Effect of DNA Groove Binder Distamycin A upon Chromatin Structure

BACKGROUND: Distamycin A is a prototype minor groove binder, which binds to B-form DNA, preferentially at A/T rich sites. Extensive work in the past few decades has characterized the binding at the level of double stranded DNA. However, effect of the same on physiological DNA, i.e. DNA complexed in chromatin, has not been well studied. Here we elucidate from a structural perspective, the interaction of distamycin with soluble chromatin, isolated from Sprague-Dawley rat. METHODOLOGY/PRINCIPAL FINDINGS: Chromatin is a hierarchical assemblage of DNA and protein. Therefore, in order to characterize the interaction of the same with distamycin, we have classified the system into various levels, according to the requirements of the method adopted, and the information to be obtained. Isothermal titration calorimetry has been employed to characterize the binding at the levels of chromatin, chromatosome and chromosomal DNA. Thermodynamic parameters obtained thereof, identify enthalpy as the driving force for the association, with comparable binding affinity and free energy for chromatin and chromosomal DNA. Reaction enthalpies at different temperatures were utilized to evaluate the change in specific heat capacity (ΔCp), which, in turn, indicated a possible binding associated structural change. Ligand induced structural alterations have been monitored by two complementary methods--dynamic light scattering, and transmission electron microscopy. They indicate compaction of chromatin. Using transmission electron microscopy, we have visualized the effect of distamycin upon chromatin architecture at di- and trinucleosome levels. Our results elucidate the simultaneous involvement of linker bending and internucleosomal angle contraction in compaction process induced by distamycin. CONCLUSIONS/SIGNIFICANCE: We summarize here, for the first time, the thermodynamic parameters for the interaction of distamycin with soluble chromatin, and elucidate its effect on chromatin architecture. The study provides insight into a ligand induced compaction phenomenon, and suggests new mechanisms of chromatin architectural alteration

Electron microscopy of soluble chromatin.

<p>Chromatin samples were incubated with DST in drug to DNA base ratio of 0.16 and processed as detailed under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026486#s2" target="_blank">Materials and Methods</a>”. (A) Soluble chromatin incubated with buffer for 1 hour. (B) Soluble chromatin incubated with DST under similar experimental conditions. Black arrowheads indicate 15 nm nanosphere standards and the scale bar indicates 200 nm.</p

Energetics of the interaction of DST with chromatin components.

<p>The thermodynamic parameters (ΔH, −TΔS and ΔG) are plotted as function of temperature for the interaction of DST with (A) soluble chromatin, (B) chromatosome and (C) chromosomal DNA. All experiments were performed in 5 mM Tris HCl (pH 7.4), 15 mM NaCl.</p

Dynamic Light Scattering (DLS) to study the influence of DST on the hydrodynamic properties of soluble chromatin, dinucleosomes and trinucleosomes.

<p>The intensity statistics of 10 measurements each are plotted for (A) soluble chromatin (300 µM DNA base), (B) dinucleosomes (300 µM DNA base) and (C) trinucleosomes (300 µM DNA base) in presence of increasing concentration of DST. Error bars indicate standard deviation. Diffusion coefficients calculated from Z_av radii are plotted as a function of DST concentration for (D) soluble chromatin, (E) dinucleosomes, and (F) trinucleosomes. All experiments were performed at 25°C.</p

Analysis of dinucleosome morphology.

<p>(A) Survey view of a glutaraldehyde fixed dinucleosome fraction, shadowed with platinum. Some clearly defined dinucleosomes have been encircled. Black arrowheads indicate 15 nm nanosphere standards and scale bar indicates 100 nm. Three representative dinucleosomes are shown in higher magnification in (B). Scale bar indicates 20 nm. Three representative DST treated dinucleosomes are shown in (C). Scale bar is 20 nm. (D, E) Statistical analysis of the center to center distances of dinucleosomes. The percentage frequency of particles is plotted against the center to center distance in (D) free dinucleosomes and (E) DST treated dinucleosomes. Frequency distributions were obtained for 5 nm bin size. The ratio of DST to DNA base was 0.16.</p

Analysis of trinucleosomes morphology.

<p>(A) Survey view of a glutaraldehyde fixed trinucleosome fraction, shadowed with platinum. Some clearly defined trinucleosomes have been encircled. Black arrowheads indicate 7 nm nanosphere standards and scale bar indicates 100 nm. Three representative trinucleosomes are shown in higher magnification in (B). Scale bar indicates 20 nm. Three representative DST treated trinucleosomes are shown in (C). Scale bar is 20 nm. (D, E) Statistical analysis of the center to center distances of trinucleosomes. The percentage frequency of particles is plotted against the center to center distance in (D) free trinucleosomes and (E) DST treated trinucleosomes. Frequency distributions were obtained for 5 nm bin size. (F,G) Statistical analysis of the internucleosomal angles of trinucleosomes. The percentage frequency of particles was plotted against the internucleosomal projection angle in (F) free trinucleosomes and (G) DST treated trinucleosomes. The bin size for the frequency distribution was 10 degrees. DST treatment was performed at drug to DNA base ratio of 0.16.</p

Minor groove binder distamycin remodels chromatin but inhibits transcription.

The condensed structure of chromatin limits access of cellular machinery towards template DNA. This in turn represses essential processes like transcription, replication, repair and recombination. The repression is alleviated by a variety of energy dependent processes, collectively known as "chromatin remodeling". In a eukaryotic cell, a fine balance between condensed and de-condensed states of chromatin helps to maintain an optimum level of gene expression. DNA binding small molecules have the potential to perturb such equilibrium. We present herein the study of an oligopeptide antibiotic distamycin, which binds to the minor groove of B-DNA. Chromatin mobility assays and circular dichroism spectroscopy have been employed to study the effect of distamycin on chromatosomes, isolated from the liver of Sprague-Dawley rats. Our results show that distamycin is capable of remodeling both chromatosomes and reconstituted nucleosomes, and the remodeling takes place in an ATP-independent manner. Binding of distamycin to the linker and nucleosomal DNA culminates in eviction of the linker histone and the formation of a population of off-centered nucleosomes. This hints at a possible corkscrew type motion of the DNA with respect to the histone octamer. Our results indicate that distamycin in spite of remodeling chromatin, inhibits transcription from both DNA and chromatin templates. Therefore, the DNA that is made accessible due to remodeling is either structurally incompetent for transcription, or bound distamycin poses a roadblock for the transcription machinery to advance

Circular Dichroism spectroscopy to study distamycin induced structural changes of chromatosomes and chromatosomal DNA.

<p>(A) Chromatosome or (B) chromatosomal DNA (50 µM nucleotide concentration) is treated with distamycin in drug to DNA base ratios of 0.08 (­­­), 0.16 (·····), and 0.25 (-·-·-).Chromatosome, chromatosomal DNA and distamycin solutions are prepared in 5 mM Tris HCl (pH 7.4), 15 mM NaCl and titrations are performed at 25°C.</p

Interaction of distamycin with histones.

<p>ITC profiles for the interaction of distamycin with (A) core histones and (B) linker histone in 5 mM Tris HCl (pH 7.4), 100 mM NaCl at 25°C.</p