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Biophysical Journal Aug 2006We here study the interactions of a polyamide with large DNA, and compare to those of minor groove binder distamycin (DST), including high ligand/DNA binding ratios....
We here study the interactions of a polyamide with large DNA, and compare to those of minor groove binder distamycin (DST), including high ligand/DNA binding ratios. Specific as well as nonspecific binding is probed using polarized-light spectroscopy combined with singular value decomposition analysis. Circular and linear dichroism data confirm binding geometries consistent with minor groove binding for both of the ligands. Interestingly, at high and intermediate ligand/DNA ratios the polyamide exhibits no significant sequence discrimination between mixed-sequence (calf thymus) and AT DNA as compared to DST. Each ligand is concluded to exhibit two different binding modes depending upon ligand/DNA ratio and nucleo-base sequence. At high binding ratios, distinct differences between the ligands are observed: circular dichroism spectra exciton effects provide evidence of bimolecular interactions of the polyamide when bound to AT-DNA, whereas no effects are seen with DST or mixed-sequence DNA. Also linear dichroism indicates that a change in binding geometry occurs at high polyamide/AT ratios, and that the effect occurs only with polyamide in contrast to DST. Since the effect is insignificant with DST, or with calf thymus DNA, it is concluded that it relates to the sizes of the ligands and the minor grooves, becoming critical in the limit of crowding.
Topics: Animals; Biophysics; Cattle; Circular Dichroism; DNA; Indicators and Reagents; Ligands; Models, Chemical; Nylons; Protein Binding; Spectrophotometry; Thymus Gland
PubMed: 16679369
DOI: 10.1529/biophysj.105.080564 -
Antimicrobial Agents and Chemotherapy Feb 2006The multidrug-resistant mutant Streptococcus pneumoniae M22 constitutively overexpresses two genes (patA and patB) that encode proteins homologous to known efflux...
The multidrug-resistant mutant Streptococcus pneumoniae M22 constitutively overexpresses two genes (patA and patB) that encode proteins homologous to known efflux proteins belonging to the ABC transporter family. It is shown here that PatA and PatB were strongly induced by quinolone antibiotics and distamycin in fluoroquinolone-sensitive strains. PatA was very important for growth of S. pneumoniae, and it could not be disrupted in strain M22. PatB appeared to control metabolic activity, particularly in amino acid biosynthesis, and it may have a pivotal role in coordination of the response to quinolone antibiotics. The induction of PatA and PatB by antibiotics showed a pattern similar to that exhibited by SP1861, a homologue of ABC-type transporters of choline and other osmoprotectants. A second group of quinolone-induced transporter genes comprising SP1587 and SP0287, which are homologues of, respectively, oxalate/formate antiporters and xanthine or uracil permeases belonging to the major facilitator family, showed a different pattern of induction by other antibiotics. There was no evidence for the involvement of PmrA, the putative proton-dependent multidrug transporter that has been implicated in norfloxacin resistance, in the response to quinolone antibiotics in either the resistant mutant or the fluoroquinolone-sensitive strains.
Topics: ATP-Binding Cassette Transporters; Anti-Infective Agents; Ciprofloxacin; Drug Resistance, Multiple, Bacterial; Fluoroquinolones; Microbial Sensitivity Tests; Mutation; Norfloxacin; Phenotype; RNA, Messenger; Reserpine; Streptococcus pneumoniae
PubMed: 16436727
DOI: 10.1128/AAC.50.2.685-693.2006 -
Nucleic Acids Research 2006We previously reported the chemical synthesis of oligonucleotides containing thymine glycol, a major form of oxidative DNA damage. In the preparation of the...
We previously reported the chemical synthesis of oligonucleotides containing thymine glycol, a major form of oxidative DNA damage. In the preparation of the phosphoramidite building block, the predominant product of the osmium tetroxide oxidation of protected thymidine was (5R,6S)-thymidine glycol. To obtain the building block of the other isomer, (5S,6R)-thymidine glycol, in an amount sufficient for oligonucleotide synthesis, the Sharpless asymmetric dihydroxylation (AD) reaction was examined. Although the reaction was very slow, (5S,6R)-thymidine glycol was obtained in preference to the (5R,6S) isomer. The ratio of (5S,6R)- and (5R,6S)-thymidine glycols was 2:1, and a trans isomer was also formed. When an ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate, was used as a co-solvent, the reaction became faster, and the yield was improved without changing the preference. The phosphoramidite building block of (5S,6R)-thymidine glycol was prepared, and oligonucleotides containing 5S-thymine glycol were synthesized. One of the oligonucleotides was used to analyze the binding of distamycin A to thymine glycol-containing DNA by Circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR) measurements. Distamycin A bound to a duplex containing either isomer of thymine glycol within the AATT target site, and its binding was observed even when the thymine glycol was placed opposite cytosine.
Topics: Anti-Bacterial Agents; Binding Sites; Circular Dichroism; DNA; DNA Damage; Distamycins; Hydroxylation; Imidazoles; Isomerism; Oligodeoxyribonucleotides; Solvents; Surface Plasmon Resonance; Thymidine; Thymine
PubMed: 16401612
DOI: 10.1093/nar/gkj443 -
Nucleic Acids Research 2005Copper-phenanthroline complexes oxidatively damage and cleave nucleic acids. Copper bis-phenanthroline and copper complexes of mono- and bis-phenanthroline conjugates...
Copper-phenanthroline complexes oxidatively damage and cleave nucleic acids. Copper bis-phenanthroline and copper complexes of mono- and bis-phenanthroline conjugates are used as research tools for studying nucleic acid structure and binding interactions. The mechanism of DNA oxidation and cleavage by these complexes was examined using two copper-phenanthroline conjugates of the sequence-specific binding molecule, distamycin. The complexes contained either one or two phenanthroline units that were bonded to the DNA-binding domain through a linker via the 3-position of the copper ligand. A duplex containing independently generated 2-deoxyribonolactone facilitated kinetic analysis of DNA cleavage. Oxidation rate constants were highly dependent upon the ligand environment but rate constants describing elimination of the alkali-labile 2-deoxyribonolactone intermediate were not. Rate constants describing DNA cleavage induced by each molecule were 11-54 times larger than the respective oxidation rate constants. The experiments indicate that DNA cleavage resulting from beta-elimination of 2-deoxyribonolactone by copper-phenanthroline complexes is a general mechanism utilized by this family of molecules. In addition, the experiments confirm that DNA damage mediated by mono- and bis-phenanthroline copper complexes proceeds through distinct species, albeit with similar outcomes.
Topics: Binding Sites; DNA; DNA Damage; Kinetics; Ligands; Nucleic Acid Conformation; Oxidation-Reduction; Phenanthrolines; Sugar Acids
PubMed: 16186134
DOI: 10.1093/nar/gki856 -
Nucleic Acids Research 2005Molecular dynamics simulations have been performed on netropsin in two different charge states and on distamycin binding to the minor groove of the DNA duplex...
Molecular dynamics simulations have been performed on netropsin in two different charge states and on distamycin binding to the minor groove of the DNA duplex d(CGCGAAAAACGCG).d(CGCGTTTTTCGCG). The relative free energy of binding of the two non-covalently interacting ligands was calculated using the thermodynamic integration method and reflects the experimental result. From 2 ns simulations of the ligands free in solution and when bound to DNA, the mobility and the hydrogen-bonding patterns of the ligands were studied, as well as their hydration. It is shown that even though distamycin is less hydrated than netropsin, the loss of ligand-solvent interactions is very similar for both ligands. The relative mobilities of the ligands in their bound and free forms indicate a larger entropic penalty for distamycin when binding to the minor groove compared with netropsin, partially explaining the lower binding affinity of the distamycin molecule. The detailed structural and energetic insights obtained from the molecular dynamics simulations allow for a better understanding of the factors determining ligand-DNA binding.
Topics: Anti-Bacterial Agents; Base Pairing; Base Sequence; Binding Sites; Computer Simulation; DNA; Distamycins; Hydrogen Bonding; Mathematical Computing; Netropsin; Oligodeoxyribonucleotides; Poly A; Thermodynamics
PubMed: 15687382
DOI: 10.1093/nar/gki195 -
Annals of Botany Jan 2005Selaginella is the largest genus of heterosporous pteridophytes, but karyologically the genus is known only by the occurrence of a dysploid series of n=7-12, and a low...
BACKGROUND AND AIMS
Selaginella is the largest genus of heterosporous pteridophytes, but karyologically the genus is known only by the occurrence of a dysploid series of n=7-12, and a low frequency of polyploids. Aiming to contribute to a better understanding of the structural chromosomal variability of this genus, different staining methods were applied in species with different chromosome numbers.
METHODS
The chromosome complements of seven species of Selaginella were analysed and, in four of them, the distribution of 45S rDNA sites was determined by fluorescent in situ hybridization. Additionally, CMA/DA/DAPI and silver nitrate staining were performed to investigate the correlation between the 45S rDNA sites, the heterochromatic bands and the number of active rDNA sites.
KEY RESULTS
The chromosome numbers observed were 2n=18, 20 and 24. The species with 2n=20 exhibited chromosome complement sizes smaller and less variable than those with 2n=18. The only species with 2n=24, S. convoluta, had relatively large and asymmetrical chromosomes. The interphase nuclei in all species were of the chromocentric type. CMA/DA/DAPI staining showed only a weak chromosomal differentiation of heterochromatic bands. In S. willdenowii and S. convoluta eight and six CMA+ bands were observed, respectively, but no DAPI+ bands. The CMA+ bands corresponded in number, size and location to the rDNA sites. In general, the number of rDNA sites correlated with the maximum number of nucleoli per nucleus. Ten rDNA sites were found in S. plana (2n=20), eight in S. willdenowii (2n=18), six in S. convoluta (2n=24) and two in S. producta (2n=20).
CONCLUSIONS
The remarkable variation in chromosome size and number and rDNA sites shows that dramatic karyological changes have occurred during the evolution of the genus at the diploid level. These data further suggest that the two putative basic numbers of the genus, x=9 and x=10, may have arisen two or more times independently.
Topics: Cell Nucleolus; Chromomycins; Chromosomes, Plant; Coloring Agents; DNA, Plant; DNA, Ribosomal; Distamycins; Evolution, Molecular; Fluorescent Dyes; Indoles; Selaginellaceae; Species Specificity
PubMed: 15567808
DOI: 10.1093/aob/mci022 -
Nucleic Acids Research 2004Histone H1 preferentially binds and aggregates scaffold-associated regions (SARs) via the numerous homopolymeric oligo(dA).oligo(dT) tracts present within these...
Histone H1 preferentially binds and aggregates scaffold-associated regions (SARs) via the numerous homopolymeric oligo(dA).oligo(dT) tracts present within these sequences. Here we show that the mammalian somatic subtypes H1a,b,c,d,e and H1 degrees and the male germline-specific subtype H1t, all preferentially bind to the Drosophila histone SAR. Experiments with the isolated domains show that whilst the C-terminal domain maintains strong and preferential binding, the N-terminal and globular domains show weak binding and poor specificity for the SAR. The preferential binding of SAR by the H1 molecule thus appears to be determined by its highly basic C-terminal domain. Salmine, a typical fish protamine, which could have its evolutionary origin in histone H1, also shows preferential binding to the SAR. The interaction of distamycin, a minor groove binder with high affinity for homopolymeric oligo(dA).oligo(dT) tracts, abolishes preferential binding of the C-terminal domain of histone H1 and protamine to the SAR, suggesting the involvement of the DNA minor groove in the interaction.
Topics: Amino Acid Sequence; Animals; Binding Sites; DNA; Distamycins; Histones; Mice; Molecular Sequence Data; Protein Binding; Protein Structure, Tertiary; Salmine
PubMed: 15562002
DOI: 10.1093/nar/gkh945 -
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 Sep 2004We have previously described a 3.5-Mb domain of enhance scaffold/matrix attachment region (S/MAR) at a human neocentromere, and normal expression of underlying genes...
We have previously described a 3.5-Mb domain of enhance scaffold/matrix attachment region (S/MAR) at a human neocentromere, and normal expression of underlying genes within this region. We also reported that partial inhibition of histone deacetylation using 33 nmtrichostatin A (TSA) resulted in a shift in the position of the CENP-A-binding domain within the neocentromere, with no noticeable effects on mitotic segregation function. In this study, 33 nM TSA caused a reduction in the size of the enhanced S/MAR domain of one-half to 1.7 Mb. Treatment with a DNA-intercalating drug distamycin A (DST) at 75 microg/ml resulted in a size reduction of the enhanced S/MAR domain at the neocentromere of two-thirds to 1.2 Mb, and that of the CENP-A-binding domain of 40%, from 330 to 196 kb, with no significant shift in the position of the latter domain. Other DST effects include mitotic chromosomal missegregation, reduction in the levels of Topo IIalpha, CENP-A, CENP-C, and HP1alpha, and an increase in mitotic checkpoint protein BubR1. TSA or DST treatment similarly resulted in a significant reduction, by approximately 20 and 50%, respectively, in the size of the enhanced S/MAR domain at the alpha-satellite DNA of a native chromosome 10 centromere. Transcriptional competence within the neocentromere is overall not noticeably altered by either TSA or DST treatment, as is evident from the absence of any significant increase or decrease in the expression levels of 47 underlying genes tested. These results suggest that a substantial contraction of the S/MAR domain may not be deleterious to centromere function, that disruption of the S/MAR domain directly affects the binding properties of a host of scaffold/matrix and centromeric/pericentric proteins, and that the overall competence and regulation of transcription at the neocentromeric chromatin is similar to those found at the corresponding normal genomic sites.
Topics: Animals; Antigens, Neoplasm; Autoantigens; CHO Cells; Centromere; Centromere Protein A; Chromobox Protein Homolog 5; Chromosomal Proteins, Non-Histone; Cricetinae; DNA Topoisomerases, Type II; DNA-Binding Proteins; Gene Expression; Humans; Hybrid Cells; Hydroxamic Acids; In Situ Hybridization, Fluorescence
PubMed: 15220334
DOI: 10.1074/jbc.M401051200 -
Journal of Virology Feb 2004Distamycin A has been described as an inhibitor of the cellular pathogenesis of vaccinia virus in culture. Distamycin is an antibiotic that specifically targets the...
Distamycin A has been described as an inhibitor of the cellular pathogenesis of vaccinia virus in culture. Distamycin is an antibiotic that specifically targets the minor groove of DNA. We show here that distamycin is a potent inhibitor of vaccinia virus replication. Pulse-labeling experiments showed that most major late proteins failed to accumulate in the presence of the antibiotic. We characterized the effect of distamycin on vaccinia virus nucleic acid biosynthesis with the goal of determining the inhibitor's target. Early gene transcription was unaffected. DNA synthesis proceeded at normal rates, but DNA accumulated in large masses in the cytoplasm with no evidence of virion assembly. Transcription from the intermediate class promoter for the I1L gene was partially reduced by distamycin; however, transcription from the intermediate promoters for the three late transcription factor genes was severely inhibited. The accumulation of the late transcripts for the viral F17R and A10L genes also was severely impaired and was shown to be a direct inhibition of late promoter activity. These results indicate that inhibition of postreplicative intermediate and late transcription is the basis for inhibition of vaccinia virus by distamycin and indicate that DNA minor-groove ligands hold promise for effective anti-poxvirus drugs.
Topics: Antiviral Agents; Cell Line; DNA, Viral; Distamycins; HeLa Cells; Humans; Ligands; Promoter Regions, Genetic; RNA, Messenger; RNA, Viral; Transcription, Genetic; Vaccinia virus; Virus Replication
PubMed: 14747579
DOI: 10.1128/jvi.78.4.2137-2141.2004