we infer that the bound guanosine triphosphate cofactor interacts with the terminal base of the transposon DNA, apparently to position the P element DNA for catalysis. Our structure provides the first view of the P element transposase superfamily, offers new insights into P element transposition and implies a transposition pathway fundamentally distinct from other cut-and-paste DNA transposases.


Assuntos
Elementos de DNA Transponíveis , Proteínas de Drosophila/química , Drosophila melanogaster/metabolismo , Guanosina Trifosfato/química , Transposases/química , Animais , Linhagem Celular , Microscopia Crioeletrônica , DNA Forma A/química , DNA de Forma B/química , Drosophila melanogaster/genética , Modelos Moleculares , Conformação Proteica
7.
J Chem Theory Comput ; 15(12): 6984-6991, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31665604

RESUMO

A double proton transfer reaction in a guanine-cytosine (GC) base pair has been proposed as a possible mechanism for rare tautomer (G*C*) formation and thus a source of spontaneous mutations. We analyze this system with free energy calculations based on extensive Quantum Mechanics/Molecular Mechanics simulations to properly consider the influence of the DNA biomolecular environment. We find that, although the G*C* rare tautomer is metastable in the gas phase, it is completely unstable in the conditions found in cells. Thus, our calculations show that a double proton reaction cannot be the source of spontaneous point mutations. We have also analyzed the intrabase H transfer reactions in guanine. Our results show that the DNA environment gives rise to a large free energy difference between the rare and canonical tautomers. These results show the key role of the DNA biological environment for the stability of the genetic code.


Assuntos
Pareamento de Bases , Citosina/química , DNA de Forma B/química , Guanina/química , Prótons , Teoria Quântica
8.
Proc Natl Acad Sci U S A ; 116(35): 17169-17174, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31413203

RESUMO

Hydrophobic base stacking is a major contributor to DNA double-helix stability. We report the discovery of specific unstacking effects in certain semihydrophobic environments. Water-miscible ethylene glycol ethers are found to modify structure, dynamics, and reactivity of DNA by mechanisms possibly related to a biologically relevant hydrophobic catalysis. Spectroscopic data and optical tweezers experiments show that base-stacking energies are reduced while base-pair hydrogen bonds are strengthened. We propose that a modulated chemical potential of water can promote "longitudinal breathing" and the formation of unstacked holes while base unpairing is suppressed. Flow linear dichroism in 20% diglyme indicates a 20 to 30% decrease in persistence length of DNA, supported by an increased flexibility in single-molecule nanochannel experiments in poly(ethylene glycol). A limited (3 to 6%) hyperchromicity but unaffected circular dichroism is consistent with transient unstacking events while maintaining an overall average B-DNA conformation. Further information about unstacking dynamics is obtained from the binding kinetics of large thread-intercalating ruthenium complexes, indicating that the hydrophobic effect provides a 10 to 100 times increased DNA unstacking frequency and an "open hole" population on the order of 10-2 compared to 10-4 in normal aqueous solution. Spontaneous DNA strand exchange catalyzed by poly(ethylene glycol) makes us propose that hydrophobic residues in the L2 loop of recombination enzymes RecA and Rad51 may assist gene recombination via modulation of water activity near the DNA helix by hydrophobic interactions, in the manner described here. We speculate that such hydrophobic interactions may have catalytic roles also in other biological contexts, such as in polymerases.


Assuntos
DNA de Forma B/química , Polietilenoglicóis/química , Rutênio/química , Catálise , Pinças Ópticas
9.
Nano Lett ; 19(9): 6600-6603, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31424224

RESUMO

Decades of crystallographic and NMR studies have produced canonical structural models of short DNA. However, no experimental method so far has been able to test these models in vivo, where DNA is long and constrained by interactions with membranes, proteins, and other molecules. Here, we employ high-resolution frequency-modulation AFM to image single long poly(dA)-poly(dT), poly(dG)-poly(dC), and lambda DNA molecules interacting with an underlying substrate that emulates the effect of biological constraints on molecular structure. We find systematic sequence-dependent variations in groove dimensions, indicating that the structure of DNA subject to realistic interactions may differ profoundly from canonical models. These findings highlight the value of AFM as a unique, single molecule characterization tool.


Assuntos
Bacteriófago lambda/química , DNA de Forma B/química , DNA Viral/química , Modelos Moleculares , Conformação de Ácido Nucleico , Poli dA-dT/química
10.
Int J Biol Macromol ; 137: 337-345, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31247230

RESUMO

The most remarkable conformational transition in nature is the B-to-Z transition of DNA which not only contributes for epigenetic regulation but also is exploited to create several advanced nanomaterials for sensing and nanomechanics. The present communication focuses on the intrinsic factors that control the La3+/Ce3+-induced B-to-Z transition in self-assembled branched DNA (bDNA) nanostructures. The transition is sensitive even to two nucleotide change in the loop length and overhang sequences. Predominantly, bDNA structures having 3 T loop length are more sensitive towards helical switching than the 5 T bearing structures. Particularly, bDNA US-17, US-19 and US-23 having 3 T in the loop are showing B-Z transition in presence of LaCl3. Interestingly, with 'GATC' overhangs both La3+/Ce3+-induced B-to-Z transition was noticed in bDNA structures US-21 and US-22 (having 3 T and 5 T in the loop, respectively). The lanthanide-induced B-Z transition in bDNA is reversed with treatment of EDTA. Isothermal titration calorimetry (ITC) experiments show that the binding mode of lanthanide salts to bDNA followed an entropically and enthalpically favorable process. Further, for the first time ITC data suggests the B-to-Z transition in bDNA is a cooperative shift from exothermic to endothermic.


Assuntos
Sequência de Bases , DNA de Forma B/química , DNA Forma Z/química , Conformação de Ácido Nucleico , Fenômenos Biofísicos , Calorimetria , Dicroísmo Circular , Termodinâmica
11.
Nucleic Acids Res ; 47(11): 5511-5521, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31073604

RESUMO

Inspired by DNA mimic proteins, we have introduced aromatic foldamers bearing phosphonate groups as synthetic mimics of the charge surface of B-DNA and competitive inhibitors of some therapeutically relevant DNA-binding enzymes: the human DNA Topoisomerase 1 (Top1) and the human HIV-1 integrase (HIV-1 IN). We now report on variants of these anionic foldamers bearing carboxylates instead of phosphonates. Several new monomers have been synthesized with protecting groups suitable for solid phase synthesis (SPS). Six hexadecaamides have been prepared using SPS. Proof of their resemblance to B-DNA was brought by the first crystal structure of one of these DNA-mimic foldamers in its polyanionic form. While some of the foldamers were found to be as active as, or even more active than, the original phosphonate oligomers, others had no activity at all or could even stimulate enzyme activity in vitro. Some foldamers were found to have differential inhibitory effects on the two enzymes. These results demonstrate a strong dependence of inhibitory activity on foldamer structure and charge distribution. They open broad avenues for the development of new classes of derivatives that could inhibit the interaction of specific proteins with their DNA target thereby influencing the cellular pathways in which they are involved.


Assuntos
Amidas/química , DNA Topoisomerases Tipo I/química , DNA de Forma B/química , Inibidores de Integrase de HIV/química , Integrase de HIV/química , Biocatálise , Materiais Biomiméticos/química , Cristalografia por Raios X , Inibidores de Integrase de HIV/síntese química , HIV-1/enzimologia , Humanos , Estrutura Molecular , Conformação Proteica , Técnicas de Síntese em Fase Sólida
12.
Phys Rev E ; 99(3-1): 032404, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30999428

RESUMO

Overstretching of B-DNA is currently understood as force-induced melting. Depending on the geometry of the stretching experiment, the force threshold for the overstretching transition is around 65 or 110 pN. Although the mechanisms behind force-induced melting have been correctly described by Rouzina and Bloomfield [Biophys. J. 80, 882 (2001)BIOJAU0006-349510.1016/S0006-3495(01)76067-5], neither force threshold has been exactly calculated by theory. In this work, a detailed analysis of the force-extension curve is presented, based on a description of single-stranded (ss) DNA in terms of the discrete Kratky-Porod model, consistent with (i) the contour length expected from the crystallographically determined monomer distance and (ii) a high value of the elastic stretch modulus arising from covalent bonding. The value estimated for the ss-DNA persistence length, λ=1.0 nm, is at the low end of currently known estimates and reflects the intrinsic stiffness of the partially, or fully stretched state, where electrostatic repulsion effects are expected to be minimal. A detailed analysis of single- and double-stranded DNA free energies provides estimates of the overstretching force thresholds. In the unconstrained geometry, the predicted threshold is 64 pN. In the constrained geometry, after allowing for the entropic penalty of the plectonemic topology of the molten state, the predicted threshold is 111 pN.


Assuntos
DNA de Forma B , DNA de Cadeia Simples , Modelos Químicos , Modelos Moleculares , Algoritmos , Fenômenos Biofísicos , Simulação por Computador , DNA de Forma B/química , DNA de Cadeia Simples/química , Módulo de Elasticidade , Modelos Genéticos , Desnaturação de Ácido Nucleico , Termodinâmica
13.
Phys Rev E ; 99(3-1): 032415, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30999536

RESUMO

We study periodic, quasiperiodic (Thue-Morse, Fibonacci, period doubling, Rudin-Shapiro), fractal (Cantor, generalized Cantor), Kolakoski, and random binary sequences using a tight-binding wire model, where a site is a monomer (e.g., in DNA, a base pair). We use B-DNA as our prototype system. All sequences have purines, guanine (G) or adenine (A), on the same strand, i.e., our prototype binary alphabet is {G,A}. Our aim is to examine the influence of sequence intricacy and magnitude of parameters on energy structure, localization, and charge transport. We study quantities such as autocorrelation function, eigenspectra, density of states, Lyapunov exponents, transmission coefficients, and current-voltage curves. We show that the degree of sequence intricacy and the presence of correlations decisively affect the aforementioned physical properties. Periodic segments have enhanced transport properties. Specifically, in homogeneous sequences transport efficiency is maximum. There are several deterministic aperiodic sequences that can support significant currents, depending on the Fermi level of the leads. Random sequences is the less efficient category.


Assuntos
Modelos Químicos , Polímeros/química , DNA de Forma B/química , Eletricidade , Modelos Genéticos , Modelos Moleculares
14.
Luminescence ; 34(1): 113-124, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30604519

RESUMO

Novel palladium(II) complexes (7a-7e) of substituted quinoline derivatives were synthesized. The complexes were characterized using various techniques such as thermogravimetric analysis (TGA), elemental analysis, conductance measurement, mass, absorption, infra-red (IR), 1 H NMR, 13 C NMR and energy-dispersive X-ray spectroscopy (EDX). Complexes for herring sperm DNA (HS DNA) binding were explored and absorption titration and the binding constant (Kb ) as well as Gibb's free energy were evaluated. Complex 7d exhibited the highest binding constant, therefore the thermodynamic parameters of 7d at different temperatures were evaluated. To support the results of the absorption titration, fluorescence titration, viscosity measurement and molecular docking studies were performed. The fluorescence quenching data as evaluated from Stern-Volmer equation were used to calculate KSV , Kf and the number of binding sites. The results of all these studies were in good agreement with the absorption study. DNA electrophoretic mobility was performed to explore the possible application of metal complexes as artificial metallonucleases. The antibacterial activity of the complexes was accessed against different pathogenic bacteria and cytotoxicity was measured using brine shrimp and S. pombe.


Assuntos
Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , DNA de Forma B/química , Paládio/química , Animais , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Artemia/efeitos dos fármacos , Sítios de Ligação , Complexos de Coordenação/síntese química , DNA de Forma B/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Eletroforese/métodos , Ensaio de Desvio de Mobilidade Eletroforética , Ligantes , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Quinolinas/química , Schizosaccharomyces/efeitos dos fármacos , Espectrometria de Fluorescência , Espectrometria por Raios X , Termodinâmica
15.
Biochem Biophys Res Commun ; 508(4): 1215-1220, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30558789

RESUMO

The crystal structure of BZ-junction reveals that left-handed Z-DNA stabilized by Z-DNA binding domain (Zα) is continuously stacked to right-handed B-DNA with AT bases' extrusion in the junction site. However, this structure might not fully represent the BZ-junction in solution due to the possibility of the junction formation either by crystal packing or Zα interaction. Therefore, we investigated BZ-junction in solution with chemical Z-DNA inducers using CD and 2-aminopurine base-extrusion assay. We confirmed the formation of Z-DNA and BZ-junction with base-extrusion by chemical Z-DNA inducers. However, neither typical Z-DNA nor base-extrusion could be detected with some inducers such as spermine, suggesting that the energy barrier for the formation of the BZ junction might vary depending on the Z-DNA induction conditions.


Assuntos
DNA de Forma B/química , DNA Forma Z/química , Conformação de Ácido Nucleico , 2-Aminopurina/química , Temperatura Baixa , Temperatura Alta , Oligonucleotídeos/química
16.
J Phys Chem B ; 122(51): 12251-12259, 2018 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-30495958

RESUMO

Double-stranded DNA may contain mismatched base pairs beyond the Watson-Crick pairs, guanine-cytosine and adenine-thymine. Such mismatches bear adverse consequences for human health. We utilize molecular dynamics and metadynamics computer simulations to study the structure and dynamics of both matched and mismatched base pairs. We discover significant differences between the matched and mismatched pairs in structure and base flip work profiles. Mismatched pairs shift more in the plane normal to the DNA strand and exhibit more noncanonical structures, including the e-motif. We discuss the potential implications on the mismatch repair enzymes' detection of DNA mismatches.


Assuntos
DNA de Forma B/química , DNA de Forma B/genética , Pareamento Incorreto de Bases , Pareamento de Bases , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Termodinâmica
17.
Nucleic Acids Res ; 46(20): 11099-11114, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30285154

RESUMO

A(syn)-U/T and G(syn)-C+ Hoogsteen (HG) base pairs (bps) are energetically more disfavored relative to Watson-Crick (WC) bps in A-RNA as compared to B-DNA by >1 kcal/mol for reasons that are not fully understood. Here, we used NMR spectroscopy, optical melting experiments, molecular dynamics simulations and modified nucleotides to identify factors that contribute to this destabilization of HG bps in A-RNA. Removing the 2'-hydroxyl at single purine nucleotides in A-RNA duplexes did not stabilize HG bps relative to WC. In contrast, loosening the A-form geometry using a bulge in A-RNA reduced the energy cost of forming HG bps at the flanking sites to B-DNA levels. A structural and thermodynamic analysis of purine-purine HG mismatches reveals that compared to B-DNA, the A-form geometry disfavors syn purines by 1.5-4 kcal/mol due to sugar-backbone rearrangements needed to sterically accommodate the syn base. Based on MD simulations, an additional penalty of 3-4 kcal/mol applies for purine-pyrimidine HG bps due to the higher energetic cost associated with moving the bases to form hydrogen bonds in A-RNA versus B-DNA. These results provide insights into a fundamental difference between A-RNA and B-DNA duplexes with important implications for how they respond to damage and post-transcriptional modifications.


Assuntos
Pareamento de Bases/fisiologia , DNA de Forma B/química , Conformação de Ácido Nucleico , Purinas/química , RNA/química , DNA/química , Metabolismo Energético , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Pirimidinas/química , Termodinâmica
18.
Nucleic Acids Res ; 46(19): 10504-10513, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30184200

RESUMO

BZ junctions, which connect B-DNA to Z-DNA, are necessary for local transformation of B-DNA to Z-DNA in the genome. However, the limited information on the junction-forming sequences and junction structures has led to a lack of understanding of the structural diversity and sequence preferences of BZ junctions. We determined three crystal structures of BZ junctions with diverse sequences followed by spectroscopic validation of DNA conformation. The structural features of the BZ junctions were well conserved regardless of sequences via the continuous base stacking through B-to-Z DNA with A-T base extrusion. However, the sequence-dependent structural heterogeneity of the junctions was also observed in base step parameters that are correlated with steric constraints imposed during Z-DNA formation. Further, circular dichroism and fluorescence-based analysis of BZ junctions revealed that a base extrusion was only found at the A-T base pair present next to a stable dinucleotide Z-DNA unit. Our findings suggest that Z-DNA formation in the genome is influenced by the sequence preference for BZ junctions.


Assuntos
Adenosina Desaminase/química , DNA de Forma B/química , DNA Forma Z/química , DNA/química , Conformação de Ácido Nucleico , Domínios Proteicos , Proteínas de Ligação a RNA/química , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Pareamento de Bases , Sequência de Bases , Dicroísmo Circular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , DNA de Forma B/genética , DNA de Forma B/metabolismo , DNA Forma Z/genética , DNA Forma Z/metabolismo , Humanos , Modelos Moleculares , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
19.
Biophys J ; 115(7): 1180-1189, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30172386

RESUMO

With almost no consensus promoter sequence in prokaryotes, recruitment of RNA polymerase (RNAP) to precise transcriptional start sites (TSSs) has remained an unsolved puzzle. Uncovering the underlying mechanism is critical for understanding the principle of gene regulation. We attempted to search the hidden code in ∼16,500 promoters of 12 prokaryotes representing two kingdoms in their structure and energetics. Twenty-eight fundamental parameters of DNA structure including backbone angles, basepair axis, and interbasepair and intrabasepair parameters were used, and information was extracted from x-ray crystallography data. Three parameters (solvation energy, hydrogen-bond energy, and stacking energy) were selected for creating energetics profiles using in-house programs. DNA of promoter regions was found to be inherently designed to undergo a change in every parameter undertaken for the study, in all prokaryotes. The change starts from some distance upstream of TSSs and continues past some distance from TSS, hence giving a signature state to promoter regions. These signature states might be the universal hidden codes recognized by RNAP. This observation was reiterated when randomly selected promoter sequences (with little sequence conservation) were subjected to structure generation; all developed into very similar three-dimensional structures quite distinct from those of conventional B-DNA and coding sequences. Fine structural details at important motifs (viz. -11, -35, and -75 positions relative to TSS) of promoters reveal novel to our knowledge and pointed insights for RNAP interaction at these locations; it could be correlated with how some particular structural changes at the -11 region may allow insertion of RNAP amino acids in interbasepair space as well as facilitate the flipping out of bases from the DNA duplex.


Assuntos
Modelos Genéticos , Células Procarióticas/metabolismo , Regiões Promotoras Genéticas/genética , DNA de Forma B/química , DNA de Forma B/genética , DNA de Forma B/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Termodinâmica , Sítio de Iniciação de Transcrição
20.
J Phys Chem B ; 122(33): 7978-7989, 2018 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-30070843

RESUMO

The structures of single-stranded DNA oligonucleotides from dimeric to hexameric sequences have been thoroughly investigated. Computations performed at the density functional level of theory including dispersion forces and solvation show that single-stranded helices adopt conformations very close to crystallographic B-DNA, with rise coordinates amounting up to 3.3 Å. Previous results, suggesting that single strands should be shorter than double helices, largely originated from the incompleteness of the adopted basis set. Although sensible deviations with respect to standard B-DNA are predicted, computations indicate that sequences rich in stacked adenines are the most ordered ones, favoring the B-DNA pattern and inducing regular arrangements also on flanking nucleobases. Several structural properties of double helices rich in adenine are indeed already reflected by the corresponding single strands.


Assuntos
DNA de Cadeia Simples/química , Oligodesoxirribonucleotídeos/química , Adenina/química , DNA de Forma B/química , Teoria da Densidade Funcional , Guanina/química , Modelos Químicos , Conformação de Ácido Nucleico , Termodinâmica
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