Comparative microcalorimetric and dilatometric analysis of the interactions of quinacrine,chloroquine, and ethidium bromide with DNA

The enthalpies of binding of chloroquine and quinacrine to DNA at different molar ratios of drug to DNA and at different ionic strengths have been measured. The limiting values obtained with quinacrine fall in the range found for typical intercalating agents (e.g., ethidium, proflavin, adriamycin), whereas the value obtained with chloroquine is always zero, independent of the ratio of drug to DNA and ionic strength. The dilatometric measurements performed on the same systems and on the ethidium–DNA system show that when ethidium and quinacrine bind to DNA at low drug/DNA ratios, a volume decrease of about 16 mL/mol of bound drug occurs. No change in volume is observed when the two drugs bind to DNA through external, electrostatic forces. The volume change can be attributed to the loss of structured water around hydrophobic moieties of the drug molecules, following intercalation. In contrast, chloroquine binding to DNA at low drug/DNA ratios is characterized by a volume change distinctly smaller than that shown by quinacrine. The low ΔV_B and ΔH_B values shown by chloroquine are discussed in terms of the mechanism of interaction with DNA.     

Comparative binding study of the interaction of quinacrine and ethidium bromide with DNA and nucleohistone

The degree of binding of quinacrine dihydrochloride and ethidium bromide to DNA and nucleohistone has been determined by direct and indirect methods.The results obtained by the equilibrium dialysis experiments have been analyzed in terms of various theoretical models and have led us to propose for the interaction of the dyes with DNA at low ionic strength a structural scheme where the external binding sites are next to the intercalative ones.The equilibrium dialysis results were used to check those obtained by the indirect methods, i.e. absorption and fluorescence titrations, and to identify the origin of the discrepancies.The comparative study of the binding of these dyes to DNA and nucleohistone has shown that the accessible part of DNA in the nucleoprotein is to some extent different from free DNA itself.     

Analysis of cooperativity and ion effects in the interaction of quinacrine with DNA

The interaction of quinacrine with calf thymus DNA was monitored at several different ionic strengths using spectrophotometric and equilibrium dialysis techniques. The binding results can be explained, assuming each base pair is a potential binding site, using a model containing two negative cooperative effects: (1) ligand exclusion at binding sites adjacent to a filled binding site and (2) ligand–ligand negative cooperativity at adjacent filled binding sites. The logarithm of the observed equilibrium constant (K_obs) determined by this model varies linearily with log[Na⁺], as predicted by the ion condensation theory for polyelectrolytes. When the log K_obs plot is correlated for sodium release by DNA in the intercalation conformational change, the predicted number of ion pairs between the ligand and DNA is approximately two, as expected for the quinacrine dication. Even though K_obs depends strongly on ionic strength, the ligand negative cooperativity parameter ω was found to be indpendent of ionic strength within experimental error. This finding is also in agreement with the ion condensation theory, which predicts a relatively constant amount of condensed counterion on the DNA double helix over this ionic strength range. Drugs would, therefore, experience a relatively constant ionic environment when complexed to DNA even though the ionic conditions of the solvent could change considerably.     

Effect of copper ions on the photochemical activity of quinacrine and its interaction with DNA

The influence of cuprum ions on the interaction between the antimalarial drug quinacrine (QA) and DNA is studied by polarized laser luminescence spectroscopy and fluorescence microscopy at molecular and cellular levels. An alteration of quinacrine luminescence intensity in complex with DNA caused by cuprum ions is explained in terms of redistribution of QA molecules from quenching GC- to fluorescent AT-DNA binding sites due to the competition of Cu2+ with the dye. Mechanisms of component interactions in the triplex "DNA-QA-Cu2+" in model and cellular systems are shown to be in qualitative agreement. QA photodynamic activity change caused by Cu2+ action is explained on the basis of the ideas being developed.     

Testing of chloroquine and quinacrine for mutagenicity in Drosophila melanogaster

To extend the data on the mutagenic effects of intercalating agents in Drosophila melanogaster, chloroquine and quinacrine were tested for the induction of genetic damage in D. melanogaster males. Sex-linked recessive lethals and sex-chromosome loss induction were studied following treatment of adult males using a feeding technique. Our results show that both intercalating compounds increase significantly the frequency of sex-linked recessive lethals, but are unable to induce sex-chromosome loss in male germ cells under the conditions of testing.     

Kinetic studies of the interaction of synthetic RNAs with quinacrine and its analogs.

Temperature-jump relaxation method has been used to study the interaction of synthetic RNAs with quinacrine (QAC) and its analog. Two relaxation times were observed. The dependence of relaxation times on the RNA concentration and optical properties of the RNA-dye complexes suggests that (1) QAC binds to poly-(rA).poly(rU) through two bimolecular reactions including isomerization from one complex form to another and (2) the 2-methoxy group of the acridine ring plays a significant role in the isomerization.     

Spectroscopic studies of the interaction of synthetic RNAs with quinacrine and its analogs.

The interaction of synthetic RNAs with quinacrine (QAC) and its analogs has been studied by absorption, fluorescence and circular dichroism (CD) measurements. The results indicate that the 2-methoxy group of the acridine ring plays an important role in the appearance of the new absorption band upon binding of QAC to poly-(rA).poly(rU).     

The interaction of Bacillus protoplasts with sonicated phosphatidylcholine liposomes

When protoplasts from Bacillus subtilis are incubated with sonicated liposomes made from egg-yolk phosphatidylcholine, this phospholipid is incorporated into the protoplast membranes. Biochemical, fluorescence and ultrastructural data suggest that incorporation occurs through membrane fusion.     

Interaction of superhelical DNA with the nonhistone protein HMG1

The interaction between nonhistone chromosomal protein HMG1 and plasmid DNA was studied by optical and hydrodynamical methods. The recombinant protein HMG1 produced by yeast Pichia pastoris strain was used. We have shown that according to the CD spectra local conformational changes in DNA helix occur in the region of DNA-protein interaction. These changes are most significant at r < 3 (w/w). Both gel-shift assay and ultracentrifugation, as well as CD data, indicate that protein-protein interactions between HMG1 molecules play a major role in the formation of DNA-protein complexes. It is suggested that the protein C-terminus may affect HMG1-DNA binding not only by a direct interaction with DNA helix, but also by protein-protein interactions.     

Energetics of the strand separation transition in superhelical DNA.

In this paper the values of three free energy parameters governing the superhelical strand separation transition are determined by analysis of available experimental data. These are the free energy, a, needed to initiate a run of separation, the torsional stiffness, C, associated with interstrand winding of the two single strands comprising a separated site and the coefficient, K, of the quadratic free energy associated to residual linking. The experimental data used in this analysis are the locations and relative amounts of strand separation occurring in the pBR322 DNA molecule and the measured residual linking, both evaluated over a range of negative linking differences. The analytic method used treats strand separation as a heteropolymeric, co-operative, two-state transition to a torsionally deformable alternative conformation, which takes place in a circular DNA molecule constrained by the constancy of its linking number. The values determined for these parameters under the experimental conditions (T = 310 K, pH = 7.0, monovalent cation concentration = 0.01 M) are a = 10.84(+/- 0.2) kcal/mol, C = 2.5(+/- 0.3) x 10(-13) erg/rad2 and K = 2350(+/- 80) RT/N, where N is the molecular length in base-pairs. In order to assess the accuracy of the author's theoretical methods, these free energy parameters are incorporated into the analysis of superhelical strand separation in different molecules and under other conditions than those used in their evaluation. First, the temperature dependence of transition is treated, then superhelical strand separation is analyzed in a series of DNA molecules having systematic sequence modifications, and the results of these theoretical analyses are compared with those from experiments. In all molecules, transition is predicted in the range of linking differences where it is seen experimentally. Moreover, it occurs at the specific sequence locations that the analysis predicts, and with approximately the predicted relative amounts of transition at each location. The known sensitivities of this transition to changes of temperature and to small sequence modifications are predicted in a quantitatively precise manner by the theoretical results. The demonstrated high-level precision of these theoretical methods provides a tool for the screening of DNA sequences for sites susceptible to superhelical strand separation, some of which may have regulatory or other biological significance.     

Studies on the interaction of H1 histone with superhelical DNA: characterization of the recognition and binding regions of H1 histones.

The very lysine rich histone, H1, isolated from a variety of sources interacts preferentially with superhelical DNA compared to relaxed DNA duplexes. The nature of this specific interaction has been investigated by studying the ability of various purified fragments of H1 histone from calf thymus to recognize and bind superhelical DNA. The data suggest that the globular region of the H1 histone molecule (amino acid residues 72-106) is involved in the recognition of superhelical DNA. Thus, the H1 histone carboxy-terminal fragment, 72-212, resembles native H1 histone both quantitatively and qualitatively in its ability to discriminate between and bind to superhelical and relaxed DNA while the H1 histone carboxy-terminal fragment, residues 106-212, has lost this specificity, binding superhelical and relaxed DNA equally well. Furthermore, under conditions in which the globular region of the intact H1 histone has been unfolded, the molecule loses its ability to discriminate between superhelical and relaxed DNA, and binds both forms of DNA equally.     

Ultraviolet light irradiation of PM2 superhelical DNA.

Superhelical PM2 DNA can be photochemically modified by u.v. irradiation. The variation of S20,w with dose shows the following characteristics. There is a linear increase from 28 to 31s produced by a low dose of u.v. irradiation (4,000 ergs/mm2). A plateau in S20,w occurs between 4,000 and 10,000 ergs/mm2. The S20,w then increases when irradiation is increased to 56,000 ergs/mm2. Thymine dimers are introduced proportional to dose throughtout the range of exposure to u.v. light. Sedimentation velocity-dye titrations reveal anomolous behavior, i.e. apparent increases in superhelix density (sigma). However, the dye-buoyant density procedure showed no change in sigma under the same conditions. The most satisfactory model for the data is preferential photochemical modification of premelted (possibly hairpin) sites as a greater rate than the introduction of photoproducts into duplex sites. The origin of the anomoly in the sedimentation velocity dye titrations is still unclear.