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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 -
Nucleic Acids Research Jul 1988Two self complementary oligonucleotides, T(GC)4AT(GC)4ACATG and C(GC)2(AT)5 (GC)3ATG, were synthesized and cloned into plasmids. Negative supercoiling causes a...
Two self complementary oligonucleotides, T(GC)4AT(GC)4ACATG and C(GC)2(AT)5 (GC)3ATG, were synthesized and cloned into plasmids. Negative supercoiling causes a structural transition in the primary helix of both inserts. The first sequence converts into the left-handed helix, whereas the second sequence undergoes a transition into a cruciform or a Z-type structure depending on the experimental conditions employed. This has been deduced from the mapping of S1 nuclease sensitive sites, OsO4-sensitive sites, DEP modification pattern and relaxation studies. In addition, the differential effect of 5-cytosine methylation and binding of the AT-specific drug distamycin on these transitions further supports this interpretation. Thus, it is demonstrated, that the same sequence which is both inverted repeat and alternating purine-pyrimidine type may adopt either the left-handed conformation or the cruciform structure in response to the superhelical stress. Formation of the Z-type helix can be transmitted through the d(AT)n region which is 10 bp in length.
Topics: 5-Methylcytosine; Cytosine; DNA; DNA, Superhelical; Distamycins; Hydrogen Bonding; Methylation; Nucleic Acid Conformation; Plasmids; Repetitive Sequences, Nucleic Acid
PubMed: 3405754
DOI: 10.1093/nar/16.14.6915 -
Proceedings of the National Academy of... May 1990Dynemicin is a hybrid containing anthraquinone and enediyne cores, which contribute to binding and cleavage of DNA, respectively. DNA strand scission by the antitumor...
Dynemicin is a hybrid containing anthraquinone and enediyne cores, which contribute to binding and cleavage of DNA, respectively. DNA strand scission by the antitumor antibiotic is significantly enhanced by the addition of NADPH or thiol compounds. The preferential cutting site of dynemicin is on the 3' side of purine bases (i.e., 5'-GC, -GT, and -AG) and is clearly different from the cutting sites of esperamicin and calicheamicin. The double-stranded and the stem regions of single-stranded DNAs are preferentially cleaved by dynemicin. Therefore, dynemicin may be a useful reagent for probing secondary structures of DNA. Pretreatment of DNA with Adriamycin and actinomycin D alters the cutting mode of dynemicin. Dynemicin-mediated DNA breakage is strongly inhibited by pretreatment of the DNA with distamycin A and anthramycin, suggesting that dynemicin interacts with the minor groove of the DNA helix. Intercalation of the anthraquinone core into the DNA followed by the attack of the phenyl diradical formed from the enediyne core is considered as a possible mechanism of action of dynemicin.
Topics: Anthraquinones; Antibiotics, Antineoplastic; Base Sequence; DNA; Enediynes; Molecular Sequence Data; Nucleic Acid Conformation; Plasmids; Restriction Mapping
PubMed: 2339123
DOI: 10.1073/pnas.87.10.3831 -
Nucleic Acids Research Sep 1995We have examined the interaction of distamycin, netropsin, Hoechst 33258 and berenil, which are AT-selective minor groove-binding ligands, with synthetic DNA fragments...
We have examined the interaction of distamycin, netropsin, Hoechst 33258 and berenil, which are AT-selective minor groove-binding ligands, with synthetic DNA fragments containing different arrangements of AT base pairs by DNase I footprinting. For fragments which contain multiple blocks of (A/T)4 quantitative DNase I footprinting reveals that AATT and AAAA are much better binding sites than TTAA and TATA. Hoechst 33258 shows that greatest discrimination between these sites with a 50-fold difference in affinity between AATT and TATA. Alone amongst these ligands, Hoechst 33258 binds to AATT better than AAAA. These differences in binding to the various AT-tracts are interpreted in terms of variations in DNA minor groove width and suggest that TpA steps within an AT-tract decrease the affinity of these ligands. The behaviour of each site also depends on the flanking sequences; adjacent pyrimidine-purine steps cause a decrease in affinity. The precise ranking order for the various binding sites is not the same for each ligand.
Topics: Base Composition; Base Sequence; Binding Sites; Bisbenzimidazole; DNA; Diminazene; Distamycins; Ligands; Molecular Sequence Data; Netropsin; Nucleic Acid Conformation; Structure-Activity Relationship
PubMed: 7567447
DOI: 10.1093/nar/23.17.3385 -
Proceedings of the National Academy of... Aug 1994We report here an effort to use molecular dynamics/free energy perturbation methodology to calculate relative binding affinities of two related drugs to DNA.... (Comparative Study)
Comparative Study
We report here an effort to use molecular dynamics/free energy perturbation methodology to calculate relative binding affinities of two related drugs to DNA. Specifically, we focus on the relative binding free energies of distamycin (Dst) and its analog, 2-imidazoledistamycin (2-ImD), to d(CGCAAGTTGGC).d(GCCAACTTGCG). The pyrrole (Dst) and the imidazole variant (2-ImD) differ only in that the C-H is substituted by an N in the central ring. The starting conformation for these calculations was the previously determined solution structure of two 2-ImD molecules in the minor groove of the above 11-residue DNA. In this complex both the ligands have the imidazole nitrogen (N3) oriented toward the amino group of G6. However only ligand 1 (site I) has N3 within the hydrogen bonding distance from N2 amino proton of G6. We have calculated the difference in free energy of binding of 2-ImD versus Dst in three different cases by mutating 2-ImD-->Dst reversibly. In the first case ligand 1 (site I) is mutated, in the second case ligand 2 (site II) is mutated, and in the third case both the ligands are mutated. These calculations show that at site I Dst has weaker binding affinity than 2-ImD by 0.7 kcal/mol, at site II Dst has stronger binding affinity than 2-ImD by 2.9 kcal/mol, and when occupying both site I and site II, Dst binds with greater affinity than 2-ImD by 1.8 kcal/mol. Recent experimental findings agree semiquantitatively (within 1 kcal/mol) with the calculations presented here. Hence the methodology presented here can be used to predict relative binding energies of two or more closely related molecules to DNA.
Topics: Base Sequence; Computer Simulation; DNA; Distamycins; Models, Chemical; Models, Molecular; Molecular Conformation; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides
PubMed: 8052641
DOI: 10.1073/pnas.91.16.7673 -
Annals of Oncology : Official Journal... Dec 1994Tallimustine, a benzoyl nitrogen mustard derivative of the antiviral agent distamycin A, is a new alkylating agent which binds to A-T rich regions of DNA in the minor... (Clinical Trial)
Clinical Trial
BACKGROUND
Tallimustine, a benzoyl nitrogen mustard derivative of the antiviral agent distamycin A, is a new alkylating agent which binds to A-T rich regions of DNA in the minor groove producing highly sequence-specific alkylations. Its main preclinical features are a significant antitumor activity in animal models and a lack of cross-resistance in vitro and in vivo with L-PAM. Myelotoxicity was dose-limiting in animals, with a more than 100-fold difference in bone marrow sensitivity between mice and dogs.
PATIENTS AND METHODS
Forty adult patients (pts) with solid malignancies were entered in the study. The drug was administered as an IV bolus every 4 weeks. CBC was repeated twice a week and serial assessments of renal function were performed in the week following the first cycle. From the starting dose of 50 micrograms/m2, corresponding to 1/3 of the highest non-toxic dose (HNTD) in dogs, there were increases through 10 dose levels, with reliance only on the features of the myelotoxicity observed.
RESULTS
The main toxic effect was neutropenia which was dose-limiting, selective and short-lasting. Only previously-untreated pts received doses of 750 micrograms/m2 or more, with grade 4 neutropenia occurring in > or = 75% of the cycles. The maximally tolerable dose (MTD) was defined as 1250 micrograms/m2, with 3 of 3 pts developing febrile neutropenia requiring IV antibiotics. A platelet count of < 100 x 10(3)/microliters was observed in only one pt. Bone marrow aspiration performed in selected pts on days 8 and 15 confirmed a highly selective impairment by tallimustine of the myeloid lineage, with rapid recovery of the proliferative compartment. Pharmacokinetic studies performed at 1000 micrograms/m2 and 1250 micrograms/m2 showed a rapid fall of the plasma levels within the first 2 hours with drug concentrations between 100 ng/ml and 400 ng/ml within the first hour. A partial response of 4 months' duration was reported in one previously-untreated pt with cutaneous recurrences of malignant mesothelioma.
CONCLUSIONS
The report of some antitumour efficacy, the high selectivity of neutropenia, the lack of significant non-hematological toxic effects and the occurrence of detectable but still low plasma drug concentrations suggest that further clinical evaluation of higher doses of tallimustine in combination with colony-stimulating factors would be justified.
Topics: Adult; Aged; Antineoplastic Agents; Distamycins; Female; Humans; Male; Middle Aged; Neoplasms; Neutropenia; Nitrogen Mustard Compounds; Remission Induction
PubMed: 7696161
DOI: 10.1093/oxfordjournals.annonc.a058728 -
Nucleic Acids Research Aug 1977The specific interaction of distamycin A and analogs with DNA's and synthetic deoxypolynucleotide duplexes were studied in detail by means of circular dichroism and the...
The specific interaction of distamycin A and analogs with DNA's and synthetic deoxypolynucleotide duplexes were studied in detail by means of circular dichroism and the data were analyzed together with viscosity results of several natural DNA's. At low ligand to nucleotide ratio the previously reported specific binding to (A-T) pairs of DNA is verified by a highly favoured interaction with (A-T)-enriched segments of distamycins containing four and five methylpyrrole carboxamide units. At higher distamycin concentration a second specific binding to (G-C) pairs most probably through hydrogen bonding is established. Viscometric results suggest a distamycin-induced local bending of the helix and could support the idea of a preferential alignment of the ligand molecule along only one strand in the groove which differs from the netropsin interaction mechanism. The possibility of an overlapping binding of the oligopeptides in the small groove is discussed.
Topics: Animals; Cattle; Chemical Phenomena; Chemistry; Circular Dichroism; DNA; DNA, Bacterial; Distamycins; Molecular Weight; Nucleic Acid Conformation; Polydeoxyribonucleotides; Pyrroles; Structure-Activity Relationship; Thymus Gland; Viscosity
PubMed: 561949
DOI: 10.1093/nar/4.8.2655 -
European Journal of Biochemistry Jun 1989It is demonstrated that DNA photofootprinting analysis of the intercalating depsipeptide echinomycin, and the minor groove-binders distamicyn,...
It is demonstrated that DNA photofootprinting analysis of the intercalating depsipeptide echinomycin, and the minor groove-binders distamicyn, 4',6-diamidino-2-phenylindole (DAPI) and Hoechst 33258 can be performed using 9-[6-(2-diazocyclopentadienylcarbonyloxy)hexylamino]acridine (DHA) [Nielsen et al. (1988) Nucleic Acids Res. 16, 3877-3888] or 2-methoxy-6-azido-9-aminoacridine (MAA) [Jeppesen et al. (1988) Nucleic Acids Res. 16, 5755-5770]. Both the extent of the drug-binding sites and their relative strength can be determined with either reagent. DNA has the advantage of giving virtually sequence-uniform DNA photocleavage. On the other hand, structural changes in the DNA are detected by MAA. Using the 232-base-pair EcoRI-PvuII pUC19 restriction fragment, it is found that cleavage protection by distamycin, DAPI and Hoechst 33258 all require an (A.T)4 sequence, whereas protection by echinomycin was confined to a G + C-rich 8-base-pair region.
Topics: Aminoacridines; Azides; Base Sequence; Bisbenzimidazole; DNA; DNA Damage; DNA Restriction Enzymes; Distamycins; Echinomycin; Indoles; Molecular Sequence Data; Molecular Structure; Plasmids; Receptors, Drug
PubMed: 2472274
DOI: 10.1111/j.1432-1033.1989.tb14850.x -
European Journal of Biochemistry Apr 1994We identify two high-affinity matrix-attachment regions (MAR elements) located in two introns of the human DNA topoisomerase I gene (TOP1). These intronic MAR elements,...
We identify two high-affinity matrix-attachment regions (MAR elements) located in two introns of the human DNA topoisomerase I gene (TOP1). These intronic MAR elements, designated MI and MII, are specifically bound by the nuclear matrix and partition with scaffolds in vitro. One of these MAR elements, MII, is part of a genomic region which is hypersensitive for endogenous nucleases. We have sequenced both DNA elements and have characterized their mode of binding to the nuclear matrix. Experiments with the minor-groove-binding ligands distamycin and chromomycin indicate that the A+T-rich regions, most likely homopolymeric A tracts, are responsible for binding of these DNA elements to the nuclear matrix. MII contains an alu-like element and a segment of curved DNA. Analysis of subfragments of MII show that the curved DNA region itself shows only weak nuclear-matrix binding, and that the high-affinity binding sites are located on subfragments on the 5' side of the curved DNA. In addition, we found that the alu-like sequence does not contribute significantly to the binding of MII and of subfragments of MII to nuclear-matrix proteins. Comparing the distribution of repetitive sequences in the cloned parts of human DNA topoisomerase I gene with the location of high-affinity matrix-binding sites we find no evidence that repetitive DNA may be located close to MAR elements as has been previously suggested.
Topics: Binding Sites; Chromomycins; DNA; DNA Topoisomerases, Type I; Deoxyribonuclease BamHI; Deoxyribonuclease I; Deoxyribonucleases, Type II Site-Specific; Distamycins; Humans; Introns; Molecular Sequence Data; Nuclear Matrix; Nucleic Acid Conformation; Repetitive Sequences, Nucleic Acid
PubMed: 8168528
DOI: 10.1111/j.1432-1033.1994.tb18753.x -
The Journal of Biological Chemistry May 2002Splice variants of the Nmp4 gene include nuclear matrix transcription factors that regulate the type I collagen alpha1(I) polypeptide chain (COL1A1) promoter and several...
Splice variants of the Nmp4 gene include nuclear matrix transcription factors that regulate the type I collagen alpha1(I) polypeptide chain (COL1A1) promoter and several matrix metalloproteinase (MMP) genes. To date, these are the only Cys(2)His(2) zinc finger proteins known to bind within the minor groove of homopolymeric (dA.dT) DNA. Nmp4 isoforms contain from 5 to 8 Cys(2)His(2) zinc fingers, an SH3-binding domain that overlaps with a putative AT-hook and a polyglutamine-alanine repeat (poly(QA)). To determine the mechanistic significance of Cys(2)His(2) zinc finger association with this unusual consensus DNA binding element, we identified the Nmp4 DNA-binding and transcriptional activation domains. Zinc fingers 2, 3, and 6 mediated association with the homopolymeric (dA.dT) COL1A1/MMP DNA consensus element. The N terminus of the Nmp4 protein exhibited a strong trans-activation capacity when fused to the GAL4 DNA-binding domain, but this activity was masked within the context of the full-length Nmp4-GAL4 DNA-binding domain chimera. However, upon binding to the COL1A1/MMP homopolymeric (dA.dT) element, the native Nmp4 protein up-regulated transcription, and the poly(QA) domain acquired a significant role in trans-activation. We propose that allosteric effects induced upon zinc finger association with the homopolymeric (dA.dT) minor groove confer context-specific functionality to this unusual family of Cys(2)His(2) transcription factors.
Topics: Antigens, Nuclear; Binding, Competitive; Cell Line; Collagen Type I; DNA-Binding Proteins; Distamycins; Gene Expression Regulation; Humans; Kinetics; Matrix Metalloproteinases; Methyl Green; Nuclear Matrix; Nuclear Proteins; Promoter Regions, Genetic; Protein Isoforms; Recombinant Proteins; Transcription Factors; Transcriptional Activation; Transfection; Zinc Fingers
PubMed: 11867614
DOI: 10.1074/jbc.M107496200