Molecular modelling study of changes induced by netropsin binding to nucleosome core particles.

It is well known that certain sequence-dependent modulators in structure appear to determine the rotational positioning of DNA on the nucleosome core particle. That preference is rather weak and could be modified by some ligands as netropsin, a minor-groove binding antibiotic. We have undertaken a molecular modelling approach to calculate the relative energy of interaction between a DNA molecule and the protein core particle. The histones particle is considered as a distribution of positive charges on the protein surface that interacts with the DNA molecule. The molecular electrostatic potentials for the DNA, simulated as a discontinuous cylinder, were calculated using the values for all the base pairs. Computing these parameters, we calculated the relative energy of interaction and the more stable rotational setting of DNA. The binding of four molecules of netropsin to this model showed that a new minimum of energy is obtained when the DNA turns toward the protein surface by about 180 degrees, so a new energetically favoured structure appears where netropsin binding sites are located facing toward the histones surface. The effect of netropsin could be explained in terms of an induced change in the phasing of DNA on the core particle. The induced rotation is considered to optimize non-bonded contacts between the netropsin molecules and the DNA backbone.     

Paradoxical hotspots for guanine oxidation by a chemical mediator of inflammation

Guanine in DNA is a major oxidation target owing to its low ionization potential (IP), and there is often an inverse correlation between damage frequency and sequence-dependent variation in guanine IP. We report that the biological oxidant nitrosoperoxycarbonate (ONOOCO2(-)) paradoxically selects guanines with the highest IP in GC-containing contexts. Along with sequence-dependent variation in damage chemistry, this behavior points to factors other than charge migration as determinants of genomic DNA oxidation.     

Sequence-dependent dynamics in duplex DNA

The submicrosecond bending dynamics of duplex DNA were measured at a single site, using a site-specific electron paramagnetic resonance active spin probe. The observed dynamics are interpreted in terms of the mean squared amplitude of bending relative to the end-to-end vector defined by the weakly bending rod model. The bending dynamics monitored at the single site varied when the length and position of a repeated AT sequence, distant from the spin probe, were changed. As the distance between the probe and the AT sequence was increased, the mean squared amplitude of bending seen by the probe due to that sequence decreased. A model for the sequence-dependent internal flexural motion of duplex DNA, which casts the mean squared bending amplitudes in terms of sequence-dependent bending parameters, has been developed. The best fit of the data to the model occurs when the (AT)(n) basepairs are assumed to be 20% more flexible than the average of the basepairs within the control sequence. These findings provide a quantitative basis for interpreting the kinetics of biological processes that depend on duplex DNA flexibility, such as protein recognition and chromatin packaging.     

Molecular dynamics simulation reveals sequence-intrinsic and protein-induced geometrical features of the OL1 DNA operator

We have carried out molecular dynamics simulation of the lambda OL1 DNA operator on the free and the protein-bound forms. Our results lead us to conclude that the binding of the repressor actually makes the N-7 atom of Gua8' more solvent exposed, thereby enhancing its reactivity to chemical methylation. This increase in solvent accessibility surface area occurs simultaneously with the formation of hydrogen bonds between Lys-4 of the nonconsensus flexible N-terminal arm and Gua6' of the nonconsensus half-site operator DNA. Calculations of protein--DNA interaction energies reveal that among the residues of the arm, Lys-4 contributes the most favorably to the interaction energies. This result is consistent with mutagenesis studies that established that lysine at position 4 is absolutely required for tight binding. We find that the nonconsensus arm and the nonconsensus monomer interacts less favorably with DNA than do their respective counterparts of the consensus monomer. Moreover, the six-residue flexible arm accounts for at least half the total protein--DNA interactions energy. These results are in agreement with previous experimental studies. In accord with the diffuse electron density map observed in crystallographic studies of the nonconsensus flexible arm, we find that our model built for this region is more flexible and exhibits more conformations than its consensus counterpart. The simulation also reveals that DNA bending observed near the outer edge of the operator site is an intrinsic sequence-dependent property. By contrast, the DNA-bending features observed toward the center of the operator are induced by the protein. On the whole, stepwise protein-induced bending is more pronounced in the consensus half-site operator. We also find that the unusually large helical twist (49 degrees ) observed in the protein-bound form near the center of the operator results from the binding of the protein at a base step with some propensity for high twists.     

Indirect recognition in sequence-specific DNA binding by Escherichia coli integration host factor: the role of DNA deformation energy

Integration host factor (IHF) is a bacterial histone-like protein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils. It does so by binding in a sequence-independent manner throughout the genome. However, unlike other structurally related bacterial histone-like proteins, IHF has evolved a sequence-dependent, high affinity DNA-binding motif. The high affinity binding sites are important for the regulation of a wide range of cellular processes. A remarkable feature of IHF is that it employs an indirect readout mechanism to bind and wrap DNA at both the nonspecific and high affinity (sequence-dependent) DNA sites. In this study we assessed the contributions of pre-formed and protein-induced DNA conformations to the energetics of IHF binding. Binding energies determined experimentally were compared with energies predicted for the IHF-induced deformation of the DNA helix (DNA deformation energy) in the IHF-DNA complex. Combinatorial sets of de novo DNA sequences were designed to systematically evaluate the influence of sequence-dependent structural characteristics of the conserved IHF recognition elements of the consensus DNA sequence. We show that IHF recognizes pre-formed conformational characteristics of the consensus DNA sequence at high affinity sites, whereas at all other sites relative affinity is determined by the deformational energy required for nearest-neighbor base pairs to adopt the DNA structure of the bound DNA-IHF complex.     

Nucleoprotein architecture and ColE1 dimer resolution: a hypothesis

Dimers of plasmid ColE1 are converted to monomers by site-specific recombination, a process that requires 240 bp of DNA ( cer ) and four host-encoded proteins (XerC, XerD, ArgR and PepA). Here, we propose structures for nucleoprotein complexes involved in cer –Xer recombination based upon existing knowledge of the structures of component proteins and computational analyses of protein structure and DNA curvature. We propose that, in the nucleoprotein complex at a single cer site, a PepA hexamer acts as an adaptor, connecting the heterodimeric recombinase (XerCD) to an ArgR hexamer. This provides a protein core around which the cer site wraps, its exact path being defined by strong sequence-specific interactions with ArgR and XerCD, weak interactions with PepA and sequence-dependent flexibility of cer . The initial association of single-site complexes (pairing) is proposed to occur via an ArgR–PepA interaction. Pairing between sites in a plasmid dimer is stabilized by DNA supercoiling and is followed by a structural isomerization to form a recombination-proficient synaptic complex. We propose that paired structures formed between sites in trans are too short-lived to permit synaptic complex formation. There is thus an energetic barrier to inappropriate recombination reactions. Our proposals are consistent with a wide range of experimental observations.     

Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains

The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway.     

Interaction of proteins located at a distance along DNA: mechanism of target immunity in the Mu DNA strand-transfer reaction

DNA molecules carrying a Mu end(s) are inefficient targets in the Mu DNA strand-transfer reaction. This target immunity is due to preferential dissociation of Mu B protein from DNA molecules that have Mu A protein bound to the Mu end; free DNA is a much poorer target than DNA with Mu B protein bound. We show that Mu B protein, which binds nonspecifically to DNA, is immobile once bound. An encounter between Mu A and Mu B proteins, bound some distance apart along DNA, is necessary to facilitate the Mu B dissociation. Experiments which show that DNA without a Mu end can acquire immunity, by catenation to DNA with a Mu end(s), are consistent with a model of Mu A-Mu B interaction by DNA looping, but not by linear movement of protein(s) along DNA.     

Crystal structure of the specific DNA-binding domain of Tc3 transposase of C.elegans in complex with transposon DNA.

The crystal structure of the complex between the N-terminal DNA-binding domain of Tc3 transposase and an oligomer of transposon DNA has been determined. The specific DNA-binding domain contains three alpha-helices, of which two form a helix-turn-helix (HTH) motif. The recognition of transposon DNA by the transposase is mediated through base-specific contacts and complementarity between protein and sequence-dependent deformations of the DNA. The HTH motif makes four base-specific contacts with the major groove, and the N-terminus makes three base-specific contacts with the minor groove. The DNA oligomer adopts a non-linear B-DNA conformation, made possible by a stretch of seven G:C base pairs at one end and a TATA sequence towards the other end. Extensive contacts (seven salt bridges and 16 hydrogen bonds) of the protein with the DNA backbone allow the protein to probe and recognize the sequence-dependent DNA deformation. The DNA-binding domain forms a dimer in the crystals. Each monomer binds a separate transposon end, implying that the dimer plays a role in synapsis, necessary for the simultaneous cleavage of both transposon termini.     

Biophysical analysis and small-angle X-ray scattering-derived structures of MeCP2-nucleosome complexes

MeCP2 is a highly abundant chromatin architectural protein with key roles in post-natal brain development in humans. Mutations in MeCP2 are associated with Rett syndrome, the main cause of mental retardation in girls. Structural information on the intrinsically disordered MeCP2 protein is restricted to the methyl-CpG binding domain; however, at least four regions capable of DNA and chromatin binding are distributed over its entire length. Here we use small angle X-ray scattering (SAXS) and other solution-state approaches to investigate the interaction of MeCP2 and a truncated, disease-causing version of MeCP2 with nucleosomes. We demonstrate that MeCP2 forms defined complexes with nucleosomes, in which all four histones are present. MeCP2 retains an extended conformation when binding nucleosomes without extra-nucleosomal DNA. In contrast, nucleosomes with extra-nucleosomal DNA engage additional DNA binding sites in MeCP2, resulting in a rather compact higher-order complex. We present ab initio envelope reconstructions of nucleosomes and their complexes with MeCP2 from SAXS data. SAXS studies also revealed unexpected sequence-dependent conformational variability in the nucleosomes themselves.     

Dual signal amplification for highly sensitive electrochemical detection of uropathogens via enzyme-based catalytic target recycling

We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences.     

Structural and functional studies of an intermediate on the pathway to operator binding by Escherichia coli MetJ

We present the results of in vitro DNA-binding assays for a mutant protein (Q44K) of the E. coli methionine repressor, MetJ, as well as the crystal structure at 2.2 A resolution of the apo-mutant bound to a 10-mer oligonucleotide encompassing an 8 bp met-box sequence. The wild-type protein binds natural operators co-operatively with respect to protein concentration forming at least a dimer of repressor dimers along operator DNAs. The minimum operator length is thus 16 bp, each MetJ dimer interacting with a single met-box site. In contrast, the Q44K mutant protein can also bind stably as a single dimer to 8 bp target sites, in part due to additional contacts made to the phosphodiester backbone outside the 8 bp target via the K44 side-chains. Protein-protein co-operativity in the mutant is reduced relative to the wild-type allowing the properties of an intermediate on the pathway to operator site saturation to be examined for the first time. The crystal structure of the decamer complex shows a unique conformation for the protein bound to the single met-box site, possibly explaining the reduced protein-protein co-operativity. In both the extended and minimal DNA complexes formed, the mutant protein makes slightly different contacts to the edges of DNA base-pairs than the wild-type, even though the site of amino acid substitution is distal from the DNA-binding motif. Quantitative binding assays suggest that this is not due to artefacts caused by the crystallisation conditions but is most likely due to the relatively small contribution of such direct contacts to the overall binding energy of DNA-protein complex formation, which is dominated by sequence-dependent distortions of the DNA duplex and by the protein-protein contact between dimers.