-
PloS One 2013The condensed structure of chromatin limits access of cellular machinery towards template DNA. This in turn represses essential processes like 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.
Topics: Adenosine Triphosphate; Animals; Chromatin; Chromatin Assembly and Disassembly; Circular Dichroism; DNA; Distamycins; Histones; Male; Nucleic Acid Conformation; Protein Binding; Rats; Rats, Sprague-Dawley; Transcription, Genetic
PubMed: 23460895
DOI: 10.1371/journal.pone.0057693 -
Journal of Molecular Biology May 2013Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally conserved but sequence-divergent...
Probing the electrostatics and pharmacological modulation of sequence-specific binding by the DNA-binding domain of the ETS family transcription factor PU.1: a binding affinity and kinetics investigation.
Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤10(5)M(-)(1)s(-)(1)), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>10(7)M(-)(1)s(-)(1)). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes.
Topics: Animals; Base Sequence; Binding Sites; Binding, Competitive; Biosensing Techniques; DNA; DNA-Binding Proteins; Distamycins; Mice; Models, Molecular; Protein Structure, Tertiary; Proto-Oncogene Proteins; Static Electricity; Surface Plasmon Resonance; Trans-Activators
PubMed: 23416556
DOI: 10.1016/j.jmb.2013.02.010 -
Analytical Biochemistry Apr 2012Polyamides (PAs) are distamycin-type ligands of DNA that bind the minor groove and are capable of sequence selective recognition. This capability provides a viable route...
Polyamides (PAs) are distamycin-type ligands of DNA that bind the minor groove and are capable of sequence selective recognition. This capability provides a viable route to their development as therapeutics. Presented here is a simple and convenient fluorescence assay for PA-DNA binding. PAs are titrated into a sample of a hairpin DNA featuring a TAMRA dye attached to an internal dU near the PA binding site. In a study of 6 PAs, PA binding leads to a steady reproducible decrease in fluorescence intensity that can be used to generate binding isotherms. The assay works equally well with both short (6- to 8-ring) and long (14-ring) PAs, and K(d) values ranging from approximately 1 nM to at least 140 nM were readily obtained using a simple monochromator or filter configuration. Competition assays provide a means to assessing possible dye interference, which can be negligible. The assay can also be used to determine PA extinction coefficients and to measure binding kinetics; thus, it is an accessible and versatile tool for the study of PA properties and PA-DNA interactions.
Topics: Base Sequence; Biological Assay; DNA; Kinetics; Nylons; Rhodamines; Spectrometry, Fluorescence
PubMed: 22342620
DOI: 10.1016/j.ab.2012.01.017 -
PloS One 2011Distamycin 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...
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.
Topics: Animals; Chromatin; DNA; Distamycins; Nucleic Acid Conformation; Nucleic Acid Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; Thermodynamics
PubMed: 22046291
DOI: 10.1371/journal.pone.0026486 -
Communicative & Integrative Biology May 2011We have recently reported the isolation and characterization of Plasmodium falciparum Dbp5/DDX19 homolog PfD66 and the results indicate that it contains ATP-dependent...
We have recently reported the isolation and characterization of Plasmodium falciparum Dbp5/DDX19 homolog PfD66 and the results indicate that it contains ATP-dependent bipolar DNA and RNA unwinding activity, intrinsic nucleic acid-dependent ATPase and RNA-binding activities. In the present study we report the effect of a number of compounds such as actinomycin D, aphidicolin, camptothecin, cyclophosphamide, 4',6'-di-amidino-2-phenylindole (DAPI), daunorubicin, distamycin, ethidium bromide, ellipticine, genistein, mitoxantrone, nalidixic acid, netropsin, nogalamycin, novobiocin and VP-16 on the DNA unwinding and ATPase activities of PfD66. The results indicate that DAPI, ethidium bromide, netropsin and nogalamycin efficiently inhibited the helicase and ATPase activities of PfD66. These studies will make an important contribution in understanding the mechanism of DNA unwinding by Plasmodium falciparum helicase PfD66.
PubMed: 21980563
DOI: 10.4161/cib.4.3.14778 -
Biochemistry Sep 2011DNA sequence-dependent conformational changes induced by the minor groove binder, distamycin, have been evaluated by polyacrylamide gel electrophoresis. The distamycin... (Comparative Study)
Comparative Study
DNA sequence-dependent conformational changes induced by the minor groove binder, distamycin, have been evaluated by polyacrylamide gel electrophoresis. The distamycin binding affinity, cooperativity, and stoichiometry with three target DNA sequences that have different sizes of alternating AT sites, ATAT, ATATA, and ATATAT, have been determined by mass spectrometry and surface plasmon resonance to help explain the conformational changes. The results show that distamycin binds strongly to and bends five or six AT base pair minor groove sites as a dimer with positive cooperativity, while it binds to ATAT as a weak, slightly anticooperative dimer. The bending direction was evaluated with an in phase A-tract reference sequence. Unlike other similar monomer minor groove binding compounds, such as netropsin, the distamycin dimer changes the directionality of the overall curvature away from the minor groove to the major groove. This distinct structural effect may allow designed distamycin derivatives to have selective therapeutic effects.
Topics: Base Sequence; DNA-Binding Proteins; Distamycins; Nucleic Acid Heteroduplexes; Protein Binding; Protein Conformation; Protein Multimerization
PubMed: 21800847
DOI: 10.1021/bi201010g -
PloS One 2011Human papillomavirus (HPV) is the main causative agent of cervical cancer, particularly high risk strains such us HPV-16, -18 and -31. The viral encoded E2 protein acts...
BACKGROUND
Human papillomavirus (HPV) is the main causative agent of cervical cancer, particularly high risk strains such us HPV-16, -18 and -31. The viral encoded E2 protein acts as a transcriptional modulator and exerts a key role in viral DNA replication. Thus, E2 constitutes an attractive target for developing antiviral agents. E2 is a homodimeric protein that interacts with the DNA target through an α-helix of each monomer. However, a peptide corresponding to the DNA recognition helix of HPV-16 E2 binds DNA with lower affinity than its full-length DNA binding domain. Therefore, in an attempt to promote the DNA binding of the isolated peptide, we have designed a conjugate compound of the E2 α-helix peptide and a derivative of the antibiotic distamycin, which involves simultaneous minor- and major-groove interactions.
METHODOLOGY/PRINCIPAL FINDINGS
An E2 α-helix peptide-distamycin conjugate was designed and synthesized. It was characterized by NMR and CD spectroscopy, and its DNA binding properties were investigated by CD, DNA melting and gel shift experiments. The coupling of E2 peptide with distamycin does not affect its structural properties. The conjugate improves significantly the affinity of the peptide for specific DNA. In addition, stoichiometric amounts of specific DNA increase meaningfully the helical population of the peptide. The conjugate enhances the DNA binding constant 50-fold, maintaining its specificity.
CONCLUSIONS/SIGNIFICANCE
These results demonstrate that peptide-distamycin conjugates are a promising tool to obtain compounds that bind the E2 target DNA-sequences with remarkable affinity and suggest that a bipartite major/minor groove binding scaffold can be a useful approach for therapeutic treatment of HPV infection.
Topics: Amino Acid Sequence; Antiviral Agents; Base Sequence; Biomimetic Materials; DNA, Viral; DNA-Binding Proteins; Distamycins; Human papillomavirus 16; Models, Molecular; Molecular Sequence Data; Oncogene Proteins, Viral; Papillomavirus Infections; Peptide Fragments; Protein Structure, Secondary; Pyrroles; Substrate Specificity
PubMed: 21799846
DOI: 10.1371/journal.pone.0022409 -
Antiviral Research Aug 2011Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease...
Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease viral DNA load and that have low toxicity. We report the potent anti-HPV activity of two N-methylpyrrole-imidazole polyamides of the hairpin type, polyamide 1 (PA1) and polyamide 25 (PA25). Both polyamides have potent anti-HPV activity against three different genotypes when tested on cells maintaining HPV episomes. The compounds were tested against HPV16 (in W12 cells), HPV18 (in Ker4-18 cells), and HPV31 (in HPV31 maintaining cells). From a library of polyamides designed to recognize AT-rich DNA sequences such as those in or near E1 or E2 binding sites of the HPV16 origin of replication (ori), four polyamides were identified that possessed apparent IC(50)s≤150nM with no evidence of cytotoxicity. We report two highly-active compounds here. Treatment of epithelia engineered in organotypic cultures with these compounds also causes a dose-dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus. Bromodeoxyuridine (BrdU) incorporation demonstrates that DNA synthesis in organotypic cultures is suppressed upon compound treatment, correlating with a loss of HPV16 and HPV18 episomes. PA1 and PA25 are currently in preclinical development as antiviral compounds for treatment of HPV-related disease, including cervical dysplasia. PA1, PA25, and related polyamides offer promise as antiviral agents and as tools to regulate HPV episomal levels in cells for the study of HPV biology. We also report that anti-HPV16 activity for Distamycin A, a natural product related to our polyamides, is accompanied by significant cellular toxicity.
Topics: Antiviral Agents; Binding Sites; Bromodeoxyuridine; Cell Line, Tumor; DNA, Viral; Distamycins; Female; Human papillomavirus 16; Human papillomavirus 18; Human papillomavirus 31; Humans; Immunohistochemistry; Inhibitory Concentration 50; Microbial Sensitivity Tests; Nylons; Papillomavirus Infections; Plasmids; Pyrroles; Replication Origin; Uterine Cervical Neoplasms; Viral Load
PubMed: 21669229
DOI: 10.1016/j.antiviral.2011.05.014 -
Molecules (Basel, Switzerland) Apr 2011A novel and straightforward solid phase synthesis of distamycin analogues containing benzene units has been developed.
A novel and straightforward solid phase synthesis of distamycin analogues containing benzene units has been developed.
Topics: Antibiotics, Antineoplastic; Chromatography, High Pressure Liquid; Combinatorial Chemistry Techniques; Distamycins; Hydrogen Bonding; Magnetic Resonance Spectroscopy
PubMed: 21483272
DOI: 10.3390/molecules16043066 -
Plant Signaling & Behavior Mar 2011Pea mini-chromosome maintenance 6 (MCM6) single subunit (93 kDa) forms homohexamer (560 kDa) and contains an ATP-dependent and replication fork stimulated 3' to 5' DNA...
Pea mini-chromosome maintenance 6 (MCM6) single subunit (93 kDa) forms homohexamer (560 kDa) and contains an ATP-dependent and replication fork stimulated 3' to 5' DNA unwinding activity along with intrinsic DNA-dependent ATPase and ATP-binding activities [Plant Mol. Biol. 2010; DOI: 10.1007/s11103-010-9675-7]. Here, we have determined the effect of various DNA-binding agents, such as actinomycin, nogalamycin, daunorubicin, doxorubicin, distamycin, camptothecin, cyclophosphamide, ellipticine, VP-16, novobiocin, netropsin, cisplatin, mitoxantrone and genistein on the DNA unwinding and ATPase activities of the pea MCM6 DNA helicase. The results show that actinomycin and nogalamycin inhibited the DNA helicase (apparent Ki values of 10 and 1 μM, respectively) and ATPase (apparent Ki values of 100 and 17 μM, respectively) activities. Although, daunorubicin and doxorubicin also inhibited the DNA helicase activity of pea MCM6, but with less efficiency; however, these could not inhibit the ATPase activity. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of MCM6, resulting in the inhibitions of the activities. This study could be useful in our better understanding of the mechanism of plant nuclear DNA helicase unwinding.
Topics: Adenosine Triphosphatases; Camptothecin; Cisplatin; DNA Helicases; Dactinomycin; Daunorubicin; Ellipticines; Enzyme Inhibitors; Etoposide; Intercalating Agents; Mitoxantrone; Nogalamycin; Novobiocin; Pisum sativum; Plant Proteins
PubMed: 21336027
DOI: 10.4161/psb.6.3.13355