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Biophysical Journal Jan 2005Mechanical properties of single double-stranded DNA (dsDNA) in the presence of different binding ligands were analyzed in optical-tweezers experiments with subpiconewton...
Mechanical properties of single double-stranded DNA (dsDNA) in the presence of different binding ligands were analyzed in optical-tweezers experiments with subpiconewton force resolution. The binding of ligands to DNA changes the overall mechanic response of the dsDNA molecule. This fundamental property can be used for discrimination and identification of different binding modes and, furthermore, may be relevant for various processes like nucleosome packing or applications like cancer therapy. We compared the effects of the minor groove binder distamycin-A, a major groove binding alpha-helical peptide, the intercalators ethidium bromide, YO-1, and daunomycin as well as the bisintercalator YOYO-1 on lambda-DNA. Binding of molecules to the minor and major groove of dsDNA induces distinct changes in the molecular elasticity compared to the free dsDNA detectable as a shift of the overstretching transition to higher forces. Intercalating molecules affect the molecular mechanics by a complete disappearance of the B-S transition and an associated increase in molecular contour length. Significant force hysteresis effects occurring during stretching/relaxation cycles with velocities >10 nm/s for YOYO-1 and >1000 nm/s for daunomycin. These indicate structural changes in the timescale of minutes for the YOYO-DNA and of seconds for the daunomycin-DNA complexes, respectively.
Topics: Benzoxazoles; Biophysics; DNA; Daunorubicin; Distamycins; Ethidium; Fluorescent Dyes; Intercalating Agents; Kinetics; Lasers; Ligands; Models, Molecular; Nucleic Acid Conformation; Peptides; Polystyrenes; Protein Binding; Protein Structure, Secondary; Quinolinium Compounds; Time Factors
PubMed: 15516529
DOI: 10.1529/biophysj.103.036293 -
The Journal of Biological Chemistry Jan 1998In budding yeast Saccharomyces cerevisiae, an ARS binding factor 1 (Abf1) binds to the sequence-specific DNA element involved in DNA replication and transcription. We...
In budding yeast Saccharomyces cerevisiae, an ARS binding factor 1 (Abf1) binds to the sequence-specific DNA element involved in DNA replication and transcription. We describe in this study how yeast Cdc6 protein stimulates Abf1 protein DNA binding activities. The Abf1 binding activity was reduced approximately 20-fold in a cdc6-1 mutant than in the wild-type strain. Introducing a copy of the wild-type CDC6 gene into the cdc6-1 mutant strain restored the Abf1 DNA binding activity. We demonstrated that recombinant Abf1 binds to ARS1 in vitro, and its DNA binding activity can be highly stimulated by the addition of a fusion glutathione S-transferase (GST)-Cdc6 protein. Deletion analysis revealed that the stimulating region is located at the amino terminus of the Cdc6 protein. However, we could not find the direct physical interaction between Cdc6 and Abf1. Instead, we found that the GST-Cdc6 can compete with distamycin A for binding to the DNA molecule. As distamycin A is a specific reagent that binds noncovalently to DNA at (A + T)-rich tracks, the stimulation of Abf1 DNA binding activity may be mediated by the Cdc6/DNA interaction. Our results favor a hypothesis that Cdc6 may function as an architectural factor in the assembly of a functional initiation replication complex.
Topics: Antiviral Agents; Cell Cycle Proteins; DNA, Fungal; DNA-Binding Proteins; Distamycins; Fungal Proteins; Phosphoproteins; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Suppression, Genetic; Transcription Factors
PubMed: 9430660
DOI: 10.1074/jbc.273.3.1298 -
FEBS Letters Nov 1998We studied the interaction of cis-diammine Pt(II)-bridged bis-netropsin, cis-diammine Pt(II)-bridged bis-distamycin and oligomethylene-bridged bis-netropsin with...
We studied the interaction of cis-diammine Pt(II)-bridged bis-netropsin, cis-diammine Pt(II)-bridged bis-distamycin and oligomethylene-bridged bis-netropsin with synthetic DNA fragments containing pseudosymmetrical AT-rich nucleotide sequences and compared it with the interaction of the parent compounds netropsin and distamycin A. For fragments containing multiple blocks of (AIT)4 and (T/A)4 separated by zero, one, two and three GC-base pairs, DNase I footprinting and CD spectroscopy studies reveal that 5'-TTTTAAAA-3' is the strongest affinity binding site for cis-diammine Pt(II)-bridged bis-netropsin and bis-distamycin. They both bind less strongly to a DNA region containing the sequence 5'-AAAATTTT-3'. Netropsin, distamycin A and oligomethylene-bridged bis-netropsin exhibit far less sequence discrimination.
Topics: Base Sequence; Binding Sites; Cloning, Molecular; DNA; DNA Footprinting; Deoxyribonuclease I; Distamycins; Molecular Sequence Data; Netropsin; Oligodeoxyribonucleotides
PubMed: 9845351
DOI: 10.1016/s0014-5793(98)01379-9 -
British Journal of Cancer Dec 1991FCE 24157 (chemically (beta-[1-methyl-4-(1-methyl-4--[1-methyl-4-(4-N,N- bis(2-chloroethyl) amino-benzene-1-carboxy-amido)...
FCE 24157 (chemically (beta-[1-methyl-4-(1-methyl-4--[1-methyl-4-(4-N,N- bis(2-chloroethyl) amino-benzene-1-carboxy-amido) pyrrole-2-carboxiamido]pyrrole-2-carboxyamido)pyrrole-2-c arboxyamido]) propionamidine, hydrochloride) is a distamycin A (Dista A) derivative bearing a benzoyl mustard moiety instead of the formyl group at the N-terminal. Contrary to Dista A, FCE 24517 has been found to display potent cytotoxic activity on human and murine tumour cell lines. The compound maintains activity on melphalan (L-PAM)-resistant cells, whereas cross-resistance is observed on doxorubicin-(DX)-resistant cells. In vivo, FCE 24517 was found to possess evident antineoplastic activity on a series of murine transplanted solid tumours and human tumour xenografts. The following neoplasms were in fact found to be sensitive to FCE 24517 treatment: M14 human melanoma xenograft, N592 human small cell lung carcinoma, MTV murine mammary carcinoma, Colon 38 murine carcinoma, PO2 murine pancreatic carcinoma and M5076 murine reticulosarcoma. Lower effectiveness was observed against the murine P388 and Gross leukaemia, Lewis lung murine carcinoma, LoVo human colon carcinoma xenografts and A459 human lung adenocarcinoma. Against the murine L1210 leukaemia, FCE 24517 displayed a clear activity only when the tumour was transplanted i.p. and treatment was given i.p., whereas only marginal activity was seen against this leukaemia if transplanted i.v. and the drug was given i.v. As true also in vitro, FCE 24517 was effective against i.p. implanted L1210 leukaemia resistant to L-PAM. The mode(s) of action of this new compound is under active investigation.
Topics: Animals; Antineoplastic Agents; Cell Division; Cell Survival; Distamycins; In Vitro Techniques; Mice; Mice, Inbred Strains; Neoplasm Transplantation; Neoplasms, Experimental; Nitrogen Mustard Compounds; Tumor Cells, Cultured
PubMed: 1764367
DOI: 10.1038/bjc.1991.463 -
Cytometry. Part a : the Journal of the... Nov 2009Drug resistant tumor "side-populations," enriched in cancer stem cells and identified by reduced accumulation of Hoechst 33342 under ABCG2-mediated efflux, may...
Drug resistant tumor "side-populations," enriched in cancer stem cells and identified by reduced accumulation of Hoechst 33342 under ABCG2-mediated efflux, may compromise therapeutic outcome. Side-population cells have predicted resistance to minor groove ligands, including the DNA topoisomerase I poison topotecan. We have used a stable Hoechst 33342-resistant murine L cell system (HoeR415) to study resistance patterns, removing the need for SP isolation before microarray analysis of gene expression and the tracking of cell cycle dynamics and cytotoxicity. The majority of HoeR415 cells displayed a side-population phenotype comparable with that of the side-population resident in the ABCG2 over-expressing A549 lung cancer cell line. Photo-crosslinking showed direct protection against minor groove ligand residence on DNA, driven by ABCG2-mediated efflux and not arising from any binding competition with endogenous polyamines. The covalent minor-groove binding properties of the drug FCE24517 (tallimustine) prevented resistance suggesting a mechanism for overcoming SP-related drug resistance. Hoechst 33342-resistant murine cells showed lower but significant crossresistance to topotecan, again attributable to enhanced ABCG2 expression, enabling cells to evade S-phase arrest. Hoechst 33342/TPT-resistant cells showed limited ancillary gene expression changes that could modify cellular capacity to cope with chronic stress including over-expression of Aldh1a1 and Mgst1, but under-expression of Plk2 and Nnt. There was no evidence to link the putative stem cell marker ALDH1A1 with any augmentation of the TPT resistance phenotype. The study has implications for the patterns of drug resistance arising during tumor repopulation and the basal resistance to minor groove-binding drugs of tumor side-populations.
Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Animals; Benzimidazoles; Cell Line, Tumor; Cell Separation; Cross-Linking Reagents; Distamycins; Enzyme Inhibitors; Flow Cytometry; Fluorescent Dyes; Humans; Ligands; Mice; Nitrogen Mustard Compounds; Polyamines; Topotecan
PubMed: 19802874
DOI: 10.1002/cyto.a.20800 -
Nucleic Acids Research May 1995The antibiotic distamycin A is a DNA minor groove binding drug (MGB) that recognizes a stretch of at least four ATs. The alkylating benzoyl mustard derivative...
The antibiotic distamycin A is a DNA minor groove binding drug (MGB) that recognizes a stretch of at least four ATs. The alkylating benzoyl mustard derivative tallimustine (FCE 24517) has powerful anti-tumor activity. Using the electrophoretic mobility shift assay (EMSA) we determined that both compounds can prevent binding of TBP and, with 10-fold higher concentration, TBP-TFIIA (DA) and TBP-TFIIA-TFIIB (DAB) to a TATA box. Once formed, the DA and DAB complexes are more resistant to MGB challenge. Both drugs can inhibit basal in vitro transcription of a minimal TATA-containing promoter and similar concentrations are necessary for binding and transcriptional inhibition. Tallimustine shows strong selectivity by decreasing only correctly initiated transcripts. Even at high doses (20 microM), however, they cannot disturb a competent pre-initiation complex or Pol II progression. This functional in vitro model will provide a way to investigate the activity of sequence-specific DNA binding drugs with potential anti-viral and anti-tumour activity and to develop novel more selective compounds.
Topics: Antineoplastic Agents; Base Sequence; DNA-Binding Proteins; Distamycins; Humans; Intercalating Agents; Molecular Sequence Data; Nitrogen Mustard Compounds; Oligodeoxyribonucleotides; Recombinant Proteins; Saccharomyces cerevisiae; TATA Box; TATA-Box Binding Protein; Transcription Factor TFIIA; Transcription Factor TFIIB; Transcription Factors; Transcription, Genetic
PubMed: 7784168
DOI: 10.1093/nar/23.10.1657 -
Nucleic Acids Research Mar 1974The binding of the antibiotics netropsin and distamycin A to DNA has been studied by thermal melting, CD and sedimentation analysis. Netropsin binds strongly at...
The binding of the antibiotics netropsin and distamycin A to DNA has been studied by thermal melting, CD and sedimentation analysis. Netropsin binds strongly at antibiotic/nucleotide ratios up to at least 0.05. CD spectra obtained using DNA model polymers reveal that netropsin binds tightly to poly (dA) . poly (dT), poly (dA-dT) . poly(dA-dT) and poly (dI-dC) . poly (dI-dC) but poorly, if at all, to poly (dG) . poly (dC). Binding curves obtained with calf thymus DNA reveal one netropsin-binding site per 6.0 nucleotides (K(a)=2.9 . 10(5) M(-1)); corresponding values for distamycin A are one site per 6.1 nucleotides with K(a)= 11.6 . 10(5) M(-1). Binding sites apparently involve predominantly A.T-rich sequences whose specific conformation determines their high affinity for the two antibiotics. It is suggested that the binding is stabilized primarily by hydrogen bonding and electrostatic interactions probably in the narrow groove of the DNA helix, but without intercalation. Any local structural deformation of the helix does not involve unwinding greater than approximately 3 degrees per bound antibiotic molecule.
Topics: Animals; Binding Sites; Cattle; Circular Dichroism; DNA; Distamycins; In Vitro Techniques; Netropsin; Nucleic Acid Conformation; Polydeoxyribonucleotides
PubMed: 10793682
DOI: 10.1093/nar/1.3.503 -
The EMBO Journal Jan 1993Histone H1 preferentially and cooperatively binds scaffold-associated regions (SARs) in vitro via specific interactions with the numerous short A + T-rich tracts...
Histone H1 preferentially and cooperatively binds scaffold-associated regions (SARs) in vitro via specific interactions with the numerous short A + T-rich tracts (A-tracts) contained in these sequences. Selective titration of A-tracts by the oligopeptide distamycin abolishes this interaction and results in a redistribution of H1. Similarly, treatment of intact cells and isolated nuclei with distamycin specifically enhances cleavage of internucleosomal linkers of SARs by topoisomerase II and restriction enzymes. The increased accessibility of these linkers is thought to result from the unfolding (or opening) of the chromatin fiber and to be due to a reduced occupancy by histone H1. Chromatin extraction and H1 assembly experiments support this view. We discuss a model whereby open, H1-depleted chromatin regions may be generated by titration of A-tracts by putative distamycin analogues; this local opening may spread to adjacent regions assuming highly cooperative H1-H1 interactions in chromatin.
Topics: Animals; Cell Fractionation; Cell Nucleus; Chromatin; Chromosomes, Human; DNA Restriction Enzymes; DNA Topoisomerases, Type II; Distamycins; Drosophila melanogaster; HeLa Cells; Histones; Humans; Metaphase; Restriction Mapping
PubMed: 8381347
DOI: 10.1002/j.1460-2075.1993.tb05637.x -
The EMBO Journal 1982The interconversion between the right (R) and left (L) helical forms of poly[d(G-C)] occurs at low concentrations of MgCl2 and EtOH, acting together in a highly...
The interconversion between the right (R) and left (L) helical forms of poly[d(G-C)] occurs at low concentrations of MgCl2 and EtOH, acting together in a highly synergistic manner. Thus, the cooperative R---L transition is induced by only 0.4 mM and 4 MM MgCl2 in combination with 20% and 10% EtOH, respectively. The L form of poly[d(G-C)] formed under these conditions has the spectroscopic properties (absorption, circular dichroism) previously demonstrated under high salt conditions (Pohl and Jovin, 1972) and thought to correspond to the left-handed Z DNA structures recently established by X-ray crystallography (Wang et al., 1979; Drew et al., 1980). However, L DNA formed in Mg2+-EtOH (which we designate as Z* DNA) has unique properties: a) it can be sedimented readily out of solution at low speed, indicative of condensation and intermolecular aggregation; b) it supports the binding of several intercalating (ethidium bromide, actinomycin D) and non-intercalating (mithramycin) drugs, although these interact preferentially with the R (i.e., B) form of DNA; and c) it functions as a template for Escherichia coli RNA polymerase. B and Z* DNAs can be generated under identical ionic conditions and compared in a number of biochemical systems. Our results suggest that left-handed DNA may form under physiological conditions and serve a biological function.
Topics: Crystallography; DNA; Dactinomycin; Distamycins; Ethanol; Ethidium; Magnesium; Magnesium Chloride; Plicamycin; Polydeoxyribonucleotides; Solutions; X-Rays
PubMed: 6232131
DOI: 10.1002/j.1460-2075.1982.tb01133.x -
Proceedings of the National Academy of... Sep 1993A number of DNA minor groove-binding ligands (MGBLs) are known to exhibit antitumor and antimicrobial activities. We show that DNA topoisomerase (Topo) I may be a...
A number of DNA minor groove-binding ligands (MGBLs) are known to exhibit antitumor and antimicrobial activities. We show that DNA topoisomerase (Topo) I may be a pharmacological target of MGBLs. In the presence of calf thymus Topo I, MGBLs induced limited but highly specific single-strand DNA breaks. The 3' ends of the broken DNA strands are covalently linked to Topo I polypeptides. Protein-linked DNA breaks are readily reversed by a brief heating to 65 degrees C or the addition of 0.5 M NaCl. These results suggest that MGBLs, like camptothecin, abort Topo I reactions by trapping reversible cleavable complexes. The sites of cleavage induced by MGBLs are distinctly different from those induced by camptothecin. Two of the major cleavage sites have been sequenced and shown to be highly A + T-rich, suggesting the possible involvement of a Topo I-drug-DNA ternary complex at the sites of cleavage. Different MGBLs also exhibit varying efficiency in inducing Topo I-cleavable complexes, and the order of efficiency is as follows: Hoechst 33342 and 33258 >> distamycin A > berenil > netropsin. The lack of correlation between DNA binding and cleavage efficiency suggest that, in addition to binding to the minor grooves of DNA, MGBLs must also interact with Topo I in trapping Topo I-cleavable complexes.
Topics: Animals; Base Sequence; Benzimidazoles; Bisbenzimidazole; Cattle; DNA; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; Diminazene; Distamycins; Indoles; Intercalating Agents; Kinetics; Ligands; Molecular Sequence Data; Oligodeoxyribonucleotides; Structure-Activity Relationship; Thymus Gland; Topoisomerase I Inhibitors
PubMed: 7690143
DOI: 10.1073/pnas.90.17.8131