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Nucleic Acids Research Jan 2004Sequence-specific binding in the minor groove of DNA by small molecules is a growing area of research with possible therapeutic relevance. By selectively binding to DNA...
Sequence-specific binding in the minor groove of DNA by small molecules is a growing area of research with possible therapeutic relevance. By selectively binding to DNA sequences required by critical transcription factors, these small molecules could potentially modulate the expression levels of disease-causing genes. Precise targeting of a critical transcription factor of a selected gene requires an understanding of the preferred sequence of the DNA binding compound. As new compounds are being synthesized, there is a need to evaluate their DNA recognition profile. We sought to establish a procedure to determine sequence preference of compounds with previously unknown binding properties. A novel procedure for determining the optimal DNA binding sequence of minor groove binding compounds is described here. The assay also allows for determination of the binding affinity to a particular sequence.
Topics: Base Sequence; Binding Sites; DNA; DNA Footprinting; DNA Glycosylases; Deoxyribonuclease I; Distamycins; Ligands; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Reproducibility of Results; Substrate Specificity; Uracil-DNA Glycosidase
PubMed: 14718553
DOI: 10.1093/nar/gng155 -
British Journal of Cancer Oct 2003Defects in DNA mismatch repair (MMR) are associated with a predisposition to tumorigenesis and with drug resistance owing to high mutation rates and failure to engage... (Comparative Study)
Comparative Study
Defects in DNA mismatch repair (MMR) are associated with a predisposition to tumorigenesis and with drug resistance owing to high mutation rates and failure to engage DNA-damage-induced apoptosis. DNA minor groove binders (MGBs) are a class of anticancer agents highly effective in a variety of human cancers. Owing to their mode of action, DNA MGB-induced DNA damage may be a substrate for DNA MMR. This study was aimed at investigating the effect of loss of MMR on the sensitivity to brostallicin (PNU-166196), a novel synthetic alpha-bromoacrylic, second-generation DNA MGB currently in Phase II clinical trials and structurally related to distamycin A. Brostallicin activity was compared to a benzoyl mustard derivative of distamycin A (tallimustine). We report that the sensitivities of MLH1-deficient and -proficient HCT116 human colon carcinoma cells were comparable after treatment with brostallicin, while tallimustine resulted in a three times lower cytotoxicity in MLH1-deficient than in -proficient cells. MSH2-deficient HEC59 parental endometrial adenocarcinoma cells were as sensitive as the proficient HEC59+ch2 cells after brostallicin treatment, but were 1.8-fold resistant after tallimustine treatment as compared to the MSH2-proficient HEC59+ch2 counterpart. In addition, p53-deficient mouse fibroblasts lacking PMS2 were as sensitive to brostallicin as PMS2-proficient cells, but were 1.6-fold resistant to tallimustine. Loss of neither ATM nor DNA-PK affected sensitivity to brostallicin in p53-deficient mouse embryonic fibroblasts, indicating that brostallicin-induced cytotoxicity in a p53-deficient genetic background does not seem to require these kinases. These data show that, unlike other DNA MGBs, MMR-deficient cells retain their sensitivity to this new alpha-bromoacrylic derivative, indicating that brostallicin-induced cytotoxicity does not depend on functional DNA MMR. Since DNA MMR deficiency is common in numerous types of tumours, brostallicin potentially offers the advantage of being effective against MMR-defective tumours that are refractory to several anticancer agents.
Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Animals; Antineoplastic Agents; Base Pair Mismatch; Carrier Proteins; Cell Death; Colonic Neoplasms; DNA Repair; DNA-Binding Proteins; Distamycins; Drug Resistance, Neoplasm; Fibroblasts; Guanidines; Humans; Mice; MutL Protein Homolog 1; MutS Homolog 2 Protein; Neoplasm Proteins; Nitrogen Mustard Compounds; Nuclear Proteins; Proto-Oncogene Proteins; Pyrroles; Tumor Cells, Cultured; Tumor Suppressor Protein p53
PubMed: 14562032
DOI: 10.1038/sj.bjc.6601316 -
Molecular and Cellular Biology Oct 2003Fragile sites are specific loci that form gaps, constrictions, and breaks on chromosomes exposed to partial replication stress and are rearranged in tumors. Fragile...
Fragile sites are specific loci that form gaps, constrictions, and breaks on chromosomes exposed to partial replication stress and are rearranged in tumors. Fragile sites are classified as rare or common, depending on their induction and frequency within the population. The molecular basis of rare fragile sites is associated with expanded repeats capable of adopting unusual non-B DNA structures that can perturb DNA replication. The molecular basis of common fragile sites was unknown. Fragile sites from R-bands are enriched in flexible sequences relative to nonfragile regions from the same chromosomal bands. Here we cloned FRA7E, a common fragile site mapped to a G-band, and revealed a significant difference between its flexibility and that of nonfragile regions mapped to G-bands, similar to the pattern found in R-bands. Thus, in the entire genome, flexible sequences might play a role in the mechanism of fragility. The flexible sequences are composed of interrupted runs of AT-dinucleotides, which have the potential to form secondary structures and hence can affect replication. These sequences show similarity to the AT-rich minisatellite repeats that underlie the fragility of the rare fragile sites FRA16B and FRA10B. We further demonstrate that the normal alleles of FRA16B and FRA10B span the same genomic regions as the common fragile sites FRA16C and FRA10E. Our results suggest that a shared molecular basis, conferred by sequences with a potential to form secondary structures that can perturb replication, may underlie the fragility of rare fragile sites harboring AT-rich minisatellite repeats and aphidicolin-induced common fragile sites.
Topics: Alleles; Antiviral Agents; Base Sequence; Bromodeoxyuridine; Cell Line, Transformed; Chromosome Banding; Chromosome Fragility; Chromosome Mapping; Cytogenetics; DNA; Databases, Genetic; Distamycins; Fibroblasts; Genome; Humans; In Situ Hybridization, Fluorescence; Minisatellite Repeats; Molecular Sequence Data; Nucleic Acid Conformation; Phylogeny; Physical Chromosome Mapping; Polymerase Chain Reaction; Software
PubMed: 14517285
DOI: 10.1128/MCB.23.20.7143-7151.2003 -
Biophysical Journal Sep 2003Changes in the elastic properties of single deoxyribonucleic acid (DNA) molecules in the presence of different DNA-binding agents are identified using atomic force...
Changes in the elastic properties of single deoxyribonucleic acid (DNA) molecules in the presence of different DNA-binding agents are identified using atomic force microscope single molecule force spectroscopy. We investigated the binding of poly(dG-dC) dsDNA with the minor groove binder distamycin A, two supposed major groove binders, an alpha-helical and a 3(10)-helical peptide, the intercalants daunomycin, ethidium bromide and YO, and the bis-intercalant YOYO. Characteristic mechanical fingerprints in the overstretching behavior of the studied single DNA-ligand complexes were observed allowing the distinction between different binding modes. Docking of ligands to the minor or major groove of DNA has the effect that the intramolecular B-S transition remains visible as a distinct plateau in the force-extension trace. By contrast, intercalation of small molecules into the double helix is characterized by the vanishing of the B-S plateau. These findings lead to the conclusion that atomic force microscope force spectroscopy can be regarded as a single molecule biosensor and is a potent tool for the characterization of binding motives of small ligands to DNA.
Topics: Amino Acid Motifs; Antibiotics, Antineoplastic; Antiviral Agents; Benzoxazoles; DNA; DNA-Binding Proteins; Daunorubicin; Distamycins; Ethidium; Intercalating Agents; Ligands; Microscopy, Atomic Force; Peptides; Protein Binding; Protein Structure, Secondary; Quinolinium Compounds
PubMed: 12944309
DOI: 10.1016/S0006-3495(03)74624-4 -
Nucleic Acids Research Sep 2003We have performed solid-state 31P-19F REDOR nuclear magnetic resonance (NMR) experiments to monitor changes in minor groove width of the oligonucleotide...
We have performed solid-state 31P-19F REDOR nuclear magnetic resonance (NMR) experiments to monitor changes in minor groove width of the oligonucleotide d(CGCAAA2'FUTGGC)*d(GCCAAT(pS)TT GCG) (A3T2) upon binding of the drug distamycin A at different stoichiometries. In the hydrated solid-state sample, the minor groove width for the unbound DNA, measured as the 2'FU7-pS19 inter-label distance, was 9.4 +/- 0.7 A, comparable to that found for similar A:T-rich DNAs. Binding of a single drug molecule is observed to cause a 2.4 A decrease in groove width. Subsequent addition of a second drug molecule results in a larger conformational change, expanding this minor groove width to 13.6 A, consistent with the results of a previous solution NMR study of the 2:1 complex. These 31P-19F REDOR results demonstrate the ability of solid-state NMR to measure distances of 7-14 A in DNA-drug complexes and provide the first example of a direct spectroscopic measurement of minor groove width in nucleic acids.
Topics: Binding Sites; DNA; Distamycins; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Structure; Nucleic Acid Conformation; Oligonucleotides
PubMed: 12930959
DOI: 10.1093/nar/gkg720 -
Nucleic Acids Research Jun 2003Distamycin binds the minor groove of duplex DNA at AT-rich regions and has been a valuable probe of protein interactions with double-stranded DNA. We find that...
Distamycin binds the minor groove of duplex DNA at AT-rich regions and has been a valuable probe of protein interactions with double-stranded DNA. We find that distamycin can also inhibit protein interactions with G-quadruplex (G4) DNA, a stable four-stranded structure in which the repeating unit is a G-quartet. Using NMR, we show that distamycin binds specifically to G4 DNA, stacking on the terminal G-quartets and contacting the flanking bases. These results demonstrate the utility of distamycin as a probe of G4 DNA-protein interactions and show that there are (at least) two distinct modes of protein-G4 DNA recognition which can be distinguished by sensitivity to distamycin.
Topics: Base Sequence; Binding Sites; Binding, Competitive; DNA; Distamycins; G-Quadruplexes; Guanine; Ligands; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Phosphoproteins; Protein Binding; Protein Structure, Tertiary; RNA-Binding Proteins; Repetitive Sequences, Nucleic Acid; Nucleolin
PubMed: 12771220
DOI: 10.1093/nar/gkg392 -
Nucleic Acids Research May 2003The interaction between DNA and a benzothiazole-quinoline cyanine dye with a trimethine bridge (TO-PRO-3) results in the formation of three noncovalent complexes.... (Comparative Study)
Comparative Study
The interaction between DNA and a benzothiazole-quinoline cyanine dye with a trimethine bridge (TO-PRO-3) results in the formation of three noncovalent complexes. Unbound TO-PRO-3 has an absorption maximum (lambda(max)) of 632 nm, while the bound dyes (with calf thymus DNA) have electronic transitions with lambda(max) = 514 nm (complex I), 584 nm (complex II) and 642 nm (complex III). The blue shifts in the electronic transitions and the bisignate shape of the circular dichroism bands indicate that TO-PRO-3 aggregates with DNA. Complex I has a high dye:base pair stoichiometry, which does not depend on base sequence or base modifications. The bound dyes exhibit strong interdye coupling, based on studies with a short oligonucleotide and on enhanced resonance scattering. From thermal dissociation studies, the complex is weakly associated with DNA. Studies with poly(dGdC)2 and poly(dIdC)2 and competitive binding with distamycin demonstrate that complex II is bound in the minor groove. This complex stabilizes the helix against dissociation. For complex III, the slightly red-shifted electronic transition and the stoichiometry are most consistent with intercalation. Using poly(dAdT)2, the complexes have the following dye mole fractions (X(dye)): X(dye) = 0.65 (complex I), 0.425 (complex II) and 0.34 (complex III).
Topics: Animals; Binding, Competitive; Carbocyanines; Cattle; Circular Dichroism; Coloring Agents; DNA; Kinetics; Ligands; Spectrophotometry; Thermodynamics; Thymus Gland
PubMed: 12736305
DOI: 10.1093/nar/gkg363 -
Molecular and Cellular Biology Apr 2003Most 5-methylcytosine in Neurospora crassa occurs in A:T-rich sequences high in TpA dinucleotides, hallmarks of repeat-induced point mutation. To investigate how such...
Most 5-methylcytosine in Neurospora crassa occurs in A:T-rich sequences high in TpA dinucleotides, hallmarks of repeat-induced point mutation. To investigate how such sequences induce methylation, we developed a sensitive in vivo system. Tests of various 25- to 100-bp synthetic DNA sequences revealed that both T and A residues were required on a given strand to induce appreciable methylation. Segments composed of (TAAA)(n) or (TTAA)(n) were the most potent signals; 25-mers induced robust methylation at the special test site, and a 75-mer induced methylation elsewhere. G:C base pairs inhibited methylation, and cytosines 5' of ApT dinucleotides were particularly inhibitory. Weak signals could be strengthened by extending their lengths. A:T tracts as short as two were found to cooperate to induce methylation. Distamycin, which, like the AT-hook DNA binding motif found in proteins such as mammalian HMG-I, binds to the minor groove of A:T-rich sequences, suppressed DNA methylation and gene silencing. We also found a correlation between the strength of methylation signals and their binding to an AT-hook protein (HMG-I) and to activities in a Neurospora extract. We propose that de novo DNA methylation in Neurospora cells is triggered by cooperative recognition of the minor groove of multiple short A:T tracts. Similarities between sequences subjected to repeat-induced point mutation in Neurospora crassa and A:T-rich repeated sequences in heterochromatin in other organisms suggest that related mechanisms control silent chromatin in fungi, plants, and animals.
Topics: AT Rich Sequence; AT-Hook Motifs; Base Pairing; Base Sequence; Biological Assay; Cytosine; DNA Methylation; DNA, Fungal; DNA, Recombinant; Electrophoretic Mobility Shift Assay; HMGB1 Protein; Molecular Sequence Data; Neurospora crassa; Point Mutation; Repetitive Sequences, Nucleic Acid; Signal Transduction; Structure-Activity Relationship
PubMed: 12640122
DOI: 10.1128/MCB.23.7.2379-2394.2003 -
Hereditas 2002We analyzed patterns of heterochromatic bands in the Neotropical stingless bee genus Melipona (Hymenoptera, Meliponini). Group I species (Melipona bicolor bicolor,...
We analyzed patterns of heterochromatic bands in the Neotropical stingless bee genus Melipona (Hymenoptera, Meliponini). Group I species (Melipona bicolor bicolor, Melipona quadrifasciata, Melipona asilvae, Melipona marginata, Melipona subnitida) were characterized by low heterochromatic content. Group II species (Melipona capixaba, Melipona compressipes, Melipona crinita, Melipona seminigra fuscopilosa e Melipona scutellaris) had high heterochromatic content. All species had 2n = 18 and n = 9. In species of Group I heterochromatin was pericentromeric and located on the short arm of acrocentric chromosomes, while in Group II species heterochromatin was distributed along most of the chromosome length. The most effective sequential staining was quinacrine mustard (QM)/distamycin (DA)/chromomycin A3(CMA3)/4-6-diamidino-2-phenylindole (DAPI). Heterochromatic and euchromatic bands varied extensively within Group I. In Group II species euchromatin was restricted to the chromosome tips and it was uniformly GC+. Patterns of restriction enzymes (EcoRI, DraI, HindIII) showed that heterochromatin was heterogeneous. In all species the first pair of homologues was of unequal size and showed heteromorphism of a GC+ pericentromeric heterochromatin. In M. asilvae (Group I) this pair bore NOR and in M. compressipes (Group II) it hybridized with a rDNA FISH probe. As for Group I species the second pair was AT+ in M. subnitida and neutral for AT and GC in the remaining species of this group. Outgroup comparison indicates that high levels of heterochromatin represent a derived condition within Melipona. The pattern of karyotypic evolution sets Melipona in an isolated position within the Meliponini.
Topics: Animals; Bees; Biological Evolution; Heterochromatin; Karyotyping; Phylogeny; Sequence Analysis, DNA
PubMed: 12184485
DOI: 10.1034/j.1601-5223.2002.1360104.x -
Virology Jul 2002The Tat protein of the human immunodeficiency virus type 1 promotes survival and growth and inhibits apoptosis of different cell types. These effects of Tat are... (Comparative Study)
Comparative Study
The Tat protein of the human immunodeficiency virus type 1 promotes survival and growth and inhibits apoptosis of different cell types. These effects of Tat are attributed to the induction of bcl-2 gene expression. In this study we show that the blocking of both intracellular and extracellular Tat correlates with a decrease of bcl-2 transcripts, leading in vitro to a lower growth rate and attenuation of the transformed phenotype and in vivo to a reduced angiogenic and oncogenic activity of Tat-expressing cells. These results support the notion that bcl-2 is an effector of Tat-induced angiogenesis and oncogenesis and indicate that the blocking of Tat functions by immunoprophylactic, pharmacological, and gene therapy approaches may help to control oncogenesis during AIDS.
Topics: Animals; Antibodies; Antisense Elements (Genetics); Apoptosis; Carcinogenicity Tests; Distamycins; Down-Regulation; Gene Expression Regulation, Viral; Gene Products, tat; HIV-1; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, Transgenic; Neovascularization, Pathologic; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Tumor Cells, Cultured; tat Gene Products, Human Immunodeficiency Virus
PubMed: 12167335
DOI: 10.1006/viro.2002.1459