Publications by authors named "Brigitte Hartmann"

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.

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Most of eukaryotic cellular DNA is packed in nucleosome core particles (NCPs), in which the DNA (DNA) 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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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Background: The B-DNA major and minor groove dimensions are crucial for DNA-protein interactions. It has long been thought that the groove dimensions depend on the DNA sequence, however this relationship has remained elusive. Here, our aim is to elucidate how the DNA sequence intrinsically shapes the grooves.

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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.

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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.

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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.

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Metal ions drive important parts of biology, yet it remains experimentally challenging to locate their binding sites. Here we present an innovative computational approach. We use interactive steering of charged ions or small molecules in an electrostatic potential map in order to identify potential binding sites.

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Understanding the preference of nonspecific proteins for certain DNA structural features requires an accurate description of the properties of free DNA, especially regarding their possible predisposition to adopt a conformation that favors the formation of a complex. Exploiting previous exhaustive NMR studies performed on free DNA oligomers, we investigated the molecular basis of DNase I sensitivity under conditions where DNase I binding limits the probability of cleavage. We showed that cleavage intensity was correlated with adjacent 3' phosphate linkage flexibility, monitored by (31)P chemical shifts.

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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.

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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.

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Understanding the recognition of DNA sequences by proteins requires an accurate description of the structural dynamics of free DNA, especially regarding indirect readout. This involves subtle sequence-dependent effects that are difficult to characterize in solution. To progress in this area, we applied NMR and extensive simulations to a DNA sequence relevant to the Jun-Fos system.

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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.

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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.

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The backbone states of B-DNA influence its helical parameters, groove dimensions, and overall curvature. Therefore, detection and fine characterization of these conformational states are desirable. Using routine NMR experiments on a nonlabeled B-DNA oligomer and analyzing high-resolution X-ray structures, we investigated the relationship between interproton distances and backbone conformational states.

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Hairpins containing hexaloops are well represented among the diverse conformations adopted by the RNA molecules. To investigate the intrinsic properties of a backbone submitted to a hexaloop fold, we present here a molecular dynamics study of an abasic hexaloop closed by an A-form 6 basepair stem. The analysis of the 23 ns trajectory made in explicit solvent shows that both the sugars and the torsion angles in the loop undergo numerous conformational transitions.

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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).

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