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.     

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.     

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.     

Studies on deoxyribonucleoprotein structure. Small-angle X-ray scattering in solutions of various ionic strengths.

The cross-sectional radius of gyration of the deoxyribonucleoprotein (DNP) threads was measured by small-angle X-ray scattering in a wide range of ionic strengths (from 0.0005 to 2 M NaCl). For DNP in a solution of low ionic strength, this value is 30 Å. The increase of ionic strength results in partial deproteinization of DNP, while the cross-sectional radius of gyration varies from 25 Å for DNP in 0.7 M NaCl to 10 Å for DNP in 2 M NaCl. It is suggested that gradual deproteinization by the increase of NaCl concentration causes conformational changes, which are associated with the alteration of the DNP superstructure. The data are interpreted on the basis of the superhelical model of DNA packing in DNP; however, the coexistence of superhelical and unfolded regions in the DNP structure is also a possibility.     

Evidence for a novel conformational transition in superhelical DNA

On the assumption that the dependence of the electrophoretic mobility of superhelical DNA upon the number of tertiary turns (Wr) is a monotonously increasing function devoid of points of inflection, it is concluded that the inflection (change of sign of the first derivative) observed on the curve gives evidence for a conformational transition in DNA secondary structure that begins long before the BZ or B cruciform transitions. The transition consumes 60% of the topological turns at native levels of supercoiling. It is proposed that the conformation produced belongs to the A-family. Provided that this transition indeed yields the A form (11 base pairs per turn), the energy of the BA conformational transition is estimatd to be 5.8–10.3 cal per base pair for different nucleotide sequences at physiological ionic strength. The energies of BZ and B cruciform transitions in superhelical DNA estimated from electrophoretic mobilities by the present method coincide perfectly with the values obtained by other authors using other methods. In addition, on the basis of the data of Brady et al. (1983) on the number of tertiary turns in superhelical DNA determined by X-ray scattering, it is concluded that the initial assumption is justified and the ratio of bending to twisting stiffnesses of superhelical DNA is estimated as 71 (in the fully supercoiled molecule containing 50% of the supposed A-conformation).     

Quasielastic light scattering by biopolymers. IV. Tertiary collapse of calf thymus DNA in 5.5M LiCl

Circular dichroism has been commonly employed to infer the conformation of DNA in solution. The basis of the conformational assignments is the work of Tunis-Schneider and Maestre, wherein CD spectra of DNA were obtained under conditions comparable to those employed in the x-ray diffraction studies of A-, B-, and C-DNA. It has recently been suggested that the CD spectrum of DNA in chromatin, which is similar to the CD spectrum of the C-form DNA, is a superposition of the normal B-DNA spectrum and a single negative band, centered at 275 nm. This negative band is qualitatively identical to the spectrum for condensed Ψ-form DNA. We have employed the hydrodynamic methods of quasielastic light scattering and sedimentation velocity to determine the extent of DNA tertiary structural alteration in 5.5M LiCl as a possible explanation of the C-form CD spectrum. These studies suggest an eightfold contraction of the Stokes hydrodynamic volume for calf thymus DNA in going from 0.4M NH₄Ac to 5.5M LiCl, with no change in molecular weight. The estimated maximum presistence length of DNA in 5.5M LiCl is estimated to be 20.0 nm compared to the “minimum” value of 44.7 nm in NaCl solutions. The value 20.0 nm corresponds to a maximum radius of 16.7 nm for a “continuously coiled” cylinder of DNA, which compares with the value 5.0 nm of DNA in the nucleosome unit of chromatin.     

Decrease in the density of DNA topological turns during in vitro aging of Syrian hamster cells

Primary cultures of Syrian hamster embryonal fibroblasts were grown until the culture flask bottom was completely covered, then passed into a fresh medium with the dilution 1:2 and grown again until complete sealing of the bottom glass surface was achieved. The period required to attain cellular confluency and the density of topological turns (‘titratable superhelical turns’) in nuclear chromosomal superhelical DNA loops were measured in the course of cultivation. Embryonal cells of Syrian hamsters were found to undergo up to twelve culture doublings (passages) in a medium containing 10% bovine serum. The period required to reach confluency increased from 1–2 days for early passage cells, to 4–8 days for late passage cells, which were often able to reach confluency only after change of growth medium. The density of topological turns in nuclear DNA loops was found to decrease, from a range of −0.076 to −0.083 turns per 10 base pairs (bp) in cells of 1–3 passages, to −0.062 turns per 10 bp in cells of 11–12 passages. This decrease in density of DNA-topological turns may play a part in switching the activity of the genes responsible for cellular proliferation and differentiation in the course of aging of primary cell culture.     

Isolation,characterization, and structure of the folded interphase genome of Drosophila melanogaster

The intact interphase genome of Drosophila melanogaster has been isolated by sucrose gradient centrifugation after gentle lysis of tissue culture cells in 0.9 M NaCl-0.4% Nonidet P40. The nonviscous folded DNA sediments as a single broad 5000S peak in a complex with RNA (a fraction of the nuclear nascent RNA) and protein (all of the four intranucleosome histones: H2A, H2B, H3, and H4).The folded DNA is supercoiled and can be relaxed to slower sedimenting forms either by intercalating ethidium or by nicking with DNAase I. Incomplete DNAase treatment gives partially relaxed complexes, indicating that each nick relaxes only a stretch of DNA (defined as a supercoiled DNA loop) without affecting the superhelical content of the rest of the genome. The concentration of superhelices in the Drosophila folded DNA is the same as in the E. coli and SV40 closed circular DNAs—that is, about one negative turn every 200 base pairs (bp) in 0.15 M NaCl at 26°C. The estimated average size of the supercoiled DNA loops, about 85,000 bp, equals the size of the larger Drosophila chromomeres.Ethidium intercalation in 0.9 M NaCl both removes the negative superhelical turns and dissociates the four histones from the DNA. The four histones are dissociated in equimolar concentrations, and the relative proportion of histones displaced from the DNA is a function of ethidium concentration. The histones are completely dissociated from the folded DNA at the ethidium concentration which removes all of the negative superhelices. Thus the data strongly suggest that the rotation of the Watson Crick helix which accompanies ethidium intercalation causes the loss of nucleosomes from the DNA.The results are interpreted in terms of a model for the folded Drosophila genome which has the DNA constrained (by both protein-DNA and RNA-DNA interactions) into independent supercoiled loops containing on the average 400 nucleosomes per loop. Each nucleosome is composed of a histone core with the DNA wound around it in a 360° left-handed toroidal supercoil; each nucleosome toroidal supercoil plus its relaxed internucleosome DNA contains, on the average, 200 bp.     

Catenation and supercoiling in the products of bacteriophage λ integrative recombination in vitro

Catenanes (interlocked circular DNA molecules) are the exclusive products of the bacteriophage λ integrative recombination reaction in vitro when the substrate is a supercoiled DNA molecule containing both the attP and attB sites. It is proposed that the catenation results from the superhelical form of the substrate DNA. We also show that both circular DNA products of a single recombination event can be recovered as superhelical molecules with a superhelical density approximately that of the substrate DNA. The recombination reaction must therefore occur as a coupled process which does not permit free rotation around single-strand breaks at any stage.     

Sedimentation coefficient of polyoma virus DNA

The sedimentation coefficient of the twisted circular form of polyoma virus DNA is calculated from the Kirkwood sedimentation–diffusion equation, the structure being assumed to be a rigid double superhelix. Agreement with the experimental sedimentation coefficient can be obtained, with the use of an experimental value for the number of superhelical turns, when the pitch of the superhelix is intermediate between its minimal and maximal possible values. Another model, which has been proposed for polyoma DNA at low ionic strengths, may be visualized as a superhelical structure wound about a torus. Calculations of sedimentation coefficients for this model agree qualitatively with experimental data at ionic strengths Below 10^?2M.     

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.     

Differential growth of some grapevine varieties in Syria in response to salt <Emphasis Type="Italic">in vitro</Emphasis>

Summary Growth sensitivity of four local grapevine (Vitis vinifera) varieties, Ashlamesh, Helwani, Kassofee, and Khoudeiry, were evaluated for salt. They were cultured on DSD1 medium until rooting stage, then they were transferred to a liquid DSD1 medium containing 0, 10, 20, 30, 40, 80, 120, or 150 mM NaCl for 30 d. The shoot length and leaf number of Ashlamesh, Helwani, and Kassofee were significantly increased at 10 and/or 30 mM NaCl, whereas, 150 mM NaCl decreased shoot length of all varieties except Kassofee. The presence of NaCl at 80 mM or higher concentrations decreased the chlorophyll content and root number of all varieties, while 30 mM NaCl increased root number of Kassofec.     

The DNA untwisting enzyme from Saccharomyces cerevisiae. Partial purification and characterization.

The DNA untwisting enzyme has been partially purified from Saccharomyces cerevisiae. The enzyme exhibits a pH optimum of 7.3 to 7.6 in phosphate buffer, appears to require 0.15 M KCl for activity as determined by a DNA filter-binding assay, and is inhibited by N-ethylmaleimide. Like the untwisting enzymes from other eucaryotic cells, it can remove both positive and negative superhelical turns. A DNA molecule containing a single strand break was shown to be an intermediate in the untwisting reaction. Thermal stabilities of the enzyme from selected conditional lethal mutants defective in DNA synthesis have been examined and were found to be indistinguishable from the wild type enzyme.     

Klebsiella sp.NIAB-I: A new diazotroph,associated with roots of kallar grass from saline sodic soils

A nitrogen fixing organism containing a plasmid has been isolated from the rhizosphere fraction ofLeptochloa fusca (L) Kunth (kallar grass) growing on saline soils in the Punjab area. This bacterium can grow aerobically in a medium containing 1M NaCl and can fix nitrogen efficiently under microaerobic conditions on semi-solid medium with glucose or sucrose as a carbon source. Maximum N₂-fixation in batch cultures occurred with 100 mM NaCl at pH 8.0 and 35°C. DNA hybridization and analysis of the protein pattern were carried out to establish its taxonomic position. On the basis of protein electrophoretic pattern, physiological characteristics, DNA relatedness, and better growth in the presence of high NaCl concentration, we regard this strain as a new species ofKlebsiella.     

Physical binding of pyrene and phenanthrene to native and denatured DNA: Measurements by spectral and coupled-column liquid chromatography methods

The physical (noncovalent) binding of pyrene and phenanthrene to calf-thymus DNA in aqueous NaCl solutions was measured by a spectral method (analysis of absorption spectra by Benesi-Hildebrand plots) and a coupled-column liquid chromatography method (equilibration of DNA solutions with solid hydrocarbon in a generator column and analysis of dissolved hydrocarbon by liquid chromatography). The measurements yielded values of an affinity constant K′ = nK, where n is the apparent number of binding sites per nucleotide and K is the apparent binding constant. The affinity of native DNA for pyrene decreases monotonically with increasing NaCl concentration, whereas the affinity of heat-denatured DNA exhibits a maximum at 0.10M NaCl.K′ for the binding of phenanthrene to native DNA is an order of magnitude lower than K′ for pyrene. The molar enthalpy for the binding of pyrene to native DNA in 10 mM NaCl is (?34.0 ± 1.0) kJ mol^?1. The spectral method data indicate that 50 is an upper limit for the average number of base pairs between intercalation sites for pyrene along the DNA helix and that only a fraction of these sites are bound at the highest binding ratios.     

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.     

The Absence of Non-Local Conformational Changes in OR3 Operator DNA on Complexing with the Cro Repressor

Abstract

The Interaction of the cro protein of λ phage with a synthetic OR3 operator having 17 base pairs in length and with its 9 bp fragment has been studied using the circular dichroism (CD) method. In both cases, a considerable change in the CD of the samples was found in the region 260-300 nm upon the addition of the cro protein. The stoichiometry obtained by the CD titration was identical for OR3 and its 9 bp fragment: one duplex per dimeric cro.

NaCl addition makes the complexes dissociate so that the 9 bp fragment becomes free at [NaCl]>0.2 M while the whole OR3 becomes free at [NaCl]>0.5 M.

The CD spectra of both the free duplexes show a typical B-form conservative pattern with a positive CD band (270 nm) and a negative one (250 nm). The specific complexing of both the duplexes results in a substantial CD depression in the positive band. The most pronounced effect occurs at 280 nm. This spectral change is quite distinct from those in the B to A transition and in the non-cooperative winding of the DNA within the B-family of forms.

The interaction of the cro protein with the non-operator DNAs, calf thymus DNA and a synthetic 10 bp duplex, reveals no visible CD changes at all.

An inference is drawn that the CD change in the specific complexes is mainly due to the induced CD in tyr-26 upon its interaction with a specific base pair in the operator or its fragment, the operator DNA conformation being conserved in a B-like form as a whole. However, some local distortions such as kinks cannot be ruled out on the basis of the CD data.     

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.