Melting of DNA-actinomycin clusters

Differential methods of scanning micro-calorimetry and UV spectrophotometry were used for understanding the interaction of natural anti-tumour antibiotic actinomycin D with cluster sites of native and fragmented DNA during thermal melting. At low (micro-molar) concentrations, the actinomycin molecules penetrate into unwound regions of DNA, but not into the double helix. Moreover, they stabilize the fragmented DNA and increase a total melting point. Actinomycin D interacts with fractions of native DNA even at very low concentrations (at the antibiotic/nucleotide ratio of 1:868) and stabilizes the most loose clusters. At high concentrations, it destabilizes the double helix.     

Effect of low-molecular amines on DNA conformation and stability of the double helix]

Interaction of low-molecular amines (cystamine, cysteamine, cystaphose, asparagine, beta-alanine) with DNA was studied. The amines change the positive circular dichroism (CD) band of DNA as well as temperature and range width of melting. Effect of amines on DNA depends on ionic strength of the solvent, concentration and structure of the ligand. Monamines cause destabilization of DNA double helix followed by stabilization as ligand concentration increases. At concentrations stabilizing the double helix DNA conformation undergoes transition from the B- to C-form. The results obtained enable to relate the stabilizing effect of low-molecular amines and conformational B leads to C-transition to the non-specific interaction of ligand amino groups with DNA phosphates, and the destabilizing effect of monoamines of low concentrations to their interaction with bases, mainly in the denaturated sites of DNA. It is proposed that a stronger effectiveness of amines as compared to monovalent metals in the conformational shift of DNA towards the C-form is due to the additional effect of disturbance of hydrophobic interactions in DNA double helix.     

Osmium-induced alteration in DNA structure

In the presence of pyridine and other ligands osmium tetroxide binds covalently to pyrimidine bases in DNA. Properties of osmium-modified native and denatured calf thymus DNA, and plasmid Co1E1 DNA were investigated by means of differential pulse polarography, absorption spectrophotometry, circular dichroism, agarose gel electrophoresis, and nuclease S1 digestion. A great difference in the reaction kinetics of native and denatured DNAs with osmium, pyridine was observed. On the ground of the slow stepwise reaction kinetics of native DNA in the initial stage of its modification by osmium it has been suggested that the primary reaction sites do not include bases contained in the intact double helix. Osmium binding to sporadic primary reaction sites (represented e.g. by bases in the vicinity of a single-strand break) in native calf thymus DNA resulted in local changes in DNA conformation limited to a close neighbourhood of the binding site. At higher osmium/nucleotide ratios disordering of the DNA structure over a region extending beyond the immediate binding site was observed. With denatured DNA the same type of structure disordering was detected already in the initial stage of the reaction at osmium/nucleotide ratios as low as 0.01. Osmium binding to the supercoiled Co1E1 DNA resulted in its relaxation without nicking and it increased its sensitivity to linearization by cleavage with nuclease S1. The behaviour of Co1E1 DNA has been explained by the formation of a denatured region in the molecule (accompanied by a coupled loss of duplex and superhelical turns). It has been suggested that osmium can be used to label and to visualize distorted regions in the DNA double helix.     

Use of the antibiotics actinomycin D and distamycin A to limit the action of restriction endonucleases and to map DNA

It is shown that distamycin A and actinomycin D protect the recognition sites of certain restriction endonucleases from the attack by these nucleases due to specific interaction of these antibiotics with double-stranded DNA. Distamycin A protects A-T containing sites and actinomycin G-C rich sites. Among Hind II recognition sites which have alternative structure (GTPyPuAC) distamycin A protects only Hpa I similar sites (GTTAAC). It is shown with several restriction endonucleases that antibiotic action depends on the nucleotide sequences in the recognition sites and in their closest environment. Proper concentrations of antibiotic give rise to larger fragments. Use of both distamycin A and actinomycin D allows to obtain a set of overlapping fragments. The data obtained with various DNAs and restriction endonucleases allow to conclude that these antibiotics may be useful for DNA mapping and for preparation of large functional fragments of DNA.     

A study of the reversibility of helix-coil transition in DNA.

The reversibility of DNA melting has been thoroughly investigated at different ionic strengths. We concentrated on those stages of the process that do not involve a complete separation of the strands of the double helix. The differential melting curves of pBR 322 DNA and a fragment of T7 phage DNA in a buffer containing 0.02M Na+ have been shown to differ substantially from the differential curves of renaturation. Electron-microscopic mapping of pBR 322 DNA at different degrees of unwinding (by a previously elaborated technique) has shown that the irreversibility of melting under real experimental conditions is connected with the stage of forming new helical regions during renaturation. In a buffer containing 0.2M Na+ the melting curves of the DNAs used (pBR322, a fragment of T7 phage DNA, a fragment of phage Lambda DNA, a fragment of phiX174 phage DNA) coincide with the renaturation curves, i.e. the process is equilibrium. We have carried out a semi-quantitative analysis of the emergence of irreversibility in the melting of a double helix. The problem of comparing theoretical and experimental melting curves is discussed.     

Studies on the structure of chemically methylated DNA

CD and melting temperature measurements on the nature of DNA with chemically methylated guanine-rich sites indicate that the stable secondary structure of DNA depicted by Ramstein et al- involves considerable distortions resulting from decreased base-base stacking interaction. Besides that quantum chemical data gained from PPP calculations are in favor of a weaker hydrogen bonding interaction in the methylated guanine-cytosine base pair. CD measurements demonstrate that methylated DNA-regions differ from the nonmethylated helical structure, since formation of a condensed conformation as occurs in the transition from B to the C-uke structure is prevented by positively charged methylated guanine residues. An increase in helix winding angle, however, can not be excluded. Binding ability of the dyes acridine orange, phenosafranine, and the antibiotic actinomycin C is lowered for methylated DNA, while binding of proflavine is, in accordance with the results of Ramstein and Leng, slightly enhanced. The reason for the opposite behavior of proflavine is at present not fully understood. In particular changes in the binding ability with dyes could not be correlated with base specificity of complex formation. It is discussed that structural changes in DNA towards a loose conformation decrease the binding tendency for acridine orange, phenosafranine, and actinomycin C.     

Interaction of the ruthenium red cation with nucleic acid double helices

The hexapositive complex cation ruthenium red very effectively stabilizes DNA and RNA double helices against thermal denaturation. In the presence of nucleic acid helices, this symmetric cation acquires an extrinsic CD spectrum near the wavelength of the dye's maximum absorbance. Competition experiments with single-stranded polyd(T) show this induced CD to be the result of selective binding to helical sites. The preferential affinity of ruthenium red for double helical binding sites is so great that it brings about biphasic absorbance- temperature profiles of polyd(A-T) at low [cation]: [polynucleotide phosphate]. The visible CD signal and fraction of helix melting at the upper transition increases with ruthenium red concentration until approximate charge neutrality is reached. These interactions, which have been studied in detail with the poly(U-U) helix as well as polyd(A-T), are likely largely electrostatic, since sufficient [NaCl] eliminates the bipliasic melting of polyd(A-T), renders the ultraviolet absorbance of poly (U) insensitive to ruthenium red, and abolishes the induced CD effects. The bipliasic melting of polyd(A-T) at intermediate [dye] is attributed to saturation of remaining double helical segments by cation migration from newly melted regions- Furthermore, virtually no change was observed in the induced CD upon melting through the first transition, whereas the effect is destroyed upon inciting through the second transition. A quantitative treatment of the data is used to obtain binding site size and association constant for the complex. The induced effect may prove useful in the exploration of exposed nucleic acid helical structure in such complex particles as nucleosomes or ribosomes.     

Nucleic acid helix-coil transitions mediated by helix-unwinding proteins from calf thymus.

We have studied nucleic acid double helix destabilization mediated by purified calf helix-unwinding proteins, measuring ultraviolet hyperchromicity to detect helix melting. Both calf unwinding protein 1 (UP1) and a high salt eluting protein fraction are found to depress strongly the helix melting temperature (Tm) of the synthetic alternating copolymers poly[d(AT)] and poly[r(AU)], indicating that both DNA and RNA are recognized by these proteins. UP1 also destabilizes natural, GC-containing DNA helices, but to a smaller extent than observed with the above polymers. A simple model is presented to aid in the qualitative interpretation of the data, outlining the expected effect on the helix-coil transition of a protein ligand with differential affinity for the helix or coil form of nucleic acid. The observed helix-destabilizing effect of UP1 is dependent on the protein to nucleic acid ratio in an expected manner. Competition studies demonstrate a low, but appreciable affinity of UP1 for native DNA, opening the possibility that protein-mediated denaturation might be initiated by protein binding to the double helix. "Hairpin" helical regions of denatured DNA are strongly destabilized by UP1. Despite the fact that removal of these hairpin helices might greatly facilitate DNA renaturation, we failed to observe renaturation from the UP1-DNA complex after a switch to helix-stabilizing conditions. Thus, UP1 shows an important difference from its presumed prokaryotic analogue, T4 gene 32-protein. Possible in vivo functions of the calf proteins are discussed in light of these observations.     

Protection of particular cleavage sites of restriction endonucleases by distamycin A and actinomycin D.

It is shown here that distamycin A and actinomycin D can protect the recognition sites of endo R.EcoRI, EcoRII, HindII, HindIII, HpaI and HpaII from the attack of these restriction endonucleases. At proper distamycin concentrations only two endo R.EcoRI sites of phage lambda DNA are available for the restriction enzyme--sRI1 and sRI4. This phenomenon results in the appearance of larger DNA fragments comprising several consecutive fragments of endo R.EcoRI complete cleavage. The distamycin fragments isolated from the agarose gels can be subsequently cleaved by endo R.EcoRI with the yield of the fragments of complete digestion. We have compared the effect of distamycin A and actinomycin D on a number of restriction endonucleases having different nucleotide sequences in the recognition sites and established that antibiotic action depends on the nucleotide sequences of the recognition sites and their closest environment     

Mechanical stability of single DNA molecules

Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In lambda-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (相似文献   

Mode of Action of Antibiotic U-20,661

Antibiotic U-20,661 was shown to inhibit predominantly deoxyribonucleic acid (DNA)-directed ribonucleic acid (RNA) synthesis by binding to the double-stranded DNA template. Specific binding to DNA was verified by difference spectroscopy, reversal of the RNA polymerase inhibitory effect by increasing concentrations of DNA template, and by moderately increasing the melting temperature of double-stranded DNA in the presence of the antibiotic. The RNA polymerase reaction primed with synthetic poly dAT was inhibited considerably, but not completely even with high concentrations of antibiotic. Thus, the agent might bind to adenine or thymidine or both bases in the double-stranded DNA helix.     

Melting of Cross-Linked DNA IV. Methods for Computer Modeling of Total Influence on DNA Melting of Monofunctional Adducts,Intrastrand and Interstrand Cross-Links Formed by Molecules of an Antitumor Drug

Abstract

A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition.     

Melting of cross-linked DNA IV. Methods for computer modeling of total influence on DNA melting of monofunctional adducts, intrastrand and interstrand cross-links formed by molecules of an antitumor drug

A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition.     

Base-stacking and base-pairing contributions into thermal stability of the DNA double helix

Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA molecules with solitary nicks and gaps we measure temperature and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temperatures from below room temperature to close to melting temperature. We also obtain DNA stacking parameters for different salt concentrations ranging from 15 to 100 mM Na⁺. From stacking parameters of individual contacts, we calculate base-stacking contribution to the stability of A•T- and G•C-containing DNA polymers. We find that temperature and salt dependences of the stacking term fully determine the temperature and the salt dependence of DNA stability parameters. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A•T pairing is always destabilizing and G•C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.     

Diethyl pyrocarbonate can detect a modified DNA structure induced by the binding of quinoxaline antibiotics.

The reactivity of the 160 bp tyrT DNA fragment towards diethyl pyrocarbonate (DEPC) has been investigated in the presence of bis-intercalating quinoxaline antibiotics and the synthetic depsipeptide TANDEM. At moderate concentrations of each ligand, specific purine residues (mainly adenosines) exhibit enhanced reactivity towards the probe, and several sites of enhancement appear to be related to the sequence selectivity of drug binding. Further experiments were performed with echinomycin at pH 5.5 and 4.6 to facilitate the protonation of cytosine required for formation of Hoogsteen GC base pairs. No significant increase in reactivity was observed under these conditions. Additionally, no protection of deoxyguanosine residues from methylation by dimethyl sulphate was observed in the presence of echinomycin. We conclude that the structural anomaly giving rise to drug-dependent enhanced DEPC reaction is not simply the formation of Hoogsteen base pairs adjacent to antibiotic binding sites. Nor is it due to a general unwinding of the double helix, since we show that conditions which are supposed to unwind the helix lead to a uniform increase in purine reactivity, regardless of the surrounding nucleotide sequence.     

Studies on the interaction of altromycin B and its platinum(II) and palladium(II) metal complexes with calf thymus DNA and nucleotides

The interaction of the anticancer antibiotic altromycin B and its isostructrural Pt(II) and Pd(II) metal complexes with native calf thymus (CT) DNA was studied using UV-thermal denaturation experiments, circular dichroism spectroscopy and temperature controlled spectrophotometric titrations. Altromycin B stabilizes the double helix by raising the T(m), mainly by intercalation of its chromophore between the base pairs and interacting electrostatically via its sugar moieties with the edges of the DNA helix. Moreover, altromycin B induces a B-->A structural transition of CT DNA. The effect on DNA stability and conformation depends on the metal ion. Pt(II) and Pd(II) complexes induce the B-->A structural transition and stabilize the double helix similarly but they present lower final hyperchromicity due to premelting effects which were caused by intra- and interstrand crosslinking. Thus, a synergic effect of the metal ions to altromycin B-CT DNA interaction is observed in both cases. Altromycin B interacts with 5'-GMP, 5'-AMP and 5'-CMP by electrophilic attack of the opened epoxide ring to the N(7)G, N(1)/N(7)A and N(3)C. Thus, covalent binding between these nucleotides and altromycin B takes place and explain the multiple binding mode suggested by the studies of the interaction of altromycin B and its complexes with DNA. The [Pd(II)-altroB] complex dissociates in the presence of the nucleotides, and various species of Pd(II)-nucleotide complexes, especially with 5'-GMP, are formed. The [Pt(II)-altroB] complex dissociates too, but only one or two species of Pt(II)-nucleotide complexes are formed, and in the case of 5'-AMP interaction the formation of a tertiary altroB-Pt(II)-5'AMP complex is proposed. 5'-TMP reacts very weakly in comparison with the other three nucleotides. These interactions were followed by 1H-NMR.     

Interaction of quercetin with DNA

I n vitro experiments to study interaction of the mutagenic flavonoid quercetin with DNA are described. Calf thymus DNA treated with quercetin for various time periods was subjected to S₁ nuclease hydrolysis. Thermal melting profles of treated DNA were also determined using St nuclease. The rate of DNA hydrolyzed after 1 hr of pre-treatment with quercetin was found to be only about 50% of that in its absence. However, after 10 and 24hrs of treatment with the drug, the rate of S₁ nuclease hydrolysis was observed to be greater than that of native DNA. Thermal melting profiles of DNA, treated with quercetin for 10 and 24 hrs, indicated a slight decrease in melting temperatures. Gel filtration of native DNA, which had been digested with S₁ nuclease after preincubation with quercetin for 24 hrs, indicated the production of various sized degraded molecules. The results suggest that the initial interaction of quercetin with DNA may have a stabilizing effect on its secondary structure, but prolonged treatment leads to an extensive disruption of the double helix.     

Daunomycin modifies the sequence-selective recognition of DNA by actinomycin.

The antitumour antibiotic actinomycin D normally binds to DNA by intercalation at sequences containing the CpG step, but in the presence of daunomycin it has been reported to interact with poly(dA-dT). This observation has neither been confirmed nor explained. Here we have used a photoreactive 7-azido derivative of actinomycin to study the effect of daunomycin on its binding to three DNA fragments. Daunomycin did indeed alter the binding of actinomycin to the DNA, such that the antibiotic was displaced from its primary GpC sites onto secondary sites in the DNA, though not to AT regions especially. These findings suggest a possible scientific explanation for the increased toxicity seen during combination chemotherapy with these two drugs.     

Ultrasonic Footprinting

Ligand binding influences the dynamics of the DNA helix in both the binding site and adjacent regions. This, in particular, is reflected in the changing pattern of cleavage of complexes under the action of ultrasound. The specificity of ultrasound-induced cleavage of the DNA sugar-phosphate backbone was studied in actinomycin D (AMD) complexes with double-stranded DNA restriction fragments. After antibiotic binding, the cleavage intensity of phosphodiester bonds between bases was shown to decrease at the chromophore intercalation site and to increase in adjacent positions. The character of cleavage depended on the sequences flanking the binding site and the presence of other AMD molecules bound in the close vicinity. A comparison of ultrasonic and DNase I cleavage patterns of AMD–DNA complexes provided more detail on the local conformation and dynamics of the DNA double helix in both binding site and adjacent regions. The results pave the way for developing a novel approach to studies of the nucleotide sequence dependence of DNA conformational dynamics and new techniques to identify functional genome regions.     

Antibiotic induced electrophoretic mobility shifts of DNA restriction fragments.

Several antibiotics, netropsin, distamycin A, actinomycin D, Hoechst 33258 and olivomycin, which demonstrate base specificity in their DNA binding properties have been found to alter the electrophoretic mobility of DNA restriction fragments in native polyacrylamide gels. The antibiotics mostly reduced the migration of larger DNA fragments, but netropsin and Hoechst 33258 were observed to increase the migration rate of several DNA fragments of intermediate size. DNA fragments of similar molecular weight which comigrate as a single gel band can at times be separated as the result of differential mobility shifts promoted by antibiotic DNA complex formations.