scholarly journals The binding of echinomycin to deoxyribonucleic acid

1976 ◽  
Vol 157 (3) ◽  
pp. 721-740 ◽  
Author(s):  
L P G Wakelin ◽  
M J Waring
Keyword(s):  
Ionic Strength ◽  
Binding Constant ◽  
Buoyant Density ◽  
Specific Interaction ◽  
Effect Of Temperature ◽  
Heat Denaturation ◽  
Peptide Antibiotic ◽  
Base Pairs ◽  
Calf Thymus ◽  
Low Ionic Strength

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.

Characterization of genomic poly(dT-dG).poly(dC-dA) sequences: structure, organization, and conformation

1984 ◽  
Vol 4 (12) ◽  
pp. 2610-2621
Author(s):  
H Hamada ◽  
M G Petrino ◽  
T Kakunaga ◽  
M Seidman ◽  
B D Stollar
Keyword(s):  
Ionic Strength ◽  
Inverted Repeats ◽  
Actin Gene ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Coding Sequences ◽  
Supercoiled Plasmid ◽  
Marked Preference ◽  
Low Ionic Strength

Hybridization studies suggest the abundant presence of poly(dT-dG).poly(dC-dA) (TG-element), a potential Z-DNA sequence, in eucaryotic genomes. We have isolated and characterized TG-elements from different locations in the human genome: from randomly isolated clones, associated with the actin gene family, and linked to another repeated element. The results indicate that the following features are typical of these TG-elements: the elements consist of 20 to 60 base pairs of (dT-dG)n.(dC-dA)n, the sequences characterized in our study were not flanked by direct or inverted repeats, the sequences are interspersed rather than in satellite blocks, the elements are not usually associated with other repeated elements, and some of the elements are found near coding sequences or in introns. Studies on the conformation of a genomic TG-element in a supercoiled plasmid indicate several distinct properties of the TG-element: it is in the Z-form only at low ionic strength, S1 nuclease recognizes its Z-form with a marked preference for one of the B-Z junctions, and the sensitive region extends for 20 base pairs near the B-Z junction. In contrast to the result with the supercoiled plasmid, S1 nuclease failed to recognize the TG-element in minichromosomes.

scholarly journals Native Ion Mobility Mass Spectrometry: when Gas Phase Ion Structures Depend on the Electrospray Charging Process

2018 ◽  
Author(s):  
Nina A. Khristenko ◽  
Jussara Amato ◽  
Sandrine Livet ◽  
Bruno Pagano ◽  
Antonio Randazzo ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Gas Phase ◽  
Ion Mobility ◽  
Droplet Surface ◽  
Dependent Manner ◽  
Base Pairs ◽  
Wide Range ◽  
A Chain ◽  
Gas Phase Ion ◽  
Low Ionic Strength

Ion mobility spectrometry (IMS) has become popular to characterize biomolecule folding. Numerous studies have shown that proteins that are folded in solution remain folded in the gas phase, whereas proteins that are unfolded in solution adopt more extended conformations in the gas phase. Here, we discuss how general this tenet is. We studied single-stranded DNAs (human telomeric cytosine-rich sequences with CCCTAA repeats), which fold into an intercalated motif (i-motif) structure in a pH-dependent manner, thanks to the formation of C‒H+‒C base pairs. As i-motif formation is favored at low ionic strength, we could investigate the ESI-IMS-MS behavior of i-motif structures at pH ~5.5 over a wide range of ammonium acetate concentrations (15 mM to 100 mM). The control experiments consisted of either the same sequence at pH ~7.5, wherein the sequence is unfolded, or sequence variants that cannot form i-motifs (CTCTAA repeats). The surprising results came from the control experiments. We found that the ionic strength of the solution had a greater effect on the compactness of the gas-phase structures than the solution folding state. This means that electrosprayed ions keep a memory of the charging process, which is influenced by the electrolyte concentration. We discuss these results in light of the analyte partitioning between the droplet interior and droplet surface, which in turn influences the probability of being ionized via a charged residue pathway or a chain extrusion pathway.<br>

scholarly journals Native Ion Mobility Mass Spectrometry: when Gas Phase Ion Structures Depend on the Electrospray Charging Process

2019 ◽  
Author(s):  
Nina A. Khristenko ◽  
Jussara Amato ◽  
Sandrine Livet ◽  
Bruno Pagano ◽  
Antonio Randazzo ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Gas Phase ◽  
Ion Mobility ◽  
Droplet Surface ◽  
Dependent Manner ◽  
Base Pairs ◽  
Wide Range ◽  
A Chain ◽  
Gas Phase Ion ◽  
Low Ionic Strength

Ion mobility spectrometry (IMS) has become popular to characterize biomolecule folding. Numerous studies have shown that proteins that are folded in solution remain folded in the gas phase, whereas proteins that are unfolded in solution adopt more extended conformations in the gas phase. Here, we discuss how general this tenet is. We studied single-stranded DNAs (human telomeric cytosine-rich sequences with CCCTAA repeats), which fold into an intercalated motif (i-motif) structure in a pH-dependent manner, thanks to the formation of C‒H+‒C base pairs. As i-motif formation is favored at low ionic strength, we could investigate the ESI-IMS-MS behavior of i-motif structures at pH ~5.5 over a wide range of ammonium acetate concentrations (15 mM to 100 mM). The control experiments consisted of either the same sequence at pH ~7.5, wherein the sequence is unfolded, or sequence variants that cannot form i-motifs (CTCTAA repeats). The surprising results came from the control experiments. We found that the ionic strength of the solution had a greater effect on the compactness of the gas-phase structures than the solution folding state. This means that electrosprayed ions keep a memory of the charging process, which is influenced by the electrolyte concentration. We discuss these results in light of the analyte partitioning between the droplet interior and droplet surface, which in turn influences the probability of being ionized via a charged residue pathway or a chain extrusion pathway.<br>

On the structure of cellular and viral chromatin

1978 ◽  
Vol 283 (997) ◽  
pp. 275-285 ◽  
Keyword(s):  
Ionic Strength ◽  
Simian Virus 40 ◽  
Histone H1 ◽  
Staphylococcal Nuclease ◽  
Dna Fragments ◽  
Base Pairs ◽  
Nuclease Digestion ◽  
Dna Fragment ◽  
Histone H5 ◽  
Low Ionic Strength

Some of the recent experimental data obtained in our laboratory are briefly reviewed. 1. A mild staphylococcal nuclease digestion of either chromatin or nuclei from mouse Ehrlich tumour cells results in chromatin subunits (mononucleosomes) of three discrete kinds. The smallest mononucleosome (MN 1 contains all histones except H1 and a DNA fragment 140 base pairs long. The intermediate mononucleosome (MN 2 ) contains all five histones and a DNA fragment 170 base pairs long. The third mononucleosome (MN 3 ) also contains all five histones, but its associated DNA is longer and somewhat heterogeneous in size (180-200 base pairs). Most of the MN 3 particles are rapidly converted to mononucleosomes MN 2 and MN 1 by nuclease digestion. However, there exists a relatively nuclease-resistant subpopulation of the MN 3 mononucleosomes. These 200 base-pair MN 3 particles contain not only histones but also non-histone proteins and are significantly more resistant to nuclease then even the smaller mononucleosomes MN 1 and MN 2 . 2. Nuclease digestion of hen erythrocyte nuclei or chromatin, in which histone H1 is partially replaced by histone H5 produces the mononucleosomes MN 1 and two electrophoretically resolvable kinds of MN 2 mononucleosomes, one containing histone H1 and the other one histone H5. A relatively nuclease-resistant subset of the mononucleosomes MN 3 is preferentially accumulated at later stages of the digestion. 3. Although pancreatic DNase (DNase I) and spleen acid DNase (DNase II) attack the DNA in chromatin in a manner different from that of staphylococcal nuclease, the deoxyribonucleoprotein (DNP) products of digestion are similar for all three enzymes under identical solvent conditions, as revealed by gel electrophoresis of the DNP at low ionic strength. 4. There are eight major kinds of staphylococcal nuclease-produced soluble subnucleosomes (i.e. particles smaller than the mononucleosomes). In particular, the subnucleosome SN 1 is a set of naked double-stranded DNA fragments ca. 20 base pairs long. Subnucleosome SN 2 is a complex of a specific highly basic non-histone protein and a DNA fragment ca. 27 base pairs long. Subnucleosomes SN 7 and SN 8 each contain all of the histones except H1 and DNA fragments ca. 100 and 120 base pairs long, respectively. 5. Nuclease digestion of isolated mono- and dinucleosomes does not produce all of the subnucleosomes. These and related findings indicate that the cleavages required to generate these subnucleosomes result from some aspect of chromatin structure which is lost upon digestion to mono- or dinucleosomes. Nuclease digestion of isolated minichromosomes of Simian virus 40 (SV40) (which contain all five histones including H1) produces mononucleosomes MN 1 and MN 2 but does not produce some of the subnucleosomes or the relatively nuclease-resistant subset of the MN 3 mononucleosomes. 6. The rate of sedimentation of the SV40 minichromosomes ( ca. 60 S ) under ‘physiological’ ionic conditions ( μ ≈ 0.15) is about two times higher than that in a low ionic strength buffer ( μ ≈ 0.005). Occurrence of the compact state of the minichromosome critically depends upon the presence of histone H1 and can be irreversibly fixed by treatment with formaldehyde.

Thermodynamics of interaction of meso-tetra-(4N-oxyethylpyridyl) porphyrin and its Cu(II)- and Co(II)- containing derivatives with A and B forms of DNA

2017 ◽  
Vol 21 (11) ◽  
pp. 731-738
Author(s):  
Ani A. Avetisyan ◽  
Ishkhan V. Vardanyan ◽  
Yeva B. Dalyan
Keyword(s):  
Ionic Strength ◽  
Spectral Data ◽  
Binding Constant ◽  
Binding Mode ◽  
Water Soluble ◽  
Binding Mechanism ◽  
Groove Binding ◽  
Low Ionic Strength ◽  
Circular Dichroïsm ◽  
Groove Binding Mode

The interaction of water soluble meso-tetra-(4N-oxyethylpyridyl) porphyrin (TOEPyP4) and its Cu(II)- and Co(II)-containing derivatives (CuTOEPyP4 and CoTOEPyP4) with A and B forms of DNA at low ionic strength was studied via UV-vis spectrophotometry and Circular Dichroism. It is shown that the binding constant of TOEPyP4 and CuTOEPyP4 with A–DNA is two times larger than with B–DNA, and the binding constant of CoTOEPyP4 does not depend on the form of DNA. The thermodynamical analysis based on spectral data indicates the preferable entropic character of porphyrins binding with both forms of DNA. This result shows that at low ionic strength the external groove binding mode is a preferred binding mechanism of these porphyrins with both forms of DNA.

Nucleosomes are assembled into discrete size structures by histone H1 in vitro

1986 ◽  
Vol 64 (5) ◽  
pp. 463-473 ◽  
Author(s):  
Teni Boulikas
Keyword(s):  
Ionic Strength ◽  
Histone H1 ◽  
Higher Order ◽  
Third Order ◽  
Calf Thymus ◽  
Exogenous Histone ◽  
Structural Subunit ◽  
Chromatin Structural ◽  
Low Ionic Strength

The involvement of histone H1 in the formation and maintenance of higher order chromatin structures in vitro was investigated biochemically. Addition of exogenous histone H1 to isolated calf thymus mononucleosomes in low ionic strength buffer resulted in the formation of electrophoretically distinct mononucleosome assemblies (supernucleosomes). The smaller super-nucleosomes were composed of about 12, 18, 24, or 30 nucleosomes and one to two molecules of histone H1 per nucleosome. It was difficult to determine accurately the size of the larger supernucleosomes, but their bands from native gels contained probably between 60 and 300 nucleosomes or more. Similar supemucleosome size classes were also obtained when oligonucleosomes instead of mononucleosomes were employed. When the assembly of mono- and oligo-nucleosomes with histone H1 took place in 0.15 M NaCl, discrete supernucleosomes containing only mono- or di-nucleosomes, but not a mixture of both, were formed. It is proposed that the small supernucleosomes containing oligomers of 6 nucleosomes may represent integral multiples of the second-order chromatin structural subunit, whereas the larger supernucleosomes containing about 60 to 300 or more nucleosomes may correspond to chromatin domains or third-order chromatin structures observed by other techniques.

scholarly journals The equilibrium constant of the phosphoglyceromutase reaction

1974 ◽  
Vol 139 (3) ◽  
pp. 491-497 ◽  
Author(s):  
John B. Clarke ◽  
Michael Birch ◽  
Hubert G. Britton
Keyword(s):  
Ionic Strength ◽  
Equilibrium Constant ◽  
Dissociation Constants ◽  
Ph Dependence ◽  
Binding Constants ◽  
Acid Dissociation ◽  
Effect Of Temperature ◽  
Acid Dissociation Constants ◽  
The Difference ◽  
Low Ionic Strength

The equilibrium constant of the phosphoglyceromutase reaction was determined over a range of pH (5.4–7.9), in solutions of different ionic strength (0.06–0.3) and in the presence of Mg2+, at 30°C and at 20°C. The values obtained (8.65–11.65) differ substantially from previously published values. The third acid dissociation constants were redetermined for 2- and 3-phosphoglycerate, and in contrast with previous reports the pK values (7.03 and 6.97 respectively at zero ionic strength) were closely similar. The Mg2+-binding constants were measured spectrophotometrically and the values, 286mm-1 and 255mm-1 for 2- and 3-phosphoglycerate at pH7 and ionic strength 0.02, were also very similar. From the relative lack of effect of temperature, pH and ionic strength it is concluded that the equilibrium constant differs from unity largely because of entropic factors. At low ionic strength, in the neutral region, the pH-dependence can be attributed to the small difference in the acid dissociation constants, but the difference in dissociation constants does not explain the pH-dependence in the acid region or at high ionic strength. Within physiological ranges of pH, Mg2+ concentration and ionic strength there will be little variation in equilibrium constant.

scholarly journals Characterization of genomic poly(dT-dG).poly(dC-dA) sequences: structure, organization, and conformation.

1984 ◽  
Vol 4 (12) ◽  
pp. 2610-2621 ◽  
Author(s):  
H Hamada ◽  
M G Petrino ◽  
T Kakunaga ◽  
M Seidman ◽  
B D Stollar
Keyword(s):  
Ionic Strength ◽  
Inverted Repeats ◽  
Actin Gene ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Coding Sequences ◽  
Supercoiled Plasmid ◽  
Marked Preference ◽  
Low Ionic Strength

Hybridization studies suggest the abundant presence of poly(dT-dG).poly(dC-dA) (TG-element), a potential Z-DNA sequence, in eucaryotic genomes. We have isolated and characterized TG-elements from different locations in the human genome: from randomly isolated clones, associated with the actin gene family, and linked to another repeated element. The results indicate that the following features are typical of these TG-elements: the elements consist of 20 to 60 base pairs of (dT-dG)n.(dC-dA)n, the sequences characterized in our study were not flanked by direct or inverted repeats, the sequences are interspersed rather than in satellite blocks, the elements are not usually associated with other repeated elements, and some of the elements are found near coding sequences or in introns. Studies on the conformation of a genomic TG-element in a supercoiled plasmid indicate several distinct properties of the TG-element: it is in the Z-form only at low ionic strength, S1 nuclease recognizes its Z-form with a marked preference for one of the B-Z junctions, and the sensitive region extends for 20 base pairs near the B-Z junction. In contrast to the result with the supercoiled plasmid, S1 nuclease failed to recognize the TG-element in minichromosomes.

scholarly journals Presence of two deoxyribonucleic acid polymerases in bull spermatozoa

1978 ◽  
Vol 175 (2) ◽  
pp. 595-600 ◽  
Author(s):  
M Philippe ◽  
P Chevaillier
Keyword(s):  
Ionic Strength ◽  
Dna Polymerase ◽  
Ethidium Bromide ◽  
Nuclear Dna ◽  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Polymerase Activity ◽  
Exogenous Dna ◽  
Middle Piece ◽  
Low Ionic Strength

A DNA polymerase-endogenous template complex was isolated from nuclear heads of bull spermatozoa. The buoyant density of the complex was 1.15 g/cm 3. The sedimentation coefficient of the nuclear DNA polymerase isolated from the complex was higher at low ionic strength, but approached 3.4S when centrifuged in a medium containing 2M-KCl. Activated exogenous DNA increased polymerase activity. Only very low activities were detected with synthetic templates such as poly(A).(dT)12-18 and poly(dT).poly(A). The nuclear reaction was stimulated by 150mM-KCl and was slightly inhibited by N-ethylmaleimide; it was resistant to actinomycin D, netropsin and ethidium bromide. Another DNA polymerase, highly sensitive to ethidium bromide, was extracted from the mitochondira-rich middle-piece fraction. Its sedimentation coefficient was close to 9S, but fell to approx. 4S in high-ionic-strength medium.

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