Supercoiled DNA folded by nonhistone proteins in cultured mouse carcinoma cells.

Upon gentle lysis of exponentially growing mouse carcinoma cells FM3A by sodium dodecyl sulfate, DNA was released as a "DNA-protein complex" in a folded conformation. No histones could be detected in the DNA-protein complex. The proteins bound to DNA were found to be composed of several kinds of nonhistone proteins with a molecular weight range of 50,000 to 60,000; they appear to play a key role in stabilizing and maintaining the compact and folded structure of the complex. Removal of the proteins by Pronase or 2-mercaptoethanol produced a more relaxed structure sedimenting about half as fast as the original complex in a neutral sucrose gradient. DNA in the folded complex is supercoiled, as indicated by the characteristic biphasic response of its sedimentation rate to increasing concentration of various intercalating agents, actinomycin D, ethidium bromide and acriflavine, with which the cells were treated before lysis. Pronase- or 2-mercaptoethanol-treated relaxed DNA still possessed the characteristic of closed-circular structure as judged from its response to intercalating agents. Nicking with gamma-ray or 4NQO broke these superhelical turns and relaxed the folded complex to slower sedimenting forms equivalent to the relaxed DNA obtained on treatment with Pronase or 2-mercaptoethanol. Viscometric observations of DNA-protein complex were consistent with the above results. A tentative model for the structure of this DNA-protein complex is proposed in which supercoiled DNA is folded into loops by several kinds of nonhistone proteins. Autoradiographic examination of the complex appeared to support this model.     

Light-scattering studies on supercoil unwinding

It has been shown previously that supercoiled [unk]X174 bacteriophage intracellular DNA (mol.wt. 3.2x10(6)) with superhelix density, sigma=-0.025 (-12 superhelical turns) at 25 degrees C is best represented as a Y shape. In this work two techniques have been used to unwind the supercoil and study the changes in tertiary structure which result from changes in the secondary structure. The molecular weights from all experiments were in the range 3.2x10(6)+/-0.12x10(6). In experiments involving temperature change little change in the Y shape was observed between sigma=-0.027 (-13 superhelical turns, 14.9 degrees C) and sigma=-0.021 (-10 superhelical turns, 53.4 degrees C) as evidenced by the root-mean-square radius and the particle-scattering factor P(theta). However, at sigma=-0.0176 (-8 superhelical turns, 74.5 degrees C) the root-mean-square radius fell to between 60 and 70nm from 90nm indicating a large structural change, as did alterations in the P(theta) function. In experiments with the intercalating dye proflavine from values of bound proflavine of 0-0.06mol of dye/mol equiv. of nucleotide which correspond to values of sigma from -0.025 to -0.0004 (-12 to 0 superhelical turns) a similar transition was found when the superhelix density was changed by the same amount, and the molecule was shown to go through a further structural change as the unwinding of the duplex proceeded. At sigma=-0.018 (-9 superhelical turns) the structure was compatible with a toroid, and at sigma=-0.0004 it was compatible with a circle but at no point in the sequence of structure transitions was the structure compatible with the conventional straight interwound model normally visualized as the shape of supercoiled DNA.     

Interaction between DNA and an Escherichia coli protein omega

An E. coli protein, designated ω, has been purified at least 1000-fold. Treatment of a eovalently closed DNA duplex containing negative superhelical turns with ω results in the loss of most of the superhelical turns. The loss of superhelical turns follows a gradual course rather than a one-hit mechanism. This reaction does not require a cofactor. No other change in the physical properties of the DNA could be detected. The DNA remains covalently closed. Its ultraviolet absorption spectrum, circular dichroism, buoyant density in CsCl, sedimentation properties in neutral media containing varying amounts of ethidium and in an alkaline medium, and its susceptibility toward Neurospora endonuclease, are not significantly different from an untreated DNA containing the same number of superhelical turns. Thus it appears that ω is capable of introducing a “swivel” reversibly into a DNA. A plausible mechanism is postulated.     

Agarose gel electrophoretic analysis of damage to supercoiled DNA by adriamycin in the presence of beta-NADH dehydrogenase

In a cell-free system, the anticancer anthracycline antibiotic adriamycin was able to convert purified covalently closed circular, superhelical, form I bacteriophage PM2 DNA to relaxed circular form II DNA in the presence of either sodium borohydride (NaBH4), NADPH cytochrome P-450 reductase or beta-NADH dehydrogenase isolated from myocardial cells. There was no detectable increase in the amount of form III linear duplex DNA formed during the reaction even at high drug concentrations. Less drug was required for the conversion of form I to form II DNA in the presence of the enzymic reducing agents than in the presence of NaBH4. Form II DNA, prepared by irradiation using a Cs-137 source, was not degraded to form III linear duplex DNA. However, form I0 DNA, covalently closed circular DNA without superhelical turns, freshly prepared using topoisomerase I, was converted to form II DNA similar to the conversion of superhelical form I to form II DNA. Again, no increase in the amount of form III linear duplex DNA could be detected.     

The effect of H1 histone on the action of DNA-relaxing enzyme.

The action of DNA-relaxing enzyme on H1-DNA complexes was investigated. Complexes of superhelical and relaxed closed circular duplex DNA with H1 were treated with mammalian relaxing enzyme, deproteinized, and electrophoresed on agarose gels. At relatively low ratios of H1 to superhelical DNA, molecules of superhelical density intermediate between those of the starting material and relaxed DNA, the normal product, were generated. At relatively high H1 histone concentrations (H1:DNA greater than 0.4 w/w), the superhelical DNA was not relaxed. Further, no superhelical turns were introduced into relaxed closed duplex DNA at any concentration of H1 tested. Thus, the binding of H1 histone to DNA prevents the action of the relaxing enzyme. Moreover, H1 histone does not appear to unwind the DNA duplex upon binding. The implications of these observations and the previously demonstrated specificity of H1 histone for superhelical DNA are discussed in relation to the structure of chromatin.     

Spectrofluorometric measurement of the binding of ethidium to superhelical DNA from cell nuclei.

Structures retaining many of the morphological features of nuclei may be released by lysing HeLa cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain few chromatin proteins. We have shown that the DNA of nucleoids is quasicircular and supercoiled by measure spectrofluorometrically the amount of the intercalating dye, ethidium, bound to unirradiated and gamma-irradiated nucleoids. Ethidium binds to nucleoids in the manner characteristic of the binding to superhelical DNA: at low concentrations more ethidium binds to unirradiated nucleoids than to their gamma-irradiated counterparts with broken DNA, and at higher concentrations less ethidium binds to the unirradiated nucleoids. The quasi-circles in nucleoids are 22 times less sensitive to gamma-irradiation than are circles of pure PM2 DNA: they must contain about 2.2 X 10(5) base pairs. The constraints that maintain the quasi-circularity of nucleoid DNA are very resistant to extremes of temperature and alkali; some remain under conditions in which the duplex is denatured. The constraints are destabilised by ethidium suggesting that they are stabilised by free energy of supercoiling. Proteolytic enzymes, but not ribonucleases, remove the constraints. Possible structures for the constraining mechanism are discussed.     

Dynamic light scattering as a probe of superhelical DNA-intercalating agent interaction

Polarized dynamic light-scattering measurements on superhelical pBR322-plasmid DNA solutions in 0.2M NaCl, 2 mM NaPi, pH 7.0, 2 mM EDTA result in a translational diffusion coefficient D = (3.77 ± 0.10) × 10^?8 cm²/s for the native molecule. Modeling the DNA, in the simplest approximation, as a 10 × 440-nm effective hydrodynamic rigid rod yields a good fit to the apparent diffusion coefficient angular-dependence data up to 70°; the model fails at higher angles, probably due to the effects of flexibility or branching of the rod. Diffusion coefficient titration experiments with a platinum complex intercalating agent (PtTS) result in a titratable superhelix density of σ = ?0.079 ± 0.008 under our experimental conditions, corresponding to about 34 superhelical turns in the native DNA. The DNA contour length predicted by our two independent results, the rod dimensions and the number of superhelical turns, is in excellent agreement with the contour length calculated from the number of base pairs, supporting the hydrodynamic approximation of an effective rodlike structure for this small DNA molecule in solution.     

Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: analogous measurements in insect and human cells

The rate of covalent photobinding of trimethylpsoralen to DNA is greater when the DNA is wound with negative superhelical tension than when it is relaxed. In vitro the rate of photobinding is directly proportional to the negative superhelical density of the DNA. Thus measurement of the rate of photobinding provides an assay for probing in vivo unrestrained tension in the winding of the DNA double helix. This approach has been applied to measure torsional tension in DNA as it is packaged in living E. coli. Drosophila and HeLa cells. A method is described for measuring the rate of photobinding to intracellular DNA and rRNA, and for using the latter measurement as an internal control of the rate of me3-psoralen photobinding in vivo. This permits more accurate and reproducible measurement of changes in the DNA-psoralen photobinding reaction. The me3-psoralen probe interacts with intracellular bacterial DNA as expected for a purified DNA duplex wound with superhelical density sigma = -0.05 +/- 0.01. This superhelical tension is relaxed in cells when multiple single-strand breaks are introduced into the chromosomal DNA by gamma-irradiation. Similar relaxation occurs when cells are treated with the DNA gyrase inhibitor coumermycin. The results suggest that the DNA double helix is wound with torsional tension in vivo and that DNA supercoils which are equilibrated with this tension are not completely restrained in nucleosome-like structures. Torsional tension in the DNA of eucaryotic cells is not detectable in analogous measurements of the packaged DNA of HeLa and Drosophila cells. The simplest interpretation of this finding is that, within the limits of detection, all superhelical turns in the DNA are restrained in nucleosomes or nucleosome-like structures in these eucaryotic cells.     

Absence of anthracycline-induced degradation of nuclear DNA in Ehrlich ascites tumour cells

The in vivo effects of anthracycline antibiotics on the integrity of Ehrlich ascites tumour cell DNA have been studied by sedimentation analysis of nuclear structures containing superhelical DNA in neutral sucrose gradients. These fast-sedimenting protein-DNA complexes may be released by gently lysing cells in solution containing non-ionic detergents and high NaCl concentrations (1.95 M). The supercoiled structure of DNA in these protein-DNA complexes is suggested by the characteristic sedimentation in the presence of intercalating agents. Apparently, no DNA damage could be detected in Ehrlich cells from 7-day-old tumours within 3 h after various doses of daunomycin (0.5–10 mg/kg of body wt.) were administered i.p. to mice. Sedimentation anomalies could not be observed even 15 or 30 h after administration of therapeutic doses of daunomycin or adriamycin. In contrast, at 30 min after administration to mice, therapeutic doses of bleomycin (2–8 mg/kg) caused extensive fragmentation of tumour cell DNA, which could be monitored as slowly sedimenting DNA structures (compared with the control). Similarly, DNA damage could be induced by procarbazine at therapeutic doses. Exposure to bleomycin or procarbazine abolished the characteristic biphasic response to ethidium bromide. The absence of anthra-cycline-induced degradation of Ehrlich ascites tumour cell DNA is apparently in contrast with the DNA damage observed in L1210 tumour cells. These observations suggest that DNA damage is not a necessary condition for antitumour activity.     

Determination of the number of superhelical turns by the hyperchromicity of partially denatured covalently-closed DNA molecules

A novel method of determining the number of superhelical turns of a covalently-closed plasmid DNA is described. It relies on the determination of the hyperchromicity, and hence the proportion of unstacked basepairs, of a partially heat-denatured sample which co-migrates during electrophoresis with nicked circular duplex DNA. The values obtained for plasmid pBR beta G DNA at 4 degrees C (-29.8 and -33.5 in the two buffers used) agree closely with the values obtained in parallel by topoisomer band-counting. Our method is less precise than band-counting but is readily applicable to determining the superhelicity of very large DNA molecules. Our results confirm earlier findings that magnesium-containing buffers cause an increase in the duplex winding angle, and hence an increase in the number of negative superhelical turns.     

Further analysis of the altered secondary structure of superhelical DNA. Sensitivity to methylmercuric hydroxide a chemical probe for unpaired bases

A previous study in our laboratory of the reaction of formaldehyde with super-helical DNAs (φX replicative form and PM2) has led to a model for superhelical DNA in which there is a region or regions of altered secondary structure containing unpaired bases. Similar experiments using the nicked circular DNA gave no evidence of interruptions of base pairing. In this study we present additional data, which support the above model as well as extending our analysis of the secondary structure of superhelical DNA and the dynamics of the early denaturation process. In a series of experiments involving the binding of methyl-mercury as a chemical probe of unpaired bases, we obtained the following results. (1) Initially, both s⁰_20w and the buoyant density of the superhelical form of phage PM2 DNA increased as a function of methylmercuric hydroxide concentration, whereas the nicked form did not. (2) This initial binding is accompanied by an increase in superhelical content τ from ?41 to ?46 turns. (3) The binding analysis allows us to estimate that 3.7% of the bases contain methylmercury in this phase of the transition. This is in excellent agreement with the extent of formylation. (4) Such a preformylated molecule shows a shift in the transition to lower mercurial concentrations. These results are interpreted as follows. The initial increase in ?τ excludes the possibility that binding occurs to normal base-paired structures, since this would produce a coupled unwinding of duplex and superhelical turns. The additive effects of formylation and methylmercury binding support the concept that both chemical probes attack the same sites and induce similar structural changes. Thus the evidence clearly supports the view that superhelical DNA contains localized region(s) of interrupted base pairing. Recent studies from other laboratories using single strand-specific endonucleases are in complete agreement with this model.     

The number of superhelical turns in native virion SV40 DNA and minicol DNA determined by the band counting method.

By a method of overlapping the results obtained after agarose gel electrophoresis under two different sets of conditions, it has become possible to determine the number of superhelical turns in a given DNA by counting the bands present after partially relaxing the DNA (Keller and Wendel, 1974) with highly purified nicking-closing (N-C) enzyme from LA9 mouse cell nuclei. Because native supercoiled DNA is heterogeneous with respect to superhelix density, an average number of superhelical turns was determined. Virion SV40 DNA contains 26 +/- 0.5 superhelical turns, and native Minicol DNA contains 19 +/- 0.5 superhelical turns. The above are values at 0.2 M NaCl and at 37 degrees C, the condition under which the enzymatic relaxations were performed. The superhelix densities determined by the band counting method have been compared with superhelix densities determined by buoyant equilibrium in PDl-CsCl gradients. The Gray, Upholt, and Vinograd (1971) calculation procedure has been used for evaluating the superhelix densities by the latter method with the new statement, however, that relaxed DNA has zero superhelical turns. Comparison of the superhelix densities obtained by both methods permits a calculation of an unwinding angle for ethidium. The mean value from experiments with SV40 DNA is 23 +/- 3 degree. The average number of superhelical turns in SV40, 26, combined with the value, 21, obtained by both Griffith (1975) and Germond et al. (1975) for the average number of nucleosomes per SV40 genome, yields an average of 1.25 superhelical turns per 1/21 of the SV40 genome. If the regions of internucleosomal DNA are fully relaxed, 1.25 correesponds to the average number of superhelical turns with a nucleosome. When analyzed under identical conditions, the limit product generated by ligating a nicked circular substrate in the presence of 0.001 M Mg2+ at 37 degrees C (ligation conditions) is slightly more positively supercoiled than the limit product obtained when the N-C reaction is performed in 0.2 M NaCl at 37 degrees C. The difference in superhelix density as measured in gels between the two sets of limit products for both Minicol and SV40 DNAs is 0.0059 +/- 0.0005. This result indicates that the DNA duplex is overwound in the ligation solvent relative to its state in 0.2 M NaCl.     

AMP-dependent DNA relaxation catalyzed by DNA ligase occurs by a nicking-closing mechanism.

In the presence of AMP and Mg2+, a covalently closed duplex DNA containing negative superhelical turns was treated with DNA ligase isolated from bacteriophage T4-infected E. coli. This resulted in the gradual and not sudden loss of superhelical turns as for example in the case of type I DNA topoisomerase. All DNA products remain covalently closed. Since T4 enzyme-mediated DNA relaxation is inhibited by both pyrophosphate and by ATP this suggests that DNA relaxing and DNA joining activities probably coincide. EDTA addition in the presence of a large excess of enzyme, induces the formation of nicked DNA products while protein denaturing treatments are not very effective. Our observations might suggest an involvement of the relaxing activity of DNA ligase during the ligation process.     

Simian virus 40 DNA extracted from infected cells with sodium deoxycholate no longer reflects its in vivo superhelix density.

When simian virus 40 DNA is extracted from infected cells with low concentrations of sodium deoxycholate, which selectively extract non-encapsidated simian virus 40 DNA, the DNA has a lower average number of superhelical turns than the DNA extracted from purified viral particles. During extraction, a partial deproteinization of the DNA by a concentration of detergent that did not inactivate a nicking-closing activity led to the removal of some superhelical turns. The DNA extracted in this way no longer reflected its in vivo number of superhelical turns.     

Supercoiled DNA is interwound in liquid crystalline solutions.

Two structures have been proposed for supercoiled DNA: it is idealized either as a toroidal ring or as a rod of two interwound duplex chains. The latter model is the most widely depicted but the evidence remains controversial. We have worked with monomers and dimers of two plasmids, pUC8 and pKS414, of similar size and natural superhelical density. pKS414 contains a bend promoting sequence whereas pUC8 does not. In concentrated solutions these plasmids form a partially ordered liquid crystalline phase which is found, using neutron diffraction, to consist of a hexagonally packed assembly of parallel rod-like particles. This shape strongly suggests an interwound conformation for which some structural parameters are deduced. The mass/unit length obtained by combining the area of the hexagonal lattice and the concentration is approximately 3.6 times that of linear DNA. This implies a shallow superhelical pitch angle approximately 36 degrees which, when combined with the known number of supercoil turns, yields the pitch approximately 360 A and radius approximately 80 A for the supercoil. Oriented X-ray fibre diffraction patterns at 92% relative humidity indicate a B type duplex structure. Nicked circular plasmids also form liquid crystals but their behaviour, as a function of concentration, differs from that of the superhelical plasmids.     

Unpaired structures in SCA10 (ATTCT)n.(AGAAT)n repeats

A number of human hereditary diseases have been associated with the instability of DNA repeats in the genome. Recently, spinocerebellar ataxia type 10 has been associated with expansion of the pentanucleotide repeat (ATTCT)(n).(AGAAT)(n) from a normal range of ten to 22 to as many as 4500 copies. The structural properties of this repeat cloned in circular plasmids were studied by a variety of methods. Two-dimensional gel electrophoresis and atomic force microscopy detected local DNA unpairing in supercoiled plasmids. Chemical probing analysis indicated that, at moderate superhelical densities, the (ATTCT)(n).(AGAAT)(n) repeat forms an unpaired region, which further extends into adjacent A+T-rich flanking sequences at higher superhelical densities. The superhelical energy required to initiate duplex unpairing is essentially length-independent from eight to 46 repeats. In plasmids containing five repeats, minimal unpairing of (ATTCT)(5).(AGAAT)(5) occurred while 2D gel analysis and chemical probing indicate greater unpairing in A+T-rich sequences in other regions of the plasmid. The observed experimental results are consistent with a statistical mechanical, computational analysis of these supercoiled plasmids. For plasmids containing 29 repeats, which is just above the normal human size range, flanked by an A+T-rich sequence, atomic force microscopy detected the formation of a locally condensed structure at high superhelical densities. However, even at high superhelical densities, DNA strands within the presumably compact A+T-rich region were accessible to small chemicals and oligonucleotide hybridization. Thus, DNA strands in this "collapsed structure" remain unpaired and accessible for interaction with other molecules. The unpaired DNA structure functioned as an aberrant replication origin, in that it supported complete plasmid replication in a HeLa cell extract. A model is proposed in which unscheduled or aberrant DNA replication is a critical step in the expansion mutation.     

Cross-linking and relaxation of supercoiled DNA by psoralen and light.

Photoreaction of 4,5',8-trimethylpsoralen with superhelical ColE1 and ColE1amp DNA was studied. Changes in mobilities in agarose gels, formation of interstrand cross-links, and DNA strand breaks were determined. Psoralen and light treatment removed negative superhelical turns, and extensive treatments failed to produce positive superhelical turns in covalently closed plasmid DNA. The rate of relaxation of superhelical turns by psoralen Photobinding appeared to be directly proportional to the number of superhelical turns remaining. A unique reaction mechanism is presented to explain these results. By this interpretation the initial rate of psoralen photobinding to superhelical DNA was estimated to be 3 times that for linear DNA, and the ratio of cross-linking to monofuctional adducts appears to be dependent on the superhelical conformation of the DNA. The estimated ratio of psoralen molecules bound to DNA strand breaks was 1.7 . 10(4):1, and 70% of this breakage is caused by the light alone.