Daunomycin disrupts nuclear assembly and the coordinate initiation of DNA replication in Xenopus egg extracts

We have used Xenopus egg extracts to investigate the effects of the antitumor drug daunomycin on DNA replication in vitro. Xenopus sperm nuclei replicated nearly synchronously in our egg extracts, thereby allowing us to determine the effects of the drug on both replication initiation and elongation. Titration experiments demonstrated that daunomycin effectively inhibited replication in the extract, with 50% inhibition at a total drug concentration of 2.7 μM. However, a high concentration of daunomycin 150 μM) also inhibited nuclear envelope assembly, a prerequisite for the initiation of replication in this system. Therefore, to bypass the effects of daunomycin on nuclear envelope assembly, sperm nuclei were preassembled in extract prior to drug addition. Initiation of replication in preassembled nuclei was also inhibited by daunomycin, with 50% inhibition at a drug concentration of 3.6 μM. At low drug concentrations, where replication did occur, the synchrony of initiations within individual nuclei was lost. This drug-induced disruption of initiation events may provide important clues regarding the mechanism(s) by which these events are coordinated in eukaryotic cells. Daunomycin also inhibited replication elongation in preassembled, preinitiated nuclei. However, the concentration of drug required for 50% inhibition of elongation was nearly fourfold higher than that required for inhibition of initiation. Taken together, these data demonstrate that Xenopus egg extract can be used to investigate the effects of DNA-binding antitumor drugs on a number of interrelated cellular processes, many of which are less tractable in whole cell systems. J. Cell. Biochem. 64:476–491. © 1997 Wiley-Liss, Inc.     

On the inhibition of the RNA polymerase-catalysed synthesis of RNA by daunomycin. Interference of the inhibitor with elongation and pre-longation steps

The inhibition of the RNA polymerase-catalyzed synthesis of RNA by daunomycin was examined. Saturation binding of daunomycin to the template leads, as expected, to complete inhibition of RNA synthesis as a result of daunomycin interference with enzyme-template interactions. However at concentrations of the inhibitor below saturation formation of the enzyme-template complex remains remarkably undisturbed, while both the transformation of this complex to an elongating complex and the elongation of the nacsent RNA chains are substantially inhibited. Clearly, daunomycin interferes with a number of different substeps of RNA synthesis and inhibits the synthesis by different mechanisms depending on the amount of inhibitor bound to the template. Elucidation of the mechanism of inhibition at low daunomycin concentrations may be a prerequisite for a better understanding of the mechanism of the pharmacological action of the drug.     

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.     

Investigation of sesquiterpene lactones as protein synthesis inhibitors of P-388 lymphocytic leukemia cells

The mode of action of helenalin and bis(helenalinyl) malonate as protein synthesis inhibitors of P-388 lymphocytic leukemia cells was investigated. The initial characterizations were carried out in crude lysates of the P-388 cells. In the lysate, there was a 4 min lag after the addition of drug before inhibition of protein synthesis occurred. Both drugs allowed run-off of preformed polysomes, but did significantly inhibit the formation of the 80 S initiation complex suggesting a preferential inhibition of one or more initiation reactions. The effect of these drugs on inhibition of both elongation and initiation reactions was further investigated using more fractionated systems prepared from P-388 cells. Poly(U)-directed polyphenylalanine synthesis was marginally inhibited by both drugs, but the degree of inhibition was not sufficient to explain the inhibition observed in either the lysate or in whole cell preparations of P-388. The formation of the ternary initiation complex was not significantly inhibited by either drug, but the conversion of this complex to the 48 and 80 S initiation complexes was inhibited. The inhibition of 48 S initiation complex formation by both drugs was sufficient to explain their inhibition of protein synthesis in whole cells.     

Differential scanning calorimetric study of the effect of intercalators and other kinds of DNA-binding drugs on the stepwise melting of plasmid DNA.

The effect of intercalating drugs (the anthracycline group of antibiotics, ethidium bromide, actinomycin D) on stepwise melting of DNA was studied by differential scanning calorimetry (DSC). The DSC DNA melting profile of plasmid pJL3-TB5 DNA (5277 base-pairs in length) consists of seven peaks, and all the intercalators caused shifting of these peaks, particularly those formed at the high temperature ranges, to the higher temperature ranges in a characteristic manner depending upon the binding strength of the drug. The analysis of the anthracycline group of antibiotics, such as aclacinomycin A, daunomycin, adriamycin and pyrarubicin, indicates that the difference in binding is due to the sugar moiety at position O-7 of the chromophore in these antibiotics. Analysis on the basis of the helix-coil transition theory suggests that the anthracycline group of antibiotics interact preferentially with the 5'-CG-3' sequences. The effect of various DNA-binding drugs other than intercalators on stepwise melting of DNA was then studied by DSC. The representative drugs examined were distamycin A, peplomycin, cis-dichlorodiamine-platinum(II) (cis-DDP or cis-Platin) and mitomycin C, which differ in their mode of interaction with DNA; namely, minor groove binding, strand cleavage and intrastrand or interstrand cross-linking. Distamycin A caused shifting of the DSC peaks at the low temperature ranges to a higher temperature range, whereas peplomycin and cis-DDP caused shifting of all the DSC peaks to form a broad peak at a lower temperature range, suggesting that the DSC DNA melting profiles are affected in a characteristic manner depending upon the interaction mode of the drug.     

Inhibition of the thermally driven B to Z transition by intercalating drugs

Poly(dG-m5dC) in phosphate buffer containing 50 mM NaCl and Mg2+ will undergo a reversible thermally driven conversion from the B to the left-handed Z conformation. The temperature at the midpoint of the thermally driven B to Z transition (denoted Tz) is dependent upon the total Mg2+ concentration, with [d(1/Tz)]/(d ln [Mg]) = 0.0134 K-1. The Mg2+ concentration at the midpoint of the equilibrium B to Z transition curve, denoted [Mg]1/2, is dependent on temperature, with (d ln [Mg]1/2)/(d ln T) = -1.02. Binding of the anticancer drug daunomycin to the polymer results in a pronounced increase in Tz, dependent on the molar ratio of added drug. Tz is increased by 71.9 degrees C with nearly saturating amounts of drug bound. Transition profiles are biphasic at less than saturating amounts of bound drug. By experiments monitoring such biphasic curves at a visible wavelength sensitive to the binding of daunomycin, it may be demonstrated that no drug is released until the later phase of the transition. These results are analogous to the effects of intercalating drugs on the thermal denaturation of DNA and indicate that drug molecules preferentially interact with B-form DNA and are redistributed to regions in the B conformation over the course of the transition. Comparative studies show that some intercalators stabilize right-handed DNA more effectively than others. At similar initial binding ratios, the following order, from most to least effective, was experimentally observed: actinomycin greater than daunomycin greater than ethidium greater than proflavin.     

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.     

Anthracycline-binding induced DNA stiffening, bending and elongation; stereochemical implications from viscometric investigations

Upon interaction of the three anthracycline antibiotics daunomycin, adriamycin, and aclacinomycin A with calf thymus DNA the relative changes of both DNA contour length, delta L/Lo, and persistence length, delta a/ao, have been determined as a function of r, the ratio of bound ligand molecules per DNA mononucleotide. From the r dependence of delta a/ao a measure for the stiffening effect and also the angle gamma of ligand-induced DNA bending could be derived. Experimental basis are titration viscometric measurements upon both low and high molecular weight DNA. It was found that the DNA contour length increases linearly with r by approximately 0.34 nm per bound drug molecule. The comparatively very high DNA stiffening effect measured in solution is understandable as a result of helix clamping by at least two anthracycline groups of sufficient long distance. The variation of gamma on DNA interaction with different anthracycline derivatives find their explanation in terms of different values of the mismatch to in-register binding prior to complex formation. From an analogous interpretation of viscosity measurements by Arcamone and coworkers upon high molecular weight DNA with many anthracycline derivatives it can be concluded that DNA interaction by both amino sugar and 9-acetyl group are responsible for the generation of strong anthracycline binding mediated DNA stiffening effects in solution. (A combined analysis of the viscosity measurements by Cohen & Eisenberg and Armstrong et al. upon DNA interaction with proflavine indicates a very small DNA stiffening effect, gamma = 6.7 sigma and a helix elongation by 0.35 nm per bound ligand molecule.)     

Selective binding of actinomycin D induces a reversible conformational transition of nucleosomes

Equilibrium and hydrodynamic studies on the complex of actinomycin D with H1-H5 depleted, 175 basepair nucleosomes are reported. By spectral titration the intrinsic affinities of actinomycin D for nucleosomes and for DNA are found strictly comparable. Sedimentation analysis shows that actinomycin can apparently unfold the nucleosome, like ethidium bromide and daunomycin, but it does so at a much lower bound drug to DNA molar ratio (about 1 drug molecule to 45 basepairs). Since about four bound actinomycin molecules are able to induce the reversible conformational transition of a nucleosome, it is suggested that the sites of interaction may correspond to the kinked DNA sites evidenced by Klug and collaborators (Richmond, T.J., Finch, J.T., Rushton, B., Rhodes, D. and Klug, A. (1984) Nature 311, 532-537) in the structure of the nucleosome. A relevance of these findings to the interaction of actinomycin with "active chromatin" is also suggested.     

Study of the complex formation of daunomycin with deoxytetranucleotides with bases of differing sequence in an aqueous solution by 1H-NMR spectroscopy

The complex formation of the antibiotic daunomycin with deoxytetranucleotides of different base sequence in the chain, 5'-d(GpCpGpC), 5'-d(CpGpCpG), and 5'-d(TpGpCpA) in aqueous salt solution was studied by 1D and 2D (2M-TOCSY and 2M-NOESY) 1H-NMR spectroscopy. Concentration and temperature dependences of proton chemical shifts of molecules were measured. Based on these dependences, reaction equilibrium constants, relative content of various complexes depending on concentration and temperature, limiting values of chemical shifts of protons of daunomycin incorporated in various complexes, and the thermodynamic parameters delta H and delta S of complex formation were calculated. The analysis of the results enables the conclusion that the sites of predominant intercalation of daunomycin are triplet nucleotide sequences, the binding sites of the antibiotic with three consecutive GC pairs in the tetranucleotide duplex being more preferential. Daunomycin exhibits no sequence specificity upon binding to the single-stranded deoxynucleotide sequence. From the calculated values of induced chemical shifts of daunomycin protons and 2M-NOE data, the most probable spatial structures of complexes (1:2) of the antibiotic with deoxytetranucleotides were constructed. The binding of the second daunomycin molecule to both the single-stranded and duplex form of tetramers is of pronounced anticooperative mode, which is explained by the presence in the antibiotic of a positively charged amino sugar residue, which poses considerable steric constraints for the insertion of the second antibiotic molecule into the short tetranucleotide sequence. The results were compared with the data obtained under identical experimental conditions for typical intercalators proflavine and ethidium bromide.     

Influence of DNA-binding drugs on chromatin condensation

We have used transient electric dichroism to study the ability of DNA-binding drugs to affect the folding of chromatin from the 10- to the 30-nm fiber, either by themselves or in conjunction with multivalent cations. Variables considered include the cationic charge of the drug, the comparative influence of intercalation and groove binding as modes of interaction, and the effect of bis-intercalation compared to mono-intercalation. In parallel with our findings with other cations, we observe that a drug must have a charge of 3+ or greater in order to condense chromatin at concentrations substantially lower than the concentration of chromatin, measured in base pairs. Drugs of low charge, whether groove binders or mono-or bis-intercalators, are unable to condense chromatin on their own. Bis-intercalators of high charge, however, are extremely efficient condensers, being able to cross-link chromatin with greater efficiency than polyamines of corresponding charge. When Mg2+ is used in combination with bis-intercalators of high charge, the order of addition of the two determines whether compaction or cross-linking is favored. Finally, the antibiotics actinomycin D, daunomycin, and distamycin, despite varied modes of binding to DNA, all inhibit the compaction of chromatin beyond a critical point in a remarkably similar manner.     

Mapping protease susceptibility sites on the Escherichia coli transcription factor sigma70.

N-terminally and C-terminally histidine-tagged versions of Escherichia coli RNA polymerase initiation factor sigma70 were subjected to limited proteolysis and electrophoretic separation. The protein fragments were transferred to nitrocellulose, and biotinylated nitrilotriacetic acid was used to detect the His-tagged ladder that resulted. Using size markers of known lengths derived from chemical cleavage of the same His-tagged sigma70, we were able to map the sites of proteolysis for sigma70 free in solution, bound to core RNA polymerase, and in the Mg2+-dependent open complex with lambdaPR promoter DNA. Numerous sites of changed susceptibility were mapped. Most of these sites mapped near residues 100 and 500. In addition, the highly acidic region around residue 190 became susceptible to cleavage in the open promoter complex. These results suggest that sigma70 undergoes significant conformational changes upon binding to core RNA polymerase and upon open promoter complex formation.     

Escherichia coli SeqA protein affects DNA topology and inhibits open complex formation at oriC.

Chromosome replication in Escherichia coli is initiated by the DnaA protein. Binding of DnaA to the origin, oriC, followed by formation of an open complex are the first steps in the initiation process. Based on in vivo studies the SeqA protein has been suggested to function negatively in the initiation of replication, possibly by inhibiting open complex formation. In vitro studies have shown that SeqA inhibits oriC-dependent replication. Here we show by KMnO(4) probing that SeqA inhibits open complex formation. The inhibition was not caused by prevention of DnaA binding to the oriC plasmids, indicating that SeqA prevented strand separation in oriC either directly, by interacting with the AT-rich region, or indirectly, by changing the topology of the oriC plasmids. SeqA was found to restrain the negative supercoils of the oriC plasmid. In comparison with the effect of HU on plasmid topology, SeqA seemed to act more cooperatively. It is likely that the inhibition of open complex formation is caused by the effect of SeqA on the topology of the plasmids. SeqA also restrained the negative supercoils of unmethylated oriC plasmids, which do not bind SeqA specifically, suggesting that the effect on topology is not dependent on binding of SeqA to a specific sequence in oriC.     

Hetero-association of caffeine and aromatic drugs and their competitive binding with a DNA oligomer

NMR spectroscopy has been used to elucidate the molecular basis of the action of caffeine (CAF) on the complexation with DNA of mutagens such as ethidium bromide, propidium iodide, proflavine and acridine orange, and anticancer drugs such as actinomycin D and daunomycin. The hetero-association of CAF and each of the aromatic ligands in 0.1 mol L(-1) phosphate buffer (pD=7.1) has been investigated as a function of concentration and temperature by 500 MHz 1H NMR spectroscopy and analysed in terms of a statistical-thermodynamic model, in which molecules form indefinite aggregates for both self-association and hetero-association. The analysis leads to determination of the equilibrium constants of hetero-association and to the values of the limiting chemical shifts of the heteroassociation of CAF with each of the aromatic molecules. The hetero-association constants between CAF and each of the aromatic drugs/dyes are found to be intermediate in magnitude between those for self-association of CAF and the corresponding drug/dye. The most probable structures of the 1:1 CAF + ligand hetero-association complexes have been determined from the calculated values of the induced limiting chemical shifts of the drug protons. Knowledge of the equilibrium constants for self-association of CAF and the aromatic ligands, for their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the CAF-ligand and CAF-ligand-d(TGCA) complexes to be calculated as a function of CAF concentration in mixed solutions. It is concluded that, on addition of CAF to the solution, the decrease in binding of drug or mutagen with DNA is due both to competition for the binding sites by CAF and the aromatic molecules, and to formation of CAF-ligand hetero-association complexes in the mixed solution; the relative importance of each process depends on the drug or mutagen being considered.     

Binding of the Lactococcal Drug Dependent Transcriptional Regulator LmrR to Its Ligands and Responsive Promoter Regions

The heterodimeric ABC transporter LmrCD from Lactococcus lactis is able to extrude several different toxic compounds from the cell, fulfilling a role in the intrinsic and induced drug resistance. The expression of the lmrCD genes is regulated by the multi-drug binding repressor LmrR, which also binds to its own promoter to autoregulate its own expression. Previously, we reported the crystal structure of LmrR in the presence and absence of the drugs Hoechst 33342 and daunomycin. Analysis of the mechanism how drugs control the repressor activity of LmrR is impeded by the fact that these drugs also bind to DNA. Here we identified, using X-ray crystallography and fluorescence, that riboflavin binds into the drug binding cavity of LmrR, adopting a similar binding mode as Hoechst 33342 and daunomycin. Microscale thermophoresis was employed to quantify the binding affinity of LmrR to its responsive promoter regions and to evaluate the cognate site of LmrR in the lmrCD promoter region. Riboflavin reduces the binding affinity of LmrR for the promoter regions. Our results support a model wherein drug binding to LmrR relieves the LmrR dependent repression of the lmrCD genes.