Copper(II) facilitates bleomycin-mediated unwinding of plasmid DNA

The unwinding of plasmid DNA by bleomycin A2 (BLM A2) was investigated by use of two-dimensional gel electrophoresis. It was found that Cu2+ ions greatly facilitated the unwinding of topoisomers of plasmid DNA by BLM A2 at concentrations where cupric ions alone had no effect on DNA supercoiling. The concentration of BLM A2 required for observable unwinding was reduced at least 100-fold in the presence of equimolar Cu2+. A plot of [Cu2+] vs extent of DNA unwinding in the presence of 10(-4) M BLM A2 gave a curve consistent with the action of cupric ions on BLM in an allosteric fashion, possibly rearranging the drug into a conformation that facilitates DNA unwinding. The participation of the metal center in enhancing DNA unwinding via direct ionic interaction with one or more negatively charged groups on the DNA duplex also seems possible. Further analysis of the structural factors required for BLM-mediated DNA unwinding was carried out with Cu2+ + BLM demethyl A2, the latter of which differs from BLM A2 only in that it lacks a methyl group, and associated positive charge, at the C-terminus. Cu(II).BLM demethyl A2 was found to be much less effective than Cu(II).BLM A2 as a DNA unwinding agent, emphasizing the strong dependence of this process on the presence of positively charged groups within the BLM molecule. These findings constitute the first direct evidence that the metal center of BLM can participate in DNA interaction, as well as in the previously recognized role of oxygen binding and activation.     

Simultaneously monitoring DNA binding and helicase-catalyzed DNA unwinding by fluorescence polarization

A new method for helicase-catalyzed DNA unwinding is described. This assay takes advantage of the substantial change in fluorescence polarization (FP) upon helicase binding and DNA unwinding. The low anisotropy value, due to the fast tumbling of the free oligonucleotide in solution, increases abruptly upon binding of helicase to the fluorescein-labeled oligonucleotide. The high anisotropy of the helicase– DNA complex decreases as the fluorescein-labeled oligonucleotide is released from the complex through helicase-catalyzed DNA unwinding. This FP signal can be measured in real time by fluorescent spectroscopy. This assay can simultaneously monitor DNA binding and helicase-catalyzed DNA unwinding. It can also be used to determine the polarity in DNA unwinding mediated by helicase. This FP assay should facilitate the study of the mechanism by which helicase unwinds duplex DNA, and also aid in screening for helicase inhibitors, which are of growing interest as potential anticancer agents.     

The effect of ionic strength on DNA-ligand unwinding angles for acridine and quinoline derivatives.

We have quantitatively examined the unwinding angles for the complexes of a related series of acridine and quinoline derivatives with DNA. Ethidium bromide was used as a control for determining superhelix densities at different ionic strengths. Relative to ethidium, 9-aminoacridine and quinacrine had an essentially constant unwinding angle of approximately 17 degrees at all ionic strengths tested. The apparent unwinding angle for chloroquine and 9-amino-1,2,3,4-tetrahydroacridine was found to be ionic strength dependent, increasing with increasing ionic strength. This suggests that competitive nonintercalative binding at low ionic strengths causes an apparent lowering of the quinoline unwinding angle. This can also explain why 4-aminoquinaldine, examined at low ionic strength, gives a quite low apparent unwinding angle. Quinacrine along with chloroquinine and 9-aminoacridine approaches a limiting value for their unwinding angle of approximately 17 degrees. 4-aminoquinaldine and 9-amino-1,2,3,4-tetrahydroacridine could not be examined at an ionic strength above 0.03 because of their very low equilibrium binding constants.     

Studies on amikhellin: I. Intercalative binding to double-stranded DNA

The present paper reports that amikhellin, a drug so far used as a coronary vasodilator, binds to double-stranded DNA by an intercalation process which does not depend upon DNA base composition. The binding to DNA was established by spectrophotometry, ultracentrifugation and competition with ethidium bromide. The parameters of the binding equilibrium were calculated by these two latter methods. Evidence for intercalation was obtained from the observation by viscosimetric experiments of the length increase of sonicated calf thymus DNA and of the untwisting of circular PM2 DNA. The unwinding angle was measured to be 6° per bound drug molecule.     

Intercalative binding to DNA of antitumour drugs derived from 3-nitro-1,8-naphthalic acid.

Two new antitumour drugs, imide derivatives of 3-nitro-1,8-naphthalic acid having different basic side chains linked to the imide nitrogen, have been shown to bind to double-helical DNA by intercalation. At ionic strength 0.01 mol/litre, pH 7, their intrinsic association constants are about 1.45 x 10(5) M-1 and each bound ligand molecule occludes about 3.4 nucleotides of the DNA lattice. They remove and reverse the supercoiling of closed circular duplex PM2 DNA with apparent unwinding angles of 11-12 degrees per bound drug molecule, referred to an assumed unwinding angle of 26 degrees for ethidium. They increase the viscosity of sonicated rod-like DNA fragments, each bound drug molecule producing a calculated increment in length of 2.2 - 2.5 A. No important differences between the DNA-binding characteristics of the two drugs were detected, though one appears marginally more active than the other in certain biological tests.     

The binding of echinomycin to deoxyribonucleic acid.

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.     

A rapid method for detecting DNA strand breaks in Mytilus galloprovincialis Lam. induced by genotoxic xenobiotic chemicals

1. In this study, DNA from haemolymph cells of Mytilus galloprovincialis Lam., as well as from L1210 (murine leukemia) mouse cells was investigated utilizing the technique of the alkaline unwinding of the double stranded DNA molecule. 2. The data show that DNA of haemolymph cells from the marine invertebrate has an unwinding time and, therefore, a molecular weight considerably lower than that of DNA of mammalian cells. 3. The exposure of the cells from mussel haemolymph and from mouse L1210 to a genotoxic compound such as dimethylsulfate results in DNA damage and consequently in a reduction of the unwinding time. 4. These results suggest that the fluorimetric DNA unwinding assay can be used in studies concerning the damage of DNA of marine organisms induced by genotoxic compounds or environmental factors.     

Processivity of nucleic acid unwinding and translocation by helicases

Helicases are a class of enzymes that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid (NA) strand separation. Besides the NA unwinding speed, another important factor for the helicase activity is the NA unwinding processivity. Here, we study the NA unwinding processivity with an analytical model that captures the phenomenology of the NA unwinding process. First, we study the processivity of the non‐hexameric helicase that can unwind NA efficiently in the form of a monomer and the processivity of the hexameric helicase that can unwind DNA effectively, providing quantitative explanations of the available single‐molecule experimental data. Then, we study the processivity of the non‐hexameric helicases, in particular UvrD, in the form of a dimer and compare with that in the form of a monomer. The available single‐molecule and some biochemical data showing that while UvrD monomer is a highly processive single‐stranded DNA translocase it is inactive in DNA unwinding, whereas other biochemical data showing that UvrD is active in both single‐stranded DNA translocation and DNA unwinding in the form of a monomer can be explained quantitatively and consistently. In addition, the recent single‐molecule data are also explained quantitatively showing that constraining the 2B subdomain in closed conformation by intramolecular cross‐linking can convert Rep monomer with a very poor DNA unwinding activity into a superhelicase that can unwind more than thousands of DNA base pairs processively, even against a large opposing force. Proteins 2016; 84:1590–1605. © 2016 Wiley Periodicals, Inc.     

Neocarzinostatin chromophore binds to deoxyribonucleic acid by intercalation

The nonprotein chromophore of neocarzinostatin was found to share many of the characteristics of classical intercalators in its interaction with DNA. Viscosity studies with PM2 DNA indicated that the DNA helix unwinding induced by the chromophore was 0.82 times that of ethidium or 21 degrees. Electric dichroism of the chromophore--DNA complex showed that each bound chromophore molecule lengthened DNA by 3.3 A and that absorbance transitions of the chromophore at 315--385 nm were oriented approximately parallel to DNA bases, as expected for an intercalated aromatic ring. Binding to DNA induced strong hypochromicity and a pronounced red shift in the absorbance spectrum of the chromophore. Spectrophotometric titrations suggested at least two types of chromophore binding sites on DNA; one type of site was saturated at rb = 0.125 chromophore molecule/nucleotide, but binding to additional sites continued to at least rb = 0.3. These physical--chemical studies were performed at pH 4--5 in order to keep the chromophore stable, but chromophore bound to an excess of DNA at pH 7 showed a stable absorbance spectrum identical with that seen at pH 4--5, suggesting that a similar type of binding occurs at neutral pH. Chromophore which had spontaneously degraded in pH 8 buffer did not bind to DNA at all, as judged by absorbance spectroscopy. The degree of protection afforded by DNA against spontaneous chromophore degradation implied a dissociation constant of approximately 5 microM for the DNA--chromophore complex at neutral pH and physiological ionic strength. Supercoiled DNA was nearly twice as effective as relaxed DNA in protecting chromophore from degradation, providing additional evidence for intercalation at neutral pH. Comparison of absorbance, fluorescence, and dichroism spectra suggests that the naphthalene ring system is the intercalating moiety.     

A rapid method for the measurement of the unwinding angle of intercalating agents and the superhelix density of circular DNAs.

By incubating covalently-closed circular DNA in the presence of calf thymus topoisomerase and unwinding ligands (intercalating drugs and proteins) DNAs of different superhelix density can be produced. These changes in superhelix density can be monitored by an ethidium fluorescence assay, since the level of ethidium binding varies with the superhelix density of a DNA. Thus the equivalence point, in a titration between an unwinding ligand and a supercoiled DNA, can be found. This forms the basis for an extremely rapid method for measuring unwinding angles and superhelix densities. Results are presented which agree well with those reported by previous authors using different techniques. The present method compares very favourably with others when evaluated in terms of rapidity, cost of materials, cost of equipment, accuracy and also applicability.     

Displacement of a DNA binding protein by Dda helicase

Bacteriophage T4 Dda helicase has recently been shown to be active as a monomer for unwinding of short duplex oligonucleotides and for displacing streptavidin from 3′-biotinylated oligonucleotides. However, its activity for streptavidin displacement and DNA unwinding has been shown to increase as the number of Dda molecules bound to the substrate molecule increases. A substrate was designed to address the ability of Dda to displace DNA binding proteins. A DNA binding site for the Escherichia coli trp repressor was introduced into an oligonucleotide substrate for Dda helicase containing single-stranded overhang. Here we show that a Dda monomer is insufficient to displace the E.coli trp repressor from dsDNA under single turnover conditions, although the substrate is unwound and the repressor displaced when the single-stranded overhang is long enough to accommodate two Dda molecules. The quantity of product formed increases when the substrate is able to accommodate more than two Dda molecules. These results indicate that multiple Dda molecules act to displace DNA binding proteins in a manner that correlates with the DNA unwinding activity and streptavidin displacement activity. We suggest a cooperative inchworm model to describe the activities of Dda helicase.     

Multiple Escherichia coli RecQ Helicase Monomers Cooperate to Unwind Long DNA Substrates: A FLUORESCENCE CROSS-CORRELATION SPECTROSCOPY STUDY*

The RecQ family helicases catalyze the DNA unwinding reaction in an ATP hydrolysis-dependent manner. We investigated the mechanism of DNA unwinding by the Escherichia coli RecQ helicase using a new sensitive helicase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. The FCCS-based assay can be used to measure the unwinding activity under both single and multiple turnover conditions with no limitation related to the size of the DNA strands constituting the DNA substrate. We found that the monomeric helicase was sufficient to perform the unwinding of short DNA substrates. However, a significant increase in the activity was observed using longer DNA substrates, under single turnover conditions, originating from the simultaneous binding of multiple helicase monomers to the same DNA molecule. This functional cooperativity was strongly dependent on several factors, including DNA substrate length, the number and size of single-stranded 3′-tails, and the temperature. Regarding the latter parameter, a strong cooperativity was observed at 37 °C, whereas only modest or no cooperativity was observed at 25 °C regardless of the nature of the DNA substrate. Consistently, the functional cooperativity was found to be tightly associated with a cooperative DNA binding mode. We also showed that the cooperative binding of helicase to the DNA substrate indirectly accounts for the sigmoidal dependence of unwinding activity on ATP concentration, which also occurs only at 37 °C but not at 25 °C. Finally, we further examined the influences of spontaneous DNA rehybridization (after helicase translocation) and the single-stranded DNA binding property of helicase on the unwinding activity as detected in the FCCS assay.     

Escherichia coli DNA helicase I catalyzes a unidirectional and highly processive unwinding reaction

Helicase I has been purified to greater than 95% homogeneity from an F+ strain of Escherichia coli, and characterized as a single-stranded DNA-dependent ATPase and a helicase. The duplex DNA unwinding reaction requires a region of ssDNA for enzyme binding and concomitant nucleoside 5'-triphosphate hydrolysis. All eight predominant nucleoside 5'-triphosphates can satisfy this requirement. Unwinding is unidirectional in the 5' to 3' direction. The length of duplex DNA unwound is independent of protein concentration suggesting that the unwinding reaction is highly processive. Kinetic analysis of the unwinding reaction indicates that the enzyme turns over very slowly from one DNA substrate molecule to another. The ATP hydrolysis reaction is continuous when circular partial duplex DNA substrates are used as DNA effectors. When linear partial duplex substrates are used ATP hydrolysis is barely detectable, although the kinetics of the unwinding reaction on linear partial duplex substrates are identical to those observed using a circular partial duplex DNA substrate. This suggests that ATP hydrolysis fuels continuous translocation of helicase I on circular single-stranded DNA while on linear single stranded DNA the enzyme translocates to the end of the DNA molecule where it must slowly dissociate from the substrate molecule and/or slowly associate with a new substrate molecule, thus resulting in a very low rate of ATP hydrolysis.     

Studies of the interaction of RecA protein with DNA

Ethidium fluorescence assays were adapted for the rapid and sensitive detection of precA; in addition, fluorescence measurements on binding precA to linear, OC and CCC PM2 DNAs have enabled the stoichiometry of precA binding as well as the precA-induced unwinding angle of DNA to be determined. The stoichiometry of binding was independently confirmed by sedimentation analysis to be one precA molecule per 3 bp. The unwinding angle was also independently confirmed by measurements of fluorescence changes induced by the binding of precA to CCC DNA which was relaxed by topoisomerase to give a precA-induced unwinding angle of 51 degrees. Electron microscopy of OC DNA molecules which bound nonsaturating amounts of precA revealed that the length increase in DNA due to precA was approximately 55%. Finally, examination of negatively stained precA complexes with a variety of linear DNAs showed that the minor groove is the primary site of interaction for this protein.     

Unwinding of chromatin by the SV40 large T antigen DNA helicase.

We have analysed the unwinding of nucleosomally organized DNA by simian virus 40 large tumour (T) antigen. Isolated T antigen can bind to existing nucleosome cores containing the viral replication origin sequence, which results in displacement of the histone octamer and unwinding of the DNA. However, specific binding to nucleosome cores is salt sensitive and nearly completely blocked under ionic conditions that otherwise support DNA replication. Once started, the progressing T antigen helicase, like an elongating RNA polymerase, is not further repressed by histone octamers, irrespective of the presence or absence of linker histone H1. Disruption of the nucleosomal structure in the process of unwinding may be assisted by the demonstrated interaction of the hexameric T antigen complex with histone proteins H1 and H3. Finally, our studies reveal the inability of topoisomerase I and/or II to continually relieve the superhelical tension of covalently closed circular minichromosomes as generated during their unwinding by T antigen. This may indicate that chromatin relaxation during the process of DNA replication can only be efficiently performed by a topoisomerase that is (trans)activated by other factors.     

Control of RecBCD Enzyme Activity by DNA Binding- and Chi Hotspot-Dependent Conformational Changes

Faithful repair of DNA double-strand breaks by homologous recombination is crucial to maintain functional genomes. The major Escherichia coli pathway of DNA break repair requires RecBCD enzyme, a complex protein machine with multiple activities. Upon encountering a Chi recombination hotspot (5′ GCTGGTGG 3′) during DNA unwinding, RecBCD's unwinding, nuclease, and RecA-loading activities change dramatically, but the physical basis for these changes is unknown. Here, we identify, during RecBCD's DNA unwinding, two Chi-stimulated conformational changes involving RecC. One produced a marked, long-lasting, Chi-dependent increase in protease sensitivity of a small patch, near the Chi recognition domain, on the solvent-exposed RecC surface. The other change was identified by crosslinking of an artificial amino acid inserted in this RecC patch to RecB. Small-angle X-ray scattering analysis confirmed a major conformational change upon binding of DNA to the enzyme and is consistent with these two changes. We propose that, upon DNA binding, the RecB nuclease domain swings from one side of RecC to the other; when RecBCD encounters Chi, the nuclease domain returns to its initial position determined by crystallography, where it nicks DNA exiting from RecC and loads RecA onto the newly generated 3′-ended single-stranded DNA during continued unwinding; a crevice between RecB and RecC increasingly narrows during these steps. This model provides a physical basis for the intramolecular “signal transduction” from Chi to RecC to RecD to RecB inferred previously from genetic and enzymatic analyses, and it accounts for the enzymatic changes that accompany Chi's stimulation of recombination.     

DNA unwinding by replication protein A is a property of the 70 kDa subunit and is facilitated by phosphorylation of the 32 kDa subunit.

Replication protein A (RP-A) is a heterotrimeric single-stranded DNA binding protein with important functions in DNA replication, DNA repair and DNA recombination. We have found that RP-A from calf thymus can unwind DNA in the absence of ATP and MgCl2, two essential cofactors for bona fide DNA helicases (Georgaki, A., Strack, B., Podust, V. and Hübscher, U. FEBS Lett. 308, 240-244, 1992). DNA unwinding by RP-A was found to be sensitive to MgCl2, ATP, heating and freezing/thawing. Escherichia coli single stranded DNA binding protein at concentrations that coat the single stranded regions had no influence on DNA unwinding by RP-A suggesting that RP-A binds fast and tightly to single-stranded DNA. DNA unwinding by RP-A did not show directionality. Experiments with monoclonal antibodies strongly suggested that the 70kDa subunit is responsible for DNA unwinding. Phosphorylation of the 32kDa subunit of RP-A by chicken cdc2 kinase facilitated DNA unwinding indicating that this posttranslational modification might be important for modulating this activity of RP-A. Finally, DNA unwinding of a primer recognition complex for DNA polymerase delta which is composed of proliferating cell nuclear antigen, replication factor C and ATP bound to a singly-primed M13DNA slightly inhibited DNA unwinding. An important role for DNA unwinding by RP-A in processes such as initiation of DNA replication, fork propagation, DNA repair and DNA recombination is discussed.     

The response of DNA length and twist to changes in ionic strength

We examine twist‐stretch coupling of unconstrained DNA using polyelectrolyte theory as applied to a line‐charge model along with published data on the ionic‐strength dependence of the twist angle. We conclude that twist‐stretch coupling is negative: environmental changes that stretch free DNA, unconstrained by externally applied pulling or twisting forces, are accompanied by unwinding of the double helix. We also analyze a helical model and conclude that the observed unwinding of the DNA helix when ionic strength is decreased is driven by radial swelling of the helix. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 223–226, 2015.     

Differences in unwinding of supercoiled DNA induced by the two enantiomers of anti-benzo[a]pyrene diol epoxide.

The unwinding of supercoiled phi X174 RFI DNA induced by the tumorigenic (+) and non-tumorigenic (-) enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) has been investigated by agarose slab-gel and ethidium titration tube gel electrophoresis. The differences in adduct conformations were verified by flow linear dichroism techniques. Both enantiomers cause a reversible unwinding by the formation of noncovalent intercalative complexes. The effects of covalently bound BPDE residues on the electrophoretic mobilities of the RF I DNA form in agarose gels were investigated in detail in the range of binding ratios rb approximately 0.0-0.06 (covalently bound BPDE residues/nucleotide). In this range of rb values, there is a striking difference in the mobilities of (+)-BPDE- and (-)-BPDE-adducted phi X174 DNA in agarose slab-gels, the covalently bound (+)-BPDE residues causing a significantly greater retardation than (-)-BPDE residues. Increasing the level of covalent adducts beyond rb approximately 0.06 in the case of the (+)-BPDE enantiomer, leads to further unwinding and a minimum in the mobilities (corresponding to comigration of the nicked form and the covalently closed relaxed modified form) at rb 0.10 +/- 0.01; at still higher rb values, rewinding of the modified DNA in the opposite sense is observed. From the minimum in the mobility, a mean unwinding angle (per BPDE residue) of theta = 12 +/- 1.5 degrees is determined, which is in good agreement the value of theta = 11 +/- 1.8 degrees obtained by the tube gel titration method. Using this latter method, values of theta = 6.8 +/- 1.7 degrees for (-)-BPDE-phi X174 adducts are observed. It is concluded that agarose slab gel techniques are not suitable for determining unwinding angles for (-)-BPDE-modified phi X174 DNA because the alterations in the tertiary structures for rb < 0.06 are too small to cause sufficiently large changes in the electrophoretic mobilities. The major trans (+)-BPDE-N2-guanosine covalent adduct is situated at external binding sites and the mechanisms of unwinding are therefore different from those relevant to noncovalent intercalative BPDE-DNA complexes or to classical intercalating drug molecules; a flexible hinge joint and a widening of the minor groove at the site of the lesion may account for the observed unwinding effects. The more heterogeneous (-)-BPDE-nucleoside adducts (involving cis and trans N2-guanosine, and adenosine adducts) are less effective in causing unwinding of supercoiled DNA for reasons which remain to be elucidated.     

Thermal stability of nucleosomes studied in situ by flow cytometry: effect of ionic strength and n-butyrate

Heating of cells permeabilized with ethanol and resuspended in aqueous media increases accessibility of DNA to intercalating dyes such as acridine orange (AO). The curves, representing increase in binding of AO as a function of rise in temperature, indicate that the transitions are cooperative. The transitions are sensitive to ionic strength and occur at lower temperatures when cells are suspended in media of increasing ionic strength. Extraction of histones raises accessibility of DNA to intercalators at room temperature, and heating has little effect on additional binding. The results are interpreted as indicating thermal destruction of nucleosomal structure in nuclear chromatin; dissociation of DNA from core histones results in its increasing ability to intercalate AO, most likely due to increased topological freedom to undergo unwinding and elongation following binding of the intercalator. Preincubation of cells with n-butyrate, known to induce histone hyperacetylation, lowers the heat stability of nucleosomes by about 5 degrees C. On the other hand, no differences are observed between chromatin of mitotic vs interphase cells tested over a wide range of ionic strengths (0.1-0.7 N NaCl). The method appears to be useful as a probe of chromatin structure at the nucleosomal level.