ABSTRACT We synthesized dimeric Hoechst dye molecules composed of two moieties of Hoechst 33258 f... more ABSTRACT We synthesized dimeric Hoechst dye molecules composed of two moieties of Hoechst 33258 fluorescent dye with the phenolic hydroxy groups tethered via pentamethylene, heptamethylene, or triethylene oxide linkers. A characteristic pattern of differential staining of chromosome preparations from human HL60 premonocytic leukemia cells was observed for all the three fluorescent dyes. The most contrasting pattern was obtained for the bisHoechst analogue with the heptamethylene linker; its quality was comparable with the picture obtained in the case of chromosome staining with 4′,6-diamidino-2-phenylindole. The ability to penetrate into live human fibroblasts was studied for the three bisHoechst compounds. The fluorescence intensity of nuclei of live and fixed cells stained with the penta- and heptamethylene-linked bisHoechst analogues was found to differ only slightly, whereas the fluorescence of the nuclei of live cells stained with triethylene oxide-linked bisHoechst was considerably weaker than that of the fixed cells. The bisHoechst molecules are new promising fluorescent dyes that can both differentially stain chromosome preparations and penetrate through cell and nuclear membranes and effectively stain cell nuclei.
The simultaneous analysis of DNAase I "footprinting" data and restriction endonucleases... more The simultaneous analysis of DNAase I "footprinting" data and restriction endonucleases inhibition data was performed on the same DNA end-labelled fragment. The inhibition induced by netropsin, a number of bis-netropsins and distamycin A was investigated. These experiments led us to the following conclusions. The restriction endonucleases inhibition by the ligands is caused by the ligand molecules binding in the close vicinity to the restriction endonuclease recognition sequence. The zone of +/- 4 bp from the center of the restriction endonuclease recognition sequence can be defined as the zone of the influence of the bounded ligand on the restriction endonuclease. But in this case the intersection of recognition sequence and the binding site occupied by a single ligand molecule is not sufficient for the inhibition to occur. Restriction endonuclease cutting sites protected by netropsin can be predicted basing upon known nucleotide sequence specificity of netropsin. Netrops...
Spectrophotometric methods are used to study the binding to DNA of Actinomycin D (AMD) and its an... more Spectrophotometric methods are used to study the binding to DNA of Actinomycin D (AMD) and its analogues: 7-nitro-AMD; 7-amino-AMD; 7-(Z-Val-Glo-NH)-AMD; 7-(AcO- . +H2-Val-Glo-NH)-AMD; 7-(AcO- . +H2-Val-Glo-Val-Glo-NH)-AMD. The binding constants are calculated from the binding isotherm of AMD and those of the AMD analogues to calf thymus DNA obtained by spectrophotometric titration. Introduction of smaller substituents such as the nitro or amino groups into position 7 of chromophore influences insignificantly the antibiotic binding to DNA, whereas bulky substituents cause a decrease in the affinity of the AMD analogues for DNA, although the spectral characteristics are not affected.
The crystal structure of the antibiotic distamycin A analog containing two pyrrol carboxamide fra... more The crystal structure of the antibiotic distamycin A analog containing two pyrrol carboxamide fragments has been determined. The space group of the crystals is P21/b; the unit-cell dimensions are a = 11.169, b = 21.535, c = 7.863 A, alpha = beta = 90 degrees, gamma = 122.45 degrees, Z = 4. The structure is solved by direct methods, and refined with the full-matrix least-squares procedure. The data on the structure of the pyrrol carboxamide backbone allow the following conclusions to be made about the molecular structure of the distamycin type antibiotics: (i) the amide groups have normal trans-configuration with slightly shortened C-C and C-N bonds adjacent to the pyrrol rings, (ii) the N-methyl groups of the pyrrol rings and the oxygen atoms of the amide groups have the same orientation with respect to the backbone. In the distamycin A analog molecule the pyrrol rings and amide group between them are approximately coplanar.
Cancer cells are characterized by the hypermethylation of promoter regions of tumor suppressor ge... more Cancer cells are characterized by the hypermethylation of promoter regions of tumor suppressor genes. DNA methyltransferase inhibitors cause re-activation of these genes that allows considering DNA methyltransferases as targets for anticancer therapy. As it was previously shown by us, dimeric bisbenzimidazoles, DB(n), differing in length of the oligomethylene linker between the two bisbenzimidazole fragments (n--number of methylene groups in linker) effectively inhibit the methylation of DNA duplexes by murine methyltransferase Dnmt3a. Here, the cytotoxicity of some of these compounds, their penetration into cells and influence on the methylation of genomic DNA in fetal lung fibroblasts line F-977 and cervical cancer cells HeLa have been studied. In the 0-60 microM concentration range, only the DB(11) displayed a significant toxic effect on the normal cells, whereas the effect of DB(n) investigated on the cancer cells was not significant. Interestingly, the DB(1) and DB(3) to a smal...
Five fluorescent symmetric dimeric bisbenzimidazoles DB(n) have been synthesized containing four ... more Five fluorescent symmetric dimeric bisbenzimidazoles DB(n) have been synthesized containing four 2,6-substited benzimidazole fragments and differ in length of oligomethylene linker (n=3, 4, 5, 7, 11) between the two bisbenzimidazole blocks. The ability of these dimeric bisbenzimidazoles to form complexes with double-stranded DNA (dsDNA) was shown by spectral methods. Upon binding to dsDNA DB(n) are localized in the minor groove. The DNA-methyltransferase Dnmt3a inhibition data are demonstrate the site-specific binding of dimeric bisbenzimidazoles DB(3) and DB(11) with oligonucleotide duplex.
Dimeric bisbenzimidazole DB(5) fluorescing in the blue spectral area (lambda(em) 476 nm) within t... more Dimeric bisbenzimidazole DB(5) fluorescing in the blue spectral area (lambda(em) 476 nm) within the DNA complex was synthesized. DB(5) is bound by a double-strand DNA and can differentially stain human and plant (flax) chromosomes. According to preliminary data, it provides considerably more intensely contrasting chromosome staining than DAPI and Hoechst 33258 dyes. It was also found that DB(5) is an in vitro inhibitor of HIV-1 integrase in the reaction of 3'-processing. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.
Dimeric Hoechst 33258 molecules [dimeric bisbenzimidazoles (DBBIs)] that, upon binding, occupy on... more Dimeric Hoechst 33258 molecules [dimeric bisbenzimidazoles (DBBIs)] that, upon binding, occupy one turn of the B form of DNA in the narrow groove were constructed by computer simulation. Three fluorescent DBBIs were synthesized; they consist of two bisbenzimidazole units tail-to-tail linked to phenolic hydroxy groups via penta- or heptamethylene or tri(ethylene glycol) spacers and have terminal positively charged N.N-dimethylaminopropyl carboxamide groups in the molecule. The absorption spectra of the DBBIs in the presence of different DNA concentrations showed a hypochromic effect and a small shift of the absorption band to longer wavelengths, which indicated the formation of a complex with DNA. The presence of an isobestic point in the spectrum indicates the formation of one type of DBBI-DNA complexes. The interaction of DBBIs with DNA was studied by CD using a cholesteric liquid-crystalline dispersion (CLD) of DNA. The appearance of a positive band in the absorption region of lig...
The kinetics of the hydrolysis of the lactone ring of a hybrid molecule containing the molecules ... more The kinetics of the hydrolysis of the lactone ring of a hybrid molecule containing the molecules of the antitumor drug camptothecin and a derivative of the antibiotic netropsines, which is highly affine and specific to the DNA A-T sequences was investigated. It was shown that intramolecular interaction significantly slows down the rate of hydrolysis but does not change the equilibrium ratio of concentrations of the lactone and carboxylate forms of the camptothecin fragment of the hybrid molecule, which corresponds to the pH value. The use of intramolecular interaction for controlling the kinetics of the lactone/carboxylate transition makes it possible to create the drugs of the camptothecin family, which preserve the biologically active lactone form under the physiological conditions for a longer time and, therefore, are more effective as anticancer agents.
Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical... more Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical methods. Topotecan absorption spectra were recorded in the pH range 0.5-11.5 and its pKa were determined. Quantum chemical calculations were made for all charge states of the topotecan molecule in lactone and carboxylate form. The calculated absorption maxima agree well with the experimental data. Protonation of the topotecan D ring (pKa = 3.6) was revealed. Comparison of experimental and calculated data showed topotecan structure with a proton at the oxygen atom at C16a rather than N4 to be the most preferable. Topotecan molecules were shown to form dimers at concentrations above 10(-5) M. Topotecan dimerization is accompanied by an increase in the pKa of hydroxy group of the A ring from 6.5 ([TPT] = 10(-6) M) to 7.1 ([TPT] = 10(-4) M), which indicates participation of this group in dimer stabilization, perhaps due to intermolecular hydrogen bonding with N1 of the B ring of a neighbori...
An analogue of netropsin has been synthesized consisting of two N-propylpyrrolcarboxamide units l... more An analogue of netropsin has been synthesized consisting of two N-propylpyrrolcarboxamide units linked covalently to a copper-chelating tripeptide Gly-Gly-L-His by means of two and three glycine residues. Binding to DNA and synthetic polynucleotides of netropsin analogue containing three glycine residues between Gly-Gly-L-His tripeptide and the N-end of netropsin analogue (His-Nt) has been studied. It is shown that this netropsin analogue chelates a copper ion with 1:1 stoichiometry, similar to a free Gly-Gly-L-His peptide. It is found that this netropsin analogue occupies 3 to 4 base pairs upon binding to poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] polymers, irrespective of whether it binds in Cu(2+)-ligated or unligated forms. Binding constants and binding site sizes have been calculated for netropsin analogue complexes with DNA, poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] polymers at the [Cu2+]/[His-Nt] ratio equal to 0 and 1.0. In the three-component system including His-Nt ...
Synthesis, DNA- and zinc ion-binding activities of the synthetic 23-residue peptide, forming a pa... more Synthesis, DNA- and zinc ion-binding activities of the synthetic 23-residue peptide, forming a part of the DNA-binding domain of yeast transcription activator GAL-4, are reported. In presence of zinc ions considerable changes in the shapes of the fluorescence and CD spectra of the peptide are observed. It is shown that the peptide forms complexes with zinc ions containing one metal ion per peptide molecule with association constants on the order of (1-2) x 10(6) M-1. Using gel filtration on a TSK-gel column we have shown that in aqueous solution at concentrations of 10(-4)-10(-6) M the peptide exists predominantly in the dimeric form. Dimerization constants were found to be 5 x 10(6) M-1 and 1.7 x 10(7) M-1 in the absence and in the presence of zinc ions, respectively. It is shown that the peptide binds to DNA. The binding approaches saturation when one peptide molecule is bound approximately to five base pairs of DNA. The shapes of the titration curves obtained from binding of the ...
Effects of sibiromicyn, distamicyn A and its analogs on binding to DNA and to poly(dA).poly(dT) a... more Effects of sibiromicyn, distamicyn A and its analogs on binding to DNA and to poly(dA).poly(dT) are reported for a 23-amino acid synthetic zinc-binding peptide, a part of the DNA-binding domain of the transcriptional activator GAL-4. Circular dichroism and fluorometry have shown that the synthetic peptide and two distamicyn A analogs compete for binding sites on DNA and on poly(dA).poly(dT). Antibiotic sibiromycin which forms a covalent bond with a guanine 2-amino group in the minor DNA groove can displace the peptide from a 19 bp self-complementary oligonucleotide serving as a specific target site for Gal-4 protein. The peptide is shown to bind to a glucosylated phage T2 DNA, but its affinity to T2 DNA is weaker than to calf thymus DNA under the same conditions. A method to estimate binding constant and size of the binding site for the synthetic peptide and poly(dA).poly(dT) is proposed based on the binding isotherms of distamycin analogs in the absence and in the presence of the p...
A technique for DNA amount determination by flow cytometry based on the use of 7-amino-actinomyci... more A technique for DNA amount determination by flow cytometry based on the use of 7-amino-actinomycin D (7-amino-AMD), a fluorescent analogue of antibiotic actinomycin has been investigated, and a particular staining procedure has been developed. The procedure includes short fixation in 70% ethanol and staining for 20 min in 10(-5)M solution of 7-amino-AMD at pH7. The results of DNA content measurements are very reproducible. The histograms obtained have a coefficient of variation less than 3%. The absorption maximum of the complex of 7-amino-AMD with DNA is situated in the green spectrum region, making this stain particularly suitable for argon laser flow cytometry.
A model is proposed for the structure of stereospecific sites in regulatory proteins. On its basi... more A model is proposed for the structure of stereospecific sites in regulatory proteins. On its basis a possible code is suggested that governs the binding of regulatory proteins at specific control sites on DNA. Stereospecific sites of regulatory proteins are assumed to contain pairs of antiparallel polypeptide chain segments which form a right-hand twisted antiparallel beta-sheet, with single-stranded regions at the ends of the beta-structure. The model predicts that binding reaction between a regulatory protein and double-helical DNA is a cooperative phenomenon and is accompanied by significant structural alteration at the stereospecific site of the protein. Half of hydrogen bonds normally existing in beta-structure are broken upon complex formation with DNA and a new set of hydrogen bonds is formed between polypeptide amide groups and DNA base pairs. In a stereospecific site, one chain (t-chain) is attached through hydrogen bonds to the carbonyl oxygens of pyramides and N3 adenines...
The anisotropy of absorbance (near 320 and 258 nm) of the DNA complexes with distamycin A and two... more The anisotropy of absorbance (near 320 and 258 nm) of the DNA complexes with distamycin A and two analogues have been studied. Measurements were made along the direction of the flow oreintation of the complexes. The comparison of two orientation effects: deltaA320=(Astop--Aflow)320 and deltaA258=(Astop--Aflow)258, gave the opportunity to estimate the inclination angle of distamycin chromophores with respect to the DNA axis: for all three analogues the angle was lying between 40 and 45 degrees. These results strongly support the model of the complex where the antibiotic is inserted into the narrow grove of the DNA helix.
ABSTRACT We synthesized dimeric Hoechst dye molecules composed of two moieties of Hoechst 33258 f... more ABSTRACT We synthesized dimeric Hoechst dye molecules composed of two moieties of Hoechst 33258 fluorescent dye with the phenolic hydroxy groups tethered via pentamethylene, heptamethylene, or triethylene oxide linkers. A characteristic pattern of differential staining of chromosome preparations from human HL60 premonocytic leukemia cells was observed for all the three fluorescent dyes. The most contrasting pattern was obtained for the bisHoechst analogue with the heptamethylene linker; its quality was comparable with the picture obtained in the case of chromosome staining with 4′,6-diamidino-2-phenylindole. The ability to penetrate into live human fibroblasts was studied for the three bisHoechst compounds. The fluorescence intensity of nuclei of live and fixed cells stained with the penta- and heptamethylene-linked bisHoechst analogues was found to differ only slightly, whereas the fluorescence of the nuclei of live cells stained with triethylene oxide-linked bisHoechst was considerably weaker than that of the fixed cells. The bisHoechst molecules are new promising fluorescent dyes that can both differentially stain chromosome preparations and penetrate through cell and nuclear membranes and effectively stain cell nuclei.
The simultaneous analysis of DNAase I "footprinting" data and restriction endonucleases... more The simultaneous analysis of DNAase I "footprinting" data and restriction endonucleases inhibition data was performed on the same DNA end-labelled fragment. The inhibition induced by netropsin, a number of bis-netropsins and distamycin A was investigated. These experiments led us to the following conclusions. The restriction endonucleases inhibition by the ligands is caused by the ligand molecules binding in the close vicinity to the restriction endonuclease recognition sequence. The zone of +/- 4 bp from the center of the restriction endonuclease recognition sequence can be defined as the zone of the influence of the bounded ligand on the restriction endonuclease. But in this case the intersection of recognition sequence and the binding site occupied by a single ligand molecule is not sufficient for the inhibition to occur. Restriction endonuclease cutting sites protected by netropsin can be predicted basing upon known nucleotide sequence specificity of netropsin. Netrops...
Spectrophotometric methods are used to study the binding to DNA of Actinomycin D (AMD) and its an... more Spectrophotometric methods are used to study the binding to DNA of Actinomycin D (AMD) and its analogues: 7-nitro-AMD; 7-amino-AMD; 7-(Z-Val-Glo-NH)-AMD; 7-(AcO- . +H2-Val-Glo-NH)-AMD; 7-(AcO- . +H2-Val-Glo-Val-Glo-NH)-AMD. The binding constants are calculated from the binding isotherm of AMD and those of the AMD analogues to calf thymus DNA obtained by spectrophotometric titration. Introduction of smaller substituents such as the nitro or amino groups into position 7 of chromophore influences insignificantly the antibiotic binding to DNA, whereas bulky substituents cause a decrease in the affinity of the AMD analogues for DNA, although the spectral characteristics are not affected.
The crystal structure of the antibiotic distamycin A analog containing two pyrrol carboxamide fra... more The crystal structure of the antibiotic distamycin A analog containing two pyrrol carboxamide fragments has been determined. The space group of the crystals is P21/b; the unit-cell dimensions are a = 11.169, b = 21.535, c = 7.863 A, alpha = beta = 90 degrees, gamma = 122.45 degrees, Z = 4. The structure is solved by direct methods, and refined with the full-matrix least-squares procedure. The data on the structure of the pyrrol carboxamide backbone allow the following conclusions to be made about the molecular structure of the distamycin type antibiotics: (i) the amide groups have normal trans-configuration with slightly shortened C-C and C-N bonds adjacent to the pyrrol rings, (ii) the N-methyl groups of the pyrrol rings and the oxygen atoms of the amide groups have the same orientation with respect to the backbone. In the distamycin A analog molecule the pyrrol rings and amide group between them are approximately coplanar.
Cancer cells are characterized by the hypermethylation of promoter regions of tumor suppressor ge... more Cancer cells are characterized by the hypermethylation of promoter regions of tumor suppressor genes. DNA methyltransferase inhibitors cause re-activation of these genes that allows considering DNA methyltransferases as targets for anticancer therapy. As it was previously shown by us, dimeric bisbenzimidazoles, DB(n), differing in length of the oligomethylene linker between the two bisbenzimidazole fragments (n--number of methylene groups in linker) effectively inhibit the methylation of DNA duplexes by murine methyltransferase Dnmt3a. Here, the cytotoxicity of some of these compounds, their penetration into cells and influence on the methylation of genomic DNA in fetal lung fibroblasts line F-977 and cervical cancer cells HeLa have been studied. In the 0-60 microM concentration range, only the DB(11) displayed a significant toxic effect on the normal cells, whereas the effect of DB(n) investigated on the cancer cells was not significant. Interestingly, the DB(1) and DB(3) to a smal...
Five fluorescent symmetric dimeric bisbenzimidazoles DB(n) have been synthesized containing four ... more Five fluorescent symmetric dimeric bisbenzimidazoles DB(n) have been synthesized containing four 2,6-substited benzimidazole fragments and differ in length of oligomethylene linker (n=3, 4, 5, 7, 11) between the two bisbenzimidazole blocks. The ability of these dimeric bisbenzimidazoles to form complexes with double-stranded DNA (dsDNA) was shown by spectral methods. Upon binding to dsDNA DB(n) are localized in the minor groove. The DNA-methyltransferase Dnmt3a inhibition data are demonstrate the site-specific binding of dimeric bisbenzimidazoles DB(3) and DB(11) with oligonucleotide duplex.
Dimeric bisbenzimidazole DB(5) fluorescing in the blue spectral area (lambda(em) 476 nm) within t... more Dimeric bisbenzimidazole DB(5) fluorescing in the blue spectral area (lambda(em) 476 nm) within the DNA complex was synthesized. DB(5) is bound by a double-strand DNA and can differentially stain human and plant (flax) chromosomes. According to preliminary data, it provides considerably more intensely contrasting chromosome staining than DAPI and Hoechst 33258 dyes. It was also found that DB(5) is an in vitro inhibitor of HIV-1 integrase in the reaction of 3'-processing. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.
Dimeric Hoechst 33258 molecules [dimeric bisbenzimidazoles (DBBIs)] that, upon binding, occupy on... more Dimeric Hoechst 33258 molecules [dimeric bisbenzimidazoles (DBBIs)] that, upon binding, occupy one turn of the B form of DNA in the narrow groove were constructed by computer simulation. Three fluorescent DBBIs were synthesized; they consist of two bisbenzimidazole units tail-to-tail linked to phenolic hydroxy groups via penta- or heptamethylene or tri(ethylene glycol) spacers and have terminal positively charged N.N-dimethylaminopropyl carboxamide groups in the molecule. The absorption spectra of the DBBIs in the presence of different DNA concentrations showed a hypochromic effect and a small shift of the absorption band to longer wavelengths, which indicated the formation of a complex with DNA. The presence of an isobestic point in the spectrum indicates the formation of one type of DBBI-DNA complexes. The interaction of DBBIs with DNA was studied by CD using a cholesteric liquid-crystalline dispersion (CLD) of DNA. The appearance of a positive band in the absorption region of lig...
The kinetics of the hydrolysis of the lactone ring of a hybrid molecule containing the molecules ... more The kinetics of the hydrolysis of the lactone ring of a hybrid molecule containing the molecules of the antitumor drug camptothecin and a derivative of the antibiotic netropsines, which is highly affine and specific to the DNA A-T sequences was investigated. It was shown that intramolecular interaction significantly slows down the rate of hydrolysis but does not change the equilibrium ratio of concentrations of the lactone and carboxylate forms of the camptothecin fragment of the hybrid molecule, which corresponds to the pH value. The use of intramolecular interaction for controlling the kinetics of the lactone/carboxylate transition makes it possible to create the drugs of the camptothecin family, which preserve the biologically active lactone form under the physiological conditions for a longer time and, therefore, are more effective as anticancer agents.
Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical... more Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical methods. Topotecan absorption spectra were recorded in the pH range 0.5-11.5 and its pKa were determined. Quantum chemical calculations were made for all charge states of the topotecan molecule in lactone and carboxylate form. The calculated absorption maxima agree well with the experimental data. Protonation of the topotecan D ring (pKa = 3.6) was revealed. Comparison of experimental and calculated data showed topotecan structure with a proton at the oxygen atom at C16a rather than N4 to be the most preferable. Topotecan molecules were shown to form dimers at concentrations above 10(-5) M. Topotecan dimerization is accompanied by an increase in the pKa of hydroxy group of the A ring from 6.5 ([TPT] = 10(-6) M) to 7.1 ([TPT] = 10(-4) M), which indicates participation of this group in dimer stabilization, perhaps due to intermolecular hydrogen bonding with N1 of the B ring of a neighbori...
An analogue of netropsin has been synthesized consisting of two N-propylpyrrolcarboxamide units l... more An analogue of netropsin has been synthesized consisting of two N-propylpyrrolcarboxamide units linked covalently to a copper-chelating tripeptide Gly-Gly-L-His by means of two and three glycine residues. Binding to DNA and synthetic polynucleotides of netropsin analogue containing three glycine residues between Gly-Gly-L-His tripeptide and the N-end of netropsin analogue (His-Nt) has been studied. It is shown that this netropsin analogue chelates a copper ion with 1:1 stoichiometry, similar to a free Gly-Gly-L-His peptide. It is found that this netropsin analogue occupies 3 to 4 base pairs upon binding to poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] polymers, irrespective of whether it binds in Cu(2+)-ligated or unligated forms. Binding constants and binding site sizes have been calculated for netropsin analogue complexes with DNA, poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] polymers at the [Cu2+]/[His-Nt] ratio equal to 0 and 1.0. In the three-component system including His-Nt ...
Synthesis, DNA- and zinc ion-binding activities of the synthetic 23-residue peptide, forming a pa... more Synthesis, DNA- and zinc ion-binding activities of the synthetic 23-residue peptide, forming a part of the DNA-binding domain of yeast transcription activator GAL-4, are reported. In presence of zinc ions considerable changes in the shapes of the fluorescence and CD spectra of the peptide are observed. It is shown that the peptide forms complexes with zinc ions containing one metal ion per peptide molecule with association constants on the order of (1-2) x 10(6) M-1. Using gel filtration on a TSK-gel column we have shown that in aqueous solution at concentrations of 10(-4)-10(-6) M the peptide exists predominantly in the dimeric form. Dimerization constants were found to be 5 x 10(6) M-1 and 1.7 x 10(7) M-1 in the absence and in the presence of zinc ions, respectively. It is shown that the peptide binds to DNA. The binding approaches saturation when one peptide molecule is bound approximately to five base pairs of DNA. The shapes of the titration curves obtained from binding of the ...
Effects of sibiromicyn, distamicyn A and its analogs on binding to DNA and to poly(dA).poly(dT) a... more Effects of sibiromicyn, distamicyn A and its analogs on binding to DNA and to poly(dA).poly(dT) are reported for a 23-amino acid synthetic zinc-binding peptide, a part of the DNA-binding domain of the transcriptional activator GAL-4. Circular dichroism and fluorometry have shown that the synthetic peptide and two distamicyn A analogs compete for binding sites on DNA and on poly(dA).poly(dT). Antibiotic sibiromycin which forms a covalent bond with a guanine 2-amino group in the minor DNA groove can displace the peptide from a 19 bp self-complementary oligonucleotide serving as a specific target site for Gal-4 protein. The peptide is shown to bind to a glucosylated phage T2 DNA, but its affinity to T2 DNA is weaker than to calf thymus DNA under the same conditions. A method to estimate binding constant and size of the binding site for the synthetic peptide and poly(dA).poly(dT) is proposed based on the binding isotherms of distamycin analogs in the absence and in the presence of the p...
A technique for DNA amount determination by flow cytometry based on the use of 7-amino-actinomyci... more A technique for DNA amount determination by flow cytometry based on the use of 7-amino-actinomycin D (7-amino-AMD), a fluorescent analogue of antibiotic actinomycin has been investigated, and a particular staining procedure has been developed. The procedure includes short fixation in 70% ethanol and staining for 20 min in 10(-5)M solution of 7-amino-AMD at pH7. The results of DNA content measurements are very reproducible. The histograms obtained have a coefficient of variation less than 3%. The absorption maximum of the complex of 7-amino-AMD with DNA is situated in the green spectrum region, making this stain particularly suitable for argon laser flow cytometry.
A model is proposed for the structure of stereospecific sites in regulatory proteins. On its basi... more A model is proposed for the structure of stereospecific sites in regulatory proteins. On its basis a possible code is suggested that governs the binding of regulatory proteins at specific control sites on DNA. Stereospecific sites of regulatory proteins are assumed to contain pairs of antiparallel polypeptide chain segments which form a right-hand twisted antiparallel beta-sheet, with single-stranded regions at the ends of the beta-structure. The model predicts that binding reaction between a regulatory protein and double-helical DNA is a cooperative phenomenon and is accompanied by significant structural alteration at the stereospecific site of the protein. Half of hydrogen bonds normally existing in beta-structure are broken upon complex formation with DNA and a new set of hydrogen bonds is formed between polypeptide amide groups and DNA base pairs. In a stereospecific site, one chain (t-chain) is attached through hydrogen bonds to the carbonyl oxygens of pyramides and N3 adenines...
The anisotropy of absorbance (near 320 and 258 nm) of the DNA complexes with distamycin A and two... more The anisotropy of absorbance (near 320 and 258 nm) of the DNA complexes with distamycin A and two analogues have been studied. Measurements were made along the direction of the flow oreintation of the complexes. The comparison of two orientation effects: deltaA320=(Astop--Aflow)320 and deltaA258=(Astop--Aflow)258, gave the opportunity to estimate the inclination angle of distamycin chromophores with respect to the DNA axis: for all three analogues the angle was lying between 40 and 45 degrees. These results strongly support the model of the complex where the antibiotic is inserted into the narrow grove of the DNA helix.
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Papers by A. Zhuze