The binding mode of the DNA bisintercalator luzopeptin investigated using atomic force microscopy

The luzopeptins are DNA bisintercalating antibiotics that contain a decadepsipeptide to which are attached two quinoline chromophores. We have used atomic force microscopy (AFM) to investigate the interaction between luzopeptin B and DNA in an attempt to shed light on the binding mode of this antibiotic. AFM images provided contour lengths which were used as a direct measure of bisintercalation. Binding of luzopeptin B was investigated using two different DNA sequences, one having a GC content of 42% and the other 59%, which revealed a higher degree of bisintercalation into the DNA sequences having the lower GC content. The measured increment in contour length was found to plateau at values corresponding to binding of one drug molecule every 40 and 72 bp to the 42 and 59% GC sequences, respectively. In addition to the length increase, a higher proportion of DNA molecules displaying complex morphology was observed as the concentration of luzopeptin was increased. Such molecules were not included in the measurements of contour length. We propose that the various manifestations of complex morphology arise from both inter- and intramolecular cross-linking of the DNA caused by binding of luzopeptin, providing direct evidence of cross-linked species by AFM imaging.     

Adjustable ‘cross-linking’ of neighboring DNA molecules in liquid-crystalline dispersions through (daunomycin-copper) polymeric chelate complexes

The interaction of daunomycin molecules with double-stranded DNA in the liquid-crystalline state was investigated. It was shown that at a certain extent of daunomycin binding a change of the mechanism of anthracycline orientation with reference to the DNA chain occurs. This is testified by the alteration of the sense of spatial packing of the DNA molecules in liquid-crystalline dispersions formed as a result of phase separation in poly(ethyleneglycol)-containing solutions, as well as by the onset of the reaction of daunomycin with divalent copper ions. Using this reaction, polymeric (daunomycin-copper) chelate cross-links between the DNA molecules fixed in the liquid-crystalline dispersions were formed. The length of such cross-links is adjusted by the distance between the DNA molecules, which, in turn, depends on the concentration of poly(ethyleneglycol) used for phase separation. The above molecular building mechanism may lead to new interesting applications.     

Interplay of Protein Binding Interactions,DNA Mechanics,and Entropy in?DNA Looping Kinetics

DNA looping plays a key role in many fundamental biological processes, including gene regulation, recombination, and chromosomal organization. The looping of DNA is often mediated by proteins whose structural features and physical interactions can alter the length scale at which the looping occurs. Looping and unlooping processes are controlled by thermodynamic contributions associated with mechanical deformation of the DNA strand and entropy arising from thermal fluctuations of the conformation. To determine how these confounding effects influence DNA looping and unlooping kinetics, we present a theoretical model that incorporates the role of the protein interactions, DNA mechanics, and conformational entropy. We show that for shorter DNA strands the interaction distance affects the transition state, resulting in a complex relationship between the looped and unlooped state lifetimes and the physical properties of the looped DNA. We explore the range of behaviors that arise with varying interaction distance and DNA length. These results demonstrate how DNA deformation and entropy dictate the scaling of the looping and unlooping kinetics versus the J-factor, establishing the connection between kinetic and equilibrium behaviors. Our results show how the twist-and-bend elasticity of the DNA chain modulates the kinetics and how the influence of the interaction distance fades away at intermediate to longer chain lengths, in agreement with previous scaling predictions.     

Single-molecule atomic force spectroscopy reveals that DnaD forms scaffolds and enhances duplex melting

The Bacillus subtilis DnaD is an essential DNA-binding protein implicated in replication and DNA remodeling. Using single-molecule atomic force spectroscopy, we have studied the interaction of DnaD and its domains with DNA. Our data reveal that binding of DnaD to immobilized single molecules of duplex DNA causes a marked reduction in the ‘end-to-end’ distance of the DNA in a concentration-dependent manner, consistent with previously reported DnaD-induced looping by scaffold formation. Native DnaD enhances partial melting of the DNA strands. The C-terminal domain (Cd) of DnaD binds to DNA and enhances partial duplex melting but does not cause DNA looping. The Cd-mediated melting is not as efficient as that caused by native DnaD. The N-terminal domain (Nd) does not affect significantly the DNA. A mixture of Nd and Cd fails to recreate the DNA looping effect of native DnaD but produces exactly the same effects as Cd on its own, consistent with the previously reported failure of the separated domains to form DNA-interacting scaffolds.     

Anthracycline-binding induced DNA stiffening, bending and elongation; stereochemical implications from viscometric investigations

Upon interaction of the three anthracycline antibiotics daunomycin, adriamycin, and aclacinomycin A with calf thymus DNA the relative changes of both DNA contour length, delta L/Lo, and persistence length, delta a/ao, have been determined as a function of r, the ratio of bound ligand molecules per DNA mononucleotide. From the r dependence of delta a/ao a measure for the stiffening effect and also the angle gamma of ligand-induced DNA bending could be derived. Experimental basis are titration viscometric measurements upon both low and high molecular weight DNA. It was found that the DNA contour length increases linearly with r by approximately 0.34 nm per bound drug molecule. The comparatively very high DNA stiffening effect measured in solution is understandable as a result of helix clamping by at least two anthracycline groups of sufficient long distance. The variation of gamma on DNA interaction with different anthracycline derivatives find their explanation in terms of different values of the mismatch to in-register binding prior to complex formation. From an analogous interpretation of viscosity measurements by Arcamone and coworkers upon high molecular weight DNA with many anthracycline derivatives it can be concluded that DNA interaction by both amino sugar and 9-acetyl group are responsible for the generation of strong anthracycline binding mediated DNA stiffening effects in solution. (A combined analysis of the viscosity measurements by Cohen & Eisenberg and Armstrong et al. upon DNA interaction with proflavine indicates a very small DNA stiffening effect, gamma = 6.7 sigma and a helix elongation by 0.35 nm per bound ligand molecule.)     

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.     

Making contacts on a nucleic acid polymer

The interaction of proteins bound at distant sites on a nucleic acid chain plays an important role in many molecular biological processes. Contact between the proteins is established by looping of the intervening polymer, which can comprise either double- or single-stranded DNA or RNA, or interphase or metaphase chromatin. The effectiveness of this process, as well as the optimal separation distance, is highly dependent on the flexibility and conformation of the linker. This article reviews how the probability of looping-mediated interactions is calculated for different nucleic acid polymers. In addition, the application of the equations to the analysis of experimental data is illustrated.     

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.