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1.
PLoS Genet ; 15(10): e1008441, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31584934

RESUMO

Replication stress poses a serious threat to genome stability. Recombination-Dependent-Replication (RDR) promotes DNA synthesis resumption from arrested forks. Despite the identification of chromatin restoration pathways after DNA repair, crosstalk coupling RDR and chromatin assembly is largely unexplored. The fission yeast Chromatin Assembly Factor-1, CAF-1, is known to promote RDR. Here, we addressed the contribution of histone deposition to RDR. We expressed a mutated histone, H3-H113D, to genetically alter replication-dependent chromatin assembly by destabilizing (H3-H4)2 tetramer. We established that DNA synthesis-dependent histone deposition, by CAF-1 and Asf1, promotes RDR by preventing Rqh1-mediated disassembly of joint-molecules. The recombination factor Rad52 promotes CAF-1 binding to sites of recombination-dependent DNA synthesis, indicating that histone deposition occurs downstream Rad52. Histone deposition and Rqh1 activity act synergistically to promote cell resistance to camptothecin, a topoisomerase I inhibitor that induces replication stress. Moreover, histone deposition favors non conservative recombination events occurring spontaneously in the absence of Rqh1, indicating that the stabilization of joint-molecules by histone deposition also occurs independently of Rqh1 activity. These results indicate that histone deposition plays an active role in promoting RDR, a benefit counterbalanced by stabilizing at-risk joint-molecules for genome stability.


Assuntos
Montagem e Desmontagem da Cromatina , Replicação do DNA , Instabilidade Genômica , Histonas/metabolismo , Recombinação Genética , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/metabolismo , Histonas/genética , Chaperonas Moleculares/metabolismo , Mutação , Multimerização Proteica/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Ribonucleases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
2.
J Struct Biol ; 211(1): 107511, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32311461

RESUMO

Most of eukaryotic cellular DNA is packed in nucleosome core particles (NCPs), in which the DNA (DNANCP) is wrapped around histones. The influence of this organization on the intrinsic local dynamics of DNA is largely unknown, in particular because capturing such information from experiments remains notoriously challenging. Given the importance of dynamical properties in DNA functions, we addressed this issue using CHARMM36 MD simulations of a nucleosome containing the NCP positioning 601 sequence and four related free dodecamers. Comparison between DNANCP and free DNA reveals a limited impact of the dense DNA-histone interface on correlated motions of dinucleotide constituents and on fluctuations of inter base pair parameters. A characteristic feature intimately associated with the DNANCP super-helical path is a set of structural periodicities that includes a marked alternation of regions enriched in backbone BI and BII conformers. This observation led to uncover a convincing correspondence between the sequence effect on BI/BII propensities in both DNANCP and free DNA, strengthening the idea that the histone preference for particular DNA sequences relies on those intrinsic structural properties. These results offer for the first time a detailed view of the DNA dynamical behavior within NCP. They show in particular that the DNANCP dynamics is substantial enough to preserve the ability to structurally adjust to external proteins, for instance remodelers. Also, fresh structural arguments highlight the relevance of relationships between DNA sequence and structural properties for NCP formation. Overall, our work offers a more rational framework to approach the functional, biological roles of NCP.


Assuntos
DNA/ultraestrutura , Histonas/ultraestrutura , Conformação de Ácido Nucleico , Nucleossomos/ultraestrutura , Sequência de Bases/genética , Cristalografia por Raios X , DNA/genética , Histonas/genética , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Modelos Moleculares , Nucleossomos/genética
3.
Nucleic Acids Res ; 44(7): 3432-47, 2016 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-26883628

RESUMO

Experimental characterization of the structural couplings in free B-DNA in solution has been elusive, because of subtle effects that are challenging to tackle. Here, the exploitation of the NMR measurements collected on four dodecamers containing a substantial set of dinucleotide sequences provides new, consistent correlations revealing the DNA intrinsic mechanics. The difference between two successive residual dipolar couplings (ΔRDCs) involving C6/8-H6/8, C3'-H3' and C4'-H4' vectors are correlated to the(31)P chemical shifts (δP), which reflect the populations of the BI and BII backbone states. The δPs are also correlated to the internucleotide distances (Dinter) involving H6/8, H2' and H2″ protons. Calculations of NMR quantities on high resolution X-ray structures and controlled models of DNA enable to interpret these couplings: the studied ΔRDCs depend mostly on roll, while Dinterare mainly sensitive to twist or slide. Overall, these relations demonstrate how δP measurements inform on key inter base parameters, in addition to probe the BI↔BII backbone equilibrium, and shed new light into coordinated motions of phosphate groups and bases in free B-DNA in solution. Inspection of the 5' and 3' ends of the dodecamers also supplies new information on the fraying events, otherwise neglected.


Assuntos
DNA de Forma B/química , Desoxirribonucleotídeos/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico
4.
PLoS Comput Biol ; 11(12): e1004631, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26657165

RESUMO

The accurate prediction of the structure and dynamics of DNA remains a major challenge in computational biology due to the dearth of precise experimental information on DNA free in solution and limitations in the DNA force-fields underpinning the simulations. A new generation of force-fields has been developed to better represent the sequence-dependent B-DNA intrinsic mechanics, in particular with respect to the BI ↔ BII backbone equilibrium, which is essential to understand the B-DNA properties. Here, the performance of MD simulations with the newly updated force-fields Parmbsc0εζOLI and CHARMM36 was tested against a large ensemble of recent NMR data collected on four DNA dodecamers involved in nucleosome positioning. We find impressive progress towards a coherent, realistic representation of B-DNA in solution, despite residual shortcomings. This improved representation allows new and deeper interpretation of the experimental observables, including regarding the behavior of facing phosphate groups in complementary dinucleotides, and their modulation by the sequence. It also provides the opportunity to extensively revisit and refine the coupling between backbone states and inter base pair parameters, which emerges as a common theme across all the complementary dinucleotides. In sum, the global agreement between simulations and experiment reveals new aspects of intrinsic DNA mechanics, a key component of DNA-protein recognition.


Assuntos
Modelos Químicos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Análise de Sequência de Proteína/métodos , Sequência de Bases , Dados de Sequência Molecular , Relação Estrutura-Atividade
5.
Biochemistry ; 53(35): 5601-12, 2014 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-25102280

RESUMO

We investigated how the intrinsic sequence-dependent properties probed via the phosphate linkages (BI ↔ BII equilibrium) influence the preferred shape of free DNA, and how this affects the nucleosome formation. First, this exploits NMR solution studies of four B-DNA dodecamers that together cover 39 base pairs of the 5' half of the sequence 601, of special interest for nucleosome formation. The results validate our previous prediction of a systematic, general sequence effect on the intrinsic backbone BII propensities. NMR provides new evidence that the backbone behavior is intimately coupled to the minor groove width. Second, application of the backbone behavior predictions to the full sequence 601 and other relevant sequences demonstrates that alternation of intrinsic low and high BII propensities, coupled to intrinsic narrow and wide minor grooves, largely coincides with the sinusoidal variations of the DNA minor groove width observed in crystallographic structures of the nucleosome. This correspondence is much poorer with low affinity sequences. Overall, the results indicate that nucleosome formation involves an indirect readout process implicating pre-existing DNA minor groove conformations. It also illustrates how the prediction of the intrinsic structural DNA behavior offers a powerful framework to gain explanatory insight on how proteins read DNA.


Assuntos
DNA de Forma B/química , Nucleossomos/química , Sequência de Bases , DNA de Forma B/genética , Humanos , Substâncias Macromoleculares/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética
6.
Nucleic Acids Res ; 38(3): e18, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19942687

RESUMO

For B-DNA, the strong linear correlation observed by nuclear magnetic resonance (NMR) between the (31)P chemical shifts (deltaP) and three recurrent internucleotide distances demonstrates the tight coupling between phosphate motions and helicoidal parameters. It allows to translate deltaP into distance restraints directly exploitable in structural refinement. It even provides a new method for refining DNA oligomers with restraints exclusively inferred from deltaP. Combined with molecular dynamics in explicit solvent, these restraints lead to a structural and dynamical view of the DNA as detailed as that obtained with conventional and more extensive restraints. Tests with the Jun-Fos oligomer show that this deltaP-based strategy can provide a simple and straightforward method to capture DNA properties in solution, from routine NMR experiments on unlabeled samples.


Assuntos
DNA/química , Ressonância Magnética Nuclear Biomolecular , Fosfatos/química , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-jun/química
7.
Nucleic Acids Res ; 38(3): 1034-47, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19920127

RESUMO

B-DNA flexibility, crucial for DNA-protein recognition, is sequence dependent. Free DNA in solution would in principle be the best reference state to uncover the relation between base sequences and their intrinsic flexibility; however, this has long been hampered by a lack of suitable experimental data. We investigated this relationship by compiling and analyzing a large dataset of NMR (31)P chemical shifts in solution. These measurements reflect the BI <--> BII equilibrium in DNA, intimately correlated to helicoidal descriptors of the curvature, winding and groove dimensions. Comparing the ten complementary DNA dinucleotide steps indicates that some steps are much more flexible than others. This malleability is primarily controlled at the dinucleotide level, modulated by the tetranucleotide environment. Our analyses provide an experimental scale called TRX that quantifies the intrinsic flexibility of the ten dinucleotide steps in terms of Twist, Roll, and X-disp (base pair displacement). Applying the TRX scale to DNA sequences optimized for nucleosome formation reveals a 10 base-pair periodic alternation of stiff and flexible regions. Thus, DNA flexibility captured by the TRX scale is relevant to nucleosome formation, suggesting that this scale may be of general interest to better understand protein-DNA recognition.


Assuntos
DNA/química , Sequência de Bases , Proteínas de Ligação a DNA/química , Histonas/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Nucleotídeos/química , Fosfatos/química
8.
PLoS One ; 17(7): e0267382, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35830437

RESUMO

Structural fluctuations of nucleosomes modulate the access to internal DNA in eukaryotic cells; clearly characterisation of this fundamental process is crucial to understanding gene regulation. Here we apply PhAST (Photochemical Analysis of Structural Transitions) to monitor at a base pair level, structural alterations induced all along the DNA upon histone binding or release. By offering the first reliable, detailed comparison of nucleosome assembly and disassembly in vitro, we reveal similarities and differences between the two processes. We identify multiple, sequential intermediate states characterised by specific PhAST signals whose localisation and amplitude reflect asymmetries of DNA/histone interactions with respect to the nucleosome pseudo dyad. These asymmetries involve not only the DNA extremities but also regions close to the pseudo dyad. Localisations of asymmetries develop in a consistent manner during both assembly and disassembly processes; they primarily reflect the DNA sequence effect on the efficiency of DNA-histone binding. More unexpectedly, the amplitude component of PhAST signals not only evolves as a function of intermediate states but does so differently between assembly and disassembly pathways. Our observation of differences between assembly and disassembly opens up new avenues to define the role of the DNA sequence in processes underlying the regulation of gene expression. Overall, we provide new insights into how the intrinsic properties of DNA are integrated into a holistic mechanism that controls chromatin structure.


Assuntos
Histonas , Nucleossomos , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Histonas/metabolismo , Ligação Proteica
9.
PLoS Comput Biol ; 6(11): e1001000, 2010 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-21124947

RESUMO

DNase I requires Ca²+ and Mg²+ for hydrolyzing double-stranded DNA. However, the number and the location of DNase I ion-binding sites remain unclear, as well as the role of these counter-ions. Using molecular dynamics simulations, we show that bovine pancreatic (bp) DNase I contains four ion-binding pockets. Two of them strongly bind Ca²+ while the other two sites coordinate Mg²+. These theoretical results are strongly supported by revisiting crystallographic structures that contain bpDNase I. One Ca²+ stabilizes the functional DNase I structure. The presence of Mg²+ in close vicinity to the catalytic pocket of bpDNase I reinforces the idea of a cation-assisted hydrolytic mechanism. Importantly, Poisson-Boltzmann-type electrostatic potential calculations demonstrate that the divalent cations collectively control the electrostatic fit between bpDNase I and DNA. These results improve our understanding of the essential role of cations in the biological function of bpDNase I. The high degree of conservation of the amino acids involved in the identified cation-binding sites across DNase I and DNase I-like proteins from various species suggests that our findings generally apply to all DNase I-DNA interactions.


Assuntos
Cálcio/química , Cátions/química , DNA/química , Desoxirribonuclease I/química , Magnésio/química , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/metabolismo , Cátions/metabolismo , Bovinos , Cristalografia por Raios X , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Hidrólise , Magnésio/metabolismo , Anotação de Sequência Molecular , Dados de Sequência Molecular , Distribuição de Poisson , Reprodutibilidade dos Testes , Alinhamento de Sequência
10.
Nucleic Acids Res ; 37(13): 4341-52, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19465391

RESUMO

The Stromelysin-1 gene promoter contains a palindrome of two Ets-binding sites (EBS) that bind the p51 and p42 isoforms of the human Ets-1-transcription factor. A previous study established that full gene transactivation is associated with a ternary complex consisting of two p51 bound to the two EBS on the promoter. p42, only able to bind one of the two EBS, induces only very weak activity. Here, we investigate the mechanism by which the Stromelysin-1 promoter discriminates between p51 and p42. The differential stoichiometry of the two Ets-1 isoforms arises from the Stromelysin-1 EBS palindrome. The ternary complex requires the presence of two inhibitory domains flanking the DNA-binding domain and the ability to form an intramolecular autoinhibition module. Most importantly, the p51-ternary and the p42-binary complexes induce DNA curvatures with opposite orientations. These results establish that differential DNA bending, via p51 and p42 differential binding, is correlated with the Stromelysin-1 promoter activation process.


Assuntos
DNA/química , Metaloproteinase 3 da Matriz/genética , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-1/metabolismo , Ativação Transcricional , Sítios de Ligação , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteína Proto-Oncogênica c-ets-1/química
11.
J Gene Med ; 11(5): 401-11, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19326361

RESUMO

BACKGROUND: The nuclear factor kappa B (NF kappaB) transcription factor, which shuttles between the cytoplasm and the nucleus under specific conditions, is a suitable intracellular target to increase the nuclear import of plasmid DNA. We report the design of an optimized and extended NF kappaB DNA binding sequence that promotes an efficient plasmid nuclear import. METHODS: On the basis of structural studies, the 5'-CTGGGGACTTTCCAGCTGGGGACTTTCCAGCTGGGGACTTTCCAGG-3' segment (termed 3NF) comprising three 10-bp kappaB sites (GGGACTTTCC) separated by a 5-bp optimized spacer (AGCTG) was selected for its capacity to ensure the best structural fit with NF kappaB and to fix simultaneously three proteins. Plasmids encoding luciferase and bearing this sequence (3NF-plasmids) were constructed and their nuclear import and gene expression efficiencies compared with that of plasmids containing classical kappaB motifs. RESULTS: A high luciferase expression was associated with plasmids containing one (p3NF-luc) or two (p3NF-luc-3NF) 3NF sequences. In situ hybridization experiments and quantitative measurement of the number of plasmid copies demonstrated that the nuclear delivery of 3NF-plasmids was more efficient than that of 3NF-free plasmids. Cross-linked immunoprecipitation showed that 3NF-plasmids were recognized by NF kappaB inside cells upon transfection. The nuclear delivery was inhibited with BAY 11-7085, an inhibitor of NF kappaB activation. Finally, p3NF-luc-3NF, the most efficient construct for in vitro transfection, had a long-lived luciferase expression in vivo. CONCLUSIONS: The results obtained in the present study demonstrate the NF kappaB-mediated nuclear delivery of 3NF-plasmids. Due to its high affinity for fixing several NF kappaB, the 3NF sequence is a very promising helper for a nonviral gene delivery system.


Assuntos
Núcleo Celular/genética , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Sequência de Bases , Sítios de Ligação , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Camundongos , Microscopia Confocal , Ligação Proteica/efeitos dos fármacos , Transfecção , Fator de Necrose Tumoral alfa/farmacologia
12.
J Mol Biol ; 431(10): 1966-1980, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30876916

RESUMO

A comprehensive view of all the structural aspects related to NCp7 is essential to understand how this protein, crucial in many steps of the HIV-1 cycle, binds and anneals nucleic acids (NAs), mainly thanks to two zinc fingers, ZF1 and ZF2. Here, we inspected the structural properties of the available experimental models of NCp7 bound to either DNA or RNA molecules, or free of ligand. Our analyses included the characterization of the relative positioning of ZF1 and ZF2, accessibility measurements and the exhaustive, quantitative mapping of the contacts between amino acids and nucleotides by a recent tessellation method, VLDM. This approach unveiled the intimate connection between NA binding process and the conformations explored by the free protein. It also provided new insights into the functional specializations of ZF1 and ZF2. The larger accessibility of ZF2 in free NCp7 and the consistency of the ZF2/NA interface in different models and conditions give ZF2 the lead of the binding process. ZF1 contributes to stabilize the complexes through various organizations of the ZF1/NA interface. This work outcome is a global binding scheme of NCp7 to DNA and RNA, and an example of how protein-NA complexes are stabilized.


Assuntos
HIV-1/metabolismo , Ácidos Nucleicos/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Sítios de Ligação , Infecções por HIV/virologia , HIV-1/química , Humanos , Modelos Moleculares , Ácidos Nucleicos/química , Ligação Proteica , Conformação Proteica , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química
13.
J Mol Biol ; 368(5): 1403-11, 2007 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-17395202

RESUMO

The influence of monovalent cations on DNA conformation and readout is an open question. This NMR study of DNA with either Na(+) or K(+) at physiological concentrations shows that the nature of the cation affects the (31)P chemical shifts (deltaP) and the sequential distances H2'(i)-H6/8(i+1), H2"(i)-H6/8(i+1), and H6/8(i)-H6/8(i+1). The deltaP and distance variations ascertain that the nature of the cation affects the DNA overall structure, i.e. both the conformational equilibria between the backbone BI (epsilon-zeta <0 degrees ) and BII (epsilon-zeta >0 degrees ) states and the helical parameters, via their strong mechanical coupling. These results reveal that Na(+) and K(+) interactions with DNA are different and sequence-dependent. These ions modulate the overall intrinsic properties of DNA, and possibly its packaging and readout.


Assuntos
DNA/química , Conformação de Ácido Nucleico , Potássio/química , Sódio/química , Soluções Tampão , Cristalografia por Raios X , Dados de Sequência Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Oligonucleotídeos/química , Oligonucleotídeos/genética , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/química , Proteínas Proto-Oncogênicas c-jun/genética
14.
Nucleic Acids Res ; 34(20): 5740-51, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17041234

RESUMO

A preferential target of antisense oligonucleotides directed against human PGY/MDR1 mRNA is a hairpin containing a stem with a G*U wobble pair, capped by the purine-rich 5'r(GGGAUG)3' hexaloop. This hairpin is studied by multidimensional NMR and restrained molecular dynamics, with special emphasis on the conformation of south sugars and non-standard phosphate linkages evidenced in both the stem and the loop. The hairpin is found to be highly structured. The G*U wobble pair, a strong counterion binding site, displays structural particularities that are characteristic of this type of mismatch. The upper part of the stem undergoes distortions that optimize its interactions with the beginning of the loop. The loop adopts a new fold in which the single-stranded GGGA purine tract is structured in A-like conformation stacked in continuity of the stem and displays an extensive hydrogen bonding surface for recognition. The remarkable hairpin stability results from classical inter- and intra-strand interactions reinforced by numerous hydrogen bonds involving unusual backbone conformations and ribose 2'-hydroxyl groups. Overall, this work emphasizes numerous features that account for the well-ordered structure of the whole hairpin and highlights the loop properties that facilitate interaction with antisense oligonucleotides.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Modelos Moleculares , Oligonucleotídeos Antissenso/química , RNA Mensageiro/química , Pareamento de Bases , Carboidratos/química , Humanos , Ligação de Hidrogênio , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Fosfatos/química , Purinas/análise , Soluções
15.
J Chem Theory Comput ; 14(2): 1045-1058, 2018 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-29262675

RESUMO

The nucleosome is the fundamental unit of eukaryotic genome packaging in the chromatin. In this complex, the DNA wraps around eight histone proteins to form a superhelical double helix. The resulting bending, stronger than anything observed in free DNA, raises the question of how such a distortion is stabilized by the proteic and solvent environments. In this work, the DNA-histone interface in solution was exhaustively analyzed from nucleosome structures generated by molecular dynamics. An original Voronoi tessellation technique, measuring the topology of interacting elements without any empirical or subjective adjustment, was used to characterize the interface in terms of contact area and occurrence. Our results revealed an interface more robust than previously known, combining extensive, long-lived nonelectrostatic and electrostatic interactions between DNA and both structured and unstructured histone regions. Cation accumulation makes the proximity of juxtaposed DNA gyres in the superhelix possible by shielding the strong electrostatic repulsion of the charged phosphate groups. Overall, this study provides new insights on the nucleosome cohesion, explaining how DNA distortions can be maintained in a nucleoprotein complex.


Assuntos
DNA/química , Histonas/química , Simulação de Dinâmica Molecular , Nucleossomos/química , Soluções , Eletricidade Estática
16.
Nucleic Acids Res ; 30(24): 5398-406, 2002 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-12490708

RESUMO

In the crystal structures of protein complexes with B-DNA, alpha and gamma DNA backbone torsion angles often exhibit non-canonical values. It is not known if these alternative backbone conformations are easily accessible in solution and can contribute to the specific recognition of DNA by proteins. We have analysed the coupled transition of the alpha and gamma torsion angles within the central GpC step of a B-DNA dodecamer by computer simulations. Five stable or metastable non-canonical alpha/gamma sub-states are found. The most favourable pathway from the canonical alpha/gamma structure to any unusual form involves a counter-rotation of alpha and gamma, via the trans conformation. However, the corresponding free energy indicates that spontaneous flipping of the torsions is improbable in free B-DNA. This is supported by an analysis of the available high resolution crystallographic structures showing that unusual alpha/gamma states are only encountered in B-DNA complexed to proteins. An analysis of the structural consequences of alpha/gamma transitions shows that the non-canonical backbone geometry influences essentially the roll and twist values and reduces the equilibrium dispersion of structural parameters. Our results support the hypothesis that unusual alpha/gamma backbones arise during protein-DNA complexation, assisting the fine structural adjustments between the two partners and playing a role in the overall complexation free energy.


Assuntos
DNA/química , Conformação de Ácido Nucleico , Cristalografia por Raios X , Termodinâmica
17.
Sci Rep ; 6: 27337, 2016 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-27263658

RESUMO

We describe a biophysical approach that enables changes in the structure of DNA to be followed during nucleosome formation in in vitro reconstitution with either the canonical "Widom" sequence or a judiciously mutated sequence. The rapid non-perturbing photochemical analysis presented here provides 'snapshots' of the DNA configuration at any given moment in time during nucleosome formation under a very broad range of reaction conditions. Changes in DNA photochemical reactivity upon protein binding are interpreted as being mainly induced by alterations in individual base pair roll angles. The results strengthen the importance of the role of an initial (H3/H4)2 histone tetramer-DNA interaction and highlight the modulation of this early event by the DNA sequence. (H3/H4)2 binding precedes and dictates subsequent H2A/H2B-DNA interactions, which are less affected by the DNA sequence, leading to the final octameric nucleosome. Overall, our results provide a novel, exciting way to investigate those biophysical properties of DNA that constitute a crucial component in nucleosome formation and stabilization.


Assuntos
Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Fenômenos Biofísicos , Fenômenos Químicos , DNA/metabolismo , Histonas/metabolismo , Humanos , Ligação Proteica
18.
J Biomol Struct Dyn ; 21(4): 489-94, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14692793

RESUMO

Molecular dynamics (MD) simulations of four water-solvated DNA duplexes were used to generate a database of approximately 27000 dinucleotide conformations. Analyzing this database, we investigated the relationship between so-called BI-BII transitions and short-range interproton distances. Four H-H distances were found particularly sensitive to BI-BII transitions: internucleotide H1'(n)-H68(n+1), H2'(n)-H68(n+1), and H2"(n)-H68(n+1), and intranucleotide H2"(n)-H68(n). Determination of these distances using classical NOESY spectroscopy can thus provide valuable indications on the existence of BII substates, complementing the existing method based on (31)P chemical shifts and (31)P-(1)H spin-spin coupling constants.


Assuntos
DNA/química , Modelos Moleculares , Conformação de Ácido Nucleico , Prótons , Simulação por Computador , Cristalografia por Raios X , DNA/classificação
19.
Methods Mol Biol ; 924: 445-68, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23034759

RESUMO

The structure and dynamics of the B-DNA double helix involves subtle sequence-dependent effects which are decisive for its function, but difficult to characterize. These structural and dynamic effects can be addressed by simulations of DNA sequences in explicit solvent. Here, we present and discuss the state-of-art of B-DNA molecular dynamics simulations with the major force fields in use today. We explain why a critical analysis of the MD trajectories is required to assess their reliability, and estimate the value and limitations of these models. Overall, simulations of DNA bear great promise towards deciphering the structural and physical subtleties of this biopolymer, where much remains to be understood.


Assuntos
DNA de Forma B/química , Simulação de Dinâmica Molecular , DNA de Forma B/metabolismo , Glicosídeos/química , Internet , Conformação de Ácido Nucleico , Fosfatos/química , Proteínas/metabolismo , Reprodutibilidade dos Testes , Software , Eletricidade Estática , Água/química
20.
PLoS One ; 7(7): e41704, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22844516

RESUMO

This study investigates the effect of Mg(2+) bound to the DNA major groove on DNA structure and dynamics. The analysis of a comprehensive dataset of B-DNA crystallographic structures shows that divalent cations are preferentially located in the DNA major groove where they interact with successive bases of (A/G)pG and the phosphate group of 5'-CpA or TpG. Based on this knowledge, molecular dynamics simulations were carried out on a DNA oligomer without or with Mg(2+) close to an ApG step. These simulations showed that the hydrated Mg(2+) forms a stable intra-strand cross-link between the two purines in solution. ApG generates an electrostatic potential in the major groove that is particularly attractive for cations; its intrinsic conformation is well-adapted to the formation of water-mediated hydrogen bonds with Mg(2+). The binding of Mg(2+) modulates the behavior of the 5'-neighboring step by increasing the BII (ε-ζ>0°) population of its phosphate group. Additional electrostatic interactions between the 5'-phosphate group and Mg(2+) strengthen both the DNA-cation binding and the BII character of the 5'-step. Cation binding in the major groove may therefore locally influence the DNA conformational landscape, suggesting a possible avenue for better understanding how strong DNA distortions can be stabilized in protein-DNA complexes.


Assuntos
DNA de Forma B/química , DNA de Forma B/metabolismo , Magnésio/farmacologia , Conformação de Ácido Nucleico/efeitos dos fármacos , Cristalografia por Raios X , Magnésio/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Eletricidade Estática
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