Footprinting reveals that nogalamycin and actinomycin shuffle between DNA binding sites.

The hypothesis that sequence-selective DNA-binding antibiotics locate their preferred binding sites by a process involving migration from nonspecific sites has been tested by footprinting with DNAase I. Footprinting patterns on the tyrT DNA fragment produced by nogalamycin and actinomycin change with time after mixing the antibiotic with the DNA. Sites of protection as well as enhanced cleavage are seen to develop in a fashion which is both temperature and concentration-dependent. At certain sites cutting is transiently enhanced, then blocked. Limited evidence for slow reaction with echinomycin and mithramycin is presented, but the kinetics of footprinting with daunomycin and distamycin appear instantaneous. The feasibility of adducing direct evidence for shuffling by footprinting seems to be governed by slow dissociation of the antibiotic-DNA complex. It may also be dependent upon the mode of binding, be it intercalative or non-intercalative in character.     

Interaction of synthetic pentapeptide with DNA: specificity of binding and compactness of DNA

Binding of synthetic pentapeptide Val-Thr-Thr-Val-Val-N2H2Dns (where Dns is a residue of 5-dimethylamino naphthyl-1-sulfonic acid) is studied by circular dichroism, electron microscopy and fluorescence methods. It is found that this peptide can self-associate in aqueous solution as revealed from the concentration-dependent changes in the UV absorbance and fluorescence spectra. At high peptide concentration (3.10(-4) M) massive peptide aggregates are formed in solution and can be visualized by electron microscopy. It is shown that pentapeptide binds to DNA predominantly in a self-associated form and exhibits preferences for certain nucleotide sequences. It binds more strongly to poly(dG).poly(dC) and poly[d(A-C)].poly[d(G-T)] than to poly(dA).poly(dT). The complex with poly(dA).poly(dT) dissociates in the presence of 0.05 M NaCl, whereas the complex with poly(dG).poly(dC) is stable even in the presence of 0.2 M NaCl. The binding is a cooperative process which is accompanied by compaction of DNA at peptide/DNA base pair ratios greater than 2. At the initial stage of the compaction process the coalescence of DNA segments covered by bound peptide molecules results in the formation of DNA loops stabilized by interaction between bound peptide molecules. Increasing peptide/DNA ratio leads to the formation of rod-like particles as revealed from electron microscopy studies. Further increase in the peptide concentration leads to folding of fibrillar macromolecular complexes into globula each containing a single DNA molecule.     

The in vitro interaction of naphthyridinomycin with deoxyribonucleic acids

The binding of naphthyridinomycin (NAP) to deoxyribonucleic acid was investigated using radioisotope labeled antibiotic. Dithiothreitol (DTT) enhances complex formation in a concentration dependent fashion but was found to be slightly inhibitory at concentrations above 10 mM. [C3H3]-NAP-DNA complexes, formed in the presence or absence of reducing reagents, were stable to Sephadex G-25 chromatography and precipitation with ethanol, indicating a strong bond formed between the drug and DNA. Time course studies showed that the difference between the binding of activated and non-activated antibiotic was a DTT-dependent burst. This was followed by a second phase of binding which was similar in both the activated and non-activated antibiotics. The activation of the antibiotic by DTT was a reversible reaction at pH 7.9. The activated form at pH 5.0 was extremely stable and did not revert to the unactivated form even after an 8-h incubation period. Antibiotic-DNA complex formation was pH independent between pH 5.0 and 7.0 for activated NAP. The non-activated antibiotic bound to DNA much better at pH 5.0 than at physiological pH values. Release of antibiotic from complexes (as followed by long term dialysis) formed in the presence of DTT and at pH 5.0 was biphasic, suggesting that the drug can bind to DNA in more than one way. A constant rate of antibiotic release was observed at pH 7.9 with or without DTT. At pH 2.0 and pH 12.0, greater than 95% of the antibiotic is released from the complexes. Most of the acid released antibiotic is NAP while most of the base released antibiotic had decomposed to a more polar compound. NAP binds well to calf thymus DNA, poly(dG) . poly(dC), and T4 DNA but shows significantly less affinity for poly(dA) . poly(dT), poly(dA . dT) . poly(dA . dT), poly(dG), poly(dC), poly(dI) . poly(dC) or poly(dG . dC) . poly(dG . dC). This specificity of NAP for DNA is similar to that observed for the pyrrolo(1,4)benzodiazepine antibiotics and saframycin A and S; all of which bind to double stranded DNA through their carbinolamine or masked carbinolamine functionalities. Two mechanisms which can explain the need for activation of NAP are also proposed.     

Kinetics of dissociation of nogalamycin from DNA: comparison with other anthracycline antibiotics

Stopped-flow spectrometry and simple mixing techniques have been employed to investigate the detergent-induced dissociation of anthracycline antibiotics from natural and synthetic DNAs. Both daunomycin and nogalamycin dissociate more slowly poly(dG-dC) than from poly(dA-dT), but the difference is much more marked for nogalamycin. With an equimolar mixture of poly(dG-dC) and poly(dA-dT), or with poly(dA-dC)·poly(dG-dT), dissociation of nogalamycin occurs very slowly. In all cases the release of antibiotic from a synthetic polynucleotide is a one-step process following a sinigle exponential. Dissociation of daunomycin, adrianmycin and iremycin from calf thymus DNA is a more complex reaction which requires a two-exponential fit, in contrast to earlier reports, but differences between the behaviour of the three antibotics are minor. Dissociation of nogalamycin from natural DNA requires a three-exponential fit, is in general far slower, and depends upon the base composition, the level of binding and the time allowed for the complex to equilibrate. It is concluded that sequence selectivity is minimal or lacking for daunomycin, whereas nogalamycin binding is sequence dependent and probably involves migration of the antibiotic between DNA binding sites. There is an inverse correlation between dissociation rate constants and antibacterial potency in simple tests.     

DNA and protein footprinting analysis of the modulation of DNA binding by the N-terminal domain of the Saccharomyces cerevisiae TATA binding protein

Recombinant full-length Saccharomyces cerevisiae TATA binding protein (TBP) and its isolated C-terminal conserved core domain (TBPc) were prepared with measured high specific DNA-binding activities. Direct, quantitative comparison of TATA box binding by TBP and TBPc reveals greater affinity by TBPc for either of two high-affinity sequences at several different experimental conditions. TBPc associates more rapidly than TBP to TATA box bearing DNA and dissociates more slowly. The structural origins of the thermodynamic and kinetic effects of the N-terminal domain on DNA binding by TBP were explored in comparative studies of TBPc and TBP by "protein footprinting" with hydroxyl radical (*OH) side chain oxidation. Some residues within TBPc and the C-terminal domain of TBP are comparably protected by DNA, consistent with solvent accessibility changes calculated from core domain crystal structures. In contrast, the reactivity of some residues located on the top surface and the DNA-binding saddle of the C-terminal domain differs between TBP and TBPc in both the presence and absence of bound DNA; these results are not predicted from the crystal structures. A strikingly different pattern of side chain oxidation is observed for TBP when a nonionic detergent is present. Taken together, these results are consistent with the N-terminal domain actively modulating TATA box binding by TBP and nonionic detergent modulating the interdomain interaction.     

Kinetics of dissociation of nogalamycin from DNA: comparison with other anthracycline antibiotics

Stopped-flow spectrometry and simple mixing techniques have been employed to investigate the detergent-induced dissociation of anthracycline antibiotics from natural and synthetic DNAs. Both daunomycin and nogalamycin dissociate more slowly from poly(dG-dC) than from poly(dA-dT) but the difference is much more marked for nogalamycin. With an equimolar mixture of poly(dG-dC) and poly(dA-dT), or with poly(dA-dC).poly(dG-dT), dissociation of nogalamycin occurs very slowly. In all cases the release of antibiotic from a synthetic polynucleotide is a one-step process following a single exponential. Dissociation of daunomycin, adriamycin and iremycin from calf thymus DNA is a more complex reaction which requires a two-exponential fit, in contrast to earlier reports, but differences between the behaviour of the three antibiotics are minor. Dissociation of nogalamycin from natural DNA requires a three-exponential fit, is in general far slower, and depends upon the base composition, the level of binding and the time allowed for the complex to equilibrate. It is concluded that sequence selectivity is minimal or lacking for daunomycin, whereas nogalamycin binding is sequence dependent and probably involves migration of the antibiotic between DNA binding sites. There is an inverse correlation between dissociation rate constants and antibacterial potency in simple tests.     

Subunit topography of RNA polymerase (E. coli) in the complex with DNA.

E. Coli RNA polymerase binding to different DNAs (from E. Coli, 5-bromodeoxyuridine (BrdUrd) substituted DNA and poly [d(BrU-A)] was induced with ultraviolet (U.V.) light to form protein-DNA crosslinked complexes. Two independent methods of analysis, polyacrylamide gel electrophoresis in SDS and chloroform extraction indicated the formation of a stable complex between the enzyme and DNA. The complexes were formed under different ionic strength conditions, at low enzyme to DNA ratios in order to approach the conditions of specific binding. In contrast there was no crosslinking of the complex in 1 M KCl solution which dissociates the enzyme from DNA. The efficiency of formation of strongly bound complex was found to be much higher with holoenzyme than with core enzyme. The following results were obtained : 1) The large subunits beta and beta' were found to be bound to DNA. 2) Relatively small amount of sigma subunit were bound to DNA while alpha subunits were essentially not attached to DNA. The high binding affinity of beta and beta' subunits was also observed in the studies of isolated subunits. These results lead to a model of enzyme-DNA complex in which the large beta and beta' subunits provide the contacts between the RNA polymerase and the DNA.     

Accessory proteins bind a primed template and mediate rapid cycling of DNA polymerase III holoenzyme from Escherichia coli

DNA polymerase III holoenzyme was assembled from pure proteins onto a primer template scaffold. The assembly process could be divided into two stages. In the time-consuming first stage, beta subunit and gamma.delta subunit complex were required in forming a tightly bound ATP-activated "preinitiation complex" with a single-stranded DNA bacteriophage circle uniquely primed with a synthetic pentadecadeoxyribonucleotide. This finding substantiates an earlier study using crude protein preparations in a homopolymer system lacking Escherichia coli single-stranded DNA binding protein (Wickner, S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3511-3515). In the second stage, the polymerase III core and the tau subunit rapidly seek out and bind the preinitiation complex to form DNA polymerase III holoenzyme capable of rapid and entirely processive replication of the circular DNA. ATP is not required beyond formation of the preinitiation complex. It is remarkable that the fully assembled DNA polymerase III holoenzyme is so stably bound to the primed DNA circle (4-min half-time of dissociation), yet upon completing a round of synthesis the polymerase cycles within 10 s to a new preinitiation complex on a challenge primed DNA circle. Efficient polymerase cycling only occurred when challenge primed DNA was endowed with a preinitiation complex implying that cycling is mediated by a polymerase subassembly which dissociates from its accessory proteins and associates with a new preinitiation complex. These subunit dynamics suggest mechanisms for polymerase cycling on the lagging strand of replication forks in a growing chromosome.     

Glucocorticoid receptor-steroid complex binding to DNA. Competition between DNA and DNA-cellulose.

The binding of the glucocorticoid receptor-steroid complex from a line of rat hepatoma tissue culture (HTC) cells to DNA has been examined. An equilibrium competition assay involving a constant, low total amount of double-stranded DNA was developed to compare the complex binding ability of DNA free in solution and bound to cellulose. This binding ability is lowered by a factor of five when DNA is associated with cellulose. Similar studies with HTC cell, calf-thymus, and Escherichia coli DNA revealed no difference in the relative number or affinity of binding sites for receptor-steroid complex in each DNA. The synthetic DNA molecules poly[d(A-T)-d(A-T)] and poly[d(G-C)-d(G-C)] bound complexes equally well but less than the three "natural" DNA molecules. This appears to be due to differences in acceptor site affinity and suggests that nucleotide complexity and/or sequence influences the affinity of HTC cell receptor-glucocorticoid complexes for DNA.     

Measurement of the absolute temporal coupling between DNA binding and base flipping

The absolute temporal couplings between DNA binding and base flipping were examined for the EcoRI DNA methyltransferase. The binding event (monitored using rhodamine-x fluorescence anisotropy) was monophasic with a second-order on-rate of 1.1 x 10(7) M-1 s-1 相似文献   

Spectroscopic studies of the multiple binding modes of a trimethine-bridged cyanine dye with DNA

The interaction between DNA and a benzothiazole-quinoline cyanine dye with a trimethine bridge (TO-PRO-3) results in the formation of three noncovalent complexes. Unbound TO-PRO-3 has an absorption maximum (λ_max) of 632 nm, while the bound dyes (with calf thymus DNA) have electronic transitions with λ_max = 514nm (complex I), 584nm (complex II) and 642 nm (complex III). The blue shifts in the electronic transitions and the bisignate shape of the circular dichroism bands indicate that TO-PRO-3 aggregates with DNA. Complex I has a high dye:base pair stoichiometry, which does not depend on base sequence or base modifications. The bound dyes exhibit strong interdye coupling, based on studies with a short oligonucleotide and on enhanced resonance scattering. From thermal dissociation studies, the complex is weakly associated with DNA. Studies with poly(dGdC)₂ and poly(dIdC)₂ and competitive binding with distamycin demonstrate that complex II is bound in the minor groove. This complex stabilizes the helix against dissociation. For complex III, the slightly red-shifted electronic transition and the stoichiometry are most consistent with intercalation. Using poly(dAdT)₂, the complexes have the following dye mole fractions (X_dye): X_dye = 0.65 (complex I), 0.425 (complex II) and 0.34 (complex III).     

Interaction of bleomycin A2 with poly(deoxyadenylylthymidylic acid). A proton nuclear magnetic resonance study of the influence of temperature, pH, and ionic strength

The binding of bleomycin A2 to poly(deoxyadenylylthymidylic acid) [poly(dA-dT)] has been monitored by proton nuclear magnetic resonance spectroscopy. This study includes an analysis of the effects of temperature, ionic strength, and pH. Sites of drug-nucleic acid interaction have been delineated on the basis of chemical shift perturbations of drug and nucleic acid resonances. The data indicate that the binding of the antibiotic occurs with partial intercalation of the aromatic bithiazole group and immobilization of the cationic dimethylsulfonium group. This complex dissociates as the nucleic acid is denatured to the single-stranded form. The absence of significant pH effects suggests that the N terminus of bleomycin A2, which contains the titratable groups, does not contribute to the interaction of the drug molecule with poly(dA-dT). The problems associated with assigning a specific geometry to the drug-nucleic acid complex are discussed.     

Synthesis and Intracellular Localization of Vaccinia Virus Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase

The time course of vaccinia deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase synthesis and its intracellular localization were studied with virus-infected HeLa cells. Viral RNA polymerase activity could be meassured shortly after viral infection in the cytoplasmic fraction of infected cells in vitro. However, unless the cells were broken in the presence of the nonionic detergent Triton-X-100, no significant synthesis of new RNA polymerase was detected during the viral growth cycle. When cells were broken in the presence of this detergent, extensive increases in viral RNA polymerase activity were observed late in the infection cycle. The onset of new RNA polymerase synthesis was dependent on prior viral DNA replication. Fluorodeoxyuridine (5 x 10(-5)m) prevented the onset of viral polymerase synthesis. Streptovitacin A, a specific and complete inhibitor of protein synthesis in HeLa cells, prevented the synthesis of RNA polymerase. Thus, the synthesis of RNA polymerase is a "late" function of the virus. The newly synthesized RNA polymerase activity was primarily bound to particles which sedimented during high-speed centrifugation. These particles have been characterized by sucrose gradient centrifugation. A major class of active RNA polymerase particles were considerably "lighter" than whole virus in sucrose gradients. These particles were entirely resistant to the action of added pancreatic deoxyribonuclease, and they were not stimulated by added calf thymus primer DNA. It is concluded that these particles are not active in RNA synthesis in vivo, and that activation occurs as a result of detergent treatment in vitro.     

Effect of isonicotinic acid hydrazide-copper complex on Rous sarcoma virus and its genome RNA.

The copper complex of the antituberculous drug, insonicotinic acid hydrazide (INH), inhibits the RNA-dependent DNA polymerase of Rous sarcoma virus and inactivates its ability to malignantly transform chick embryo cells. The INH-copper complex binds to the 70S genome RNA of Rous sarcoma virus (RSV), which may account for its ability to inhibit the RNA-dependent DNA polymerase. The complex binds RNA more effectively than DNA in contrast to M-IBT-copper complexes, which bind both types of nucleic acids equally. The homopolymers, poly rA and poly rU, are bound by the INH-copper complex to a greater extent than poly rC. Isonicotinic acid hydrazide alone and CuSO4 alone bind neither DNA, RNA, poly (rA), poly (rU), nor poly (rC). However, CuSO4 alone binds poly (rI); INH alone does not. In addition to viral DNA synthesis, chick-embryo cell DNA synthesis is inhibited by the INH-copper complex. The extent of inhibition of cellular DNA synthesis is greater than that of cellular RNA and protein synthesis. No selective inhibition of transformation in cells previously infected with Rous sarcoma virus is observed.     

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.     

Mechanisms of telomerase binding to telomeres

There are essentially two alternative mechanisms for the binding of telomerase to telomeres, assuming that a protective component is initially bound to the telomerase binding region on the telomeres. Either the protective (or blocking) agent first dissociates and telomerase binds thereafter, or telomerase binds first and the protective agent then dissociates from the ternary complex. In the limit, this second possibility permits the ternary complex to become a transition complex (creating another possible mechanism). Numerical simulation of both rapid mixing and chemical relaxation is used to study these alternatives. We aim to determine how the mechanisms may be distinguished experimentally and identify an appropriate experimental design. We show that rapid mixing experiments are better than chemical relaxation experiments, since the latter are more affected by the statistics of single molecule kinetics. However, hidden fast steps can only be revealed by chemical relaxation. The detection of mechanistic changes hinges on linking fluorescence reporters to the reaction components, either directly (chemically) or indirectly (via an indicator reaction). Fluorescence is excited by two-photon absorption in a small reaction volume. Various detection strategies and design issues are examined, including limitations imposed by diffusion. Constant rather than stopped flow is shown to be preferable.     

Spacing of polypyrimidine regions in mouse DNA as determined by poly(adenylate,guanylate) binding

The DNA from many higher organisms contains unexpectedly long runs of pyrimidine nucleotides (polypyrimidines). In mouse cells, these range in size from about 25 to 250 nucleotides and account for about 0.5% of the total DNA. They contain both thymine and cytosine in close to equimolar amounts but few or none are pure thymine or cytosine runs. When native DNA from mouse cells was banded in a CsCl gradient, polypyrimidines were part of DNA molecules of all base compositions. This indicates that, unlike Drosophila melanogaster DNA (Birnboim & Sederoff, 1975), they are not clustered as a satellite component. A procedure for isolating fragments of single-stranded DNA which contain polypyrimidines has been developed. Polypyrimidine sites in DNA are complexed with poly(A,G) and the DNA/poly(A,G) complex is bound to hydroxyapatite. The percentage of DNA which binds is a function of its chain length. A maximum of 60% of the DNA was bound, at lengths of > 15 × 10³ bases². We conclude that polypyrimidine sites are distributed throughout the majority of the mouse genome at intervals of 12 to 15 × 10³ bases.     

The binding of CC-1065 to thymidine and deoxyadenosine oligonucleotides and to poly(dA).poly(dT)

In this work, we report on the binding of the novel antitumor agent CC-1065 to poly(dA).poly(dT) and to mixtures of dA and dT oligomers as determined by electronic absorption and circular dichroism (CD) methods. In addition, the DNA binding properties of CC-1065 and its binding mechanism are compared to those of netropsin. CC-1065 binds to the polymer by at least three mechanisms to produce one irreversibly and two reversibly bound species. One reversibly bound species is moderately stable, but in time (days), it converts to the irreversibly bound species. Both of these species bind within the minor groove of the polymer and exhibit intense CC-1065 induced CD spectra. The other reversibly bound species does not acquire an induced CD. CC-1065 forces B-form duplex formation between mixtures of single strand dA and dT oligomers and binds irreversibly to the duplexes without showing the presence of an intermediate, reversibly bound species. The induced CD increases with increasing length of the oligomer, from the 5-mer (barely detectable CD) to the 14-mer (intense CD). The 7-, 10- and 14-mer mixtures bind about 1, between 1 and 2, and between 2 and 3 CC-1065 molecules, respectively. Computer graphic models of the CC-1065-DNA complex show that the covalent adduct of CC-1065 and unreacted CC-1065 can attain the same close van der Waals contacts between adenine C2 hydrogens and antibiotic CH groups that were observed in the crystal structure of the netropsin-DNA complex. These contacts may account for the dA-dT base pair binding specificity of CC-1065 and for the stability of the reversibly bound CC-1065 species.     

Anomalous excitonic CD of meso-tetrakis(3-N-methylpyridiniumyl)porphyrin bound to poly[d(A-T)(2)

When meso-tetrakis(3-N-methylpyridiniumyl)porphyrin (m-TMPyP) formed a complex with poly[d(A-T)(2)], an intense bisignate excitonic CD in the Soret absorption region was observed. The excitonic CD of the m-TMPyP-poly[d(A-T)(2)] complex is unique in that no other combination of the related porphyrin, namely, meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (where n = 2, 4), and polynucleotide including calf thymus DNA, poly[d(G-C)(2)], poly[d(I-C)(2)], and poly(dA).poly(dT), exhibits a comparable CD spectrum. From the [drug]/[DNA] ratio-dependence of the intensity and the shape of the CD spectrum, this porphyrin species is assigned to an extensively aggregated form. The extensively aggregated porphyrin disperses in 1 h after mixing to form moderately stacked porphyrin at a low mixing ratio. The magnitude of linear dichroism of the extensively aggregated porphyrin was small and the sign was negative in the Soret band, which indicated that the molecular plane of porphyrin in the complex is strongly tilted. On the other hand, the molecular plane of porphyrin is almost parallel to the DNA base plane (perpendicular to the DNA helix axis) in the moderately stacked form.