Raman spectroscopy of supercoiled and nicked ColE1 plasmid

Native supercoiled and nicked ColE1 DNA were examined using laser Raman spectroscopy. ColE1 contains 6646 base pairs (bp) and, when supercoiled, approximately 47 negative supercoils. An analytical buoyant density gradient centrifugation technique developed by Burke and Bauer was scaled to preparative quantities, and used to isolate the supercoiled plasmid fraction from its nicked counterpart. This procedure allowed enriched fractions of the supercoiled plasmid to be extracted without the use of the optical contaminant ethidium bromide. The intensities of several Raman bands were altered between the spectra of the two topological forms. Notably absent were any changes in bands arising from cytosine and guanine vibrations. The observed changes are interpreted in terms of the polymorphic structures which have been observed in many DNA structural studies. The results of this study suggest that accommodation of supercoiling takes place chiefly in A-T base pairs and backbone moieties, without substantial modification of G-C base-pair structure. Premelting effects may account for the observed changes, including a slight shift to lower frequency of a band known to be responsive to base-pair disruption. Heteronomous ribose sugar pucker is evident in both supercoiled and nicked plasmid species. No gross conformational transitions were detected for native supercoiled DNA, and consequently, subtle rearrangements appear sufficient to absorb the supercoiling deformations.     

Light scattering studies of supercoiled and nicked DNA

Static and dynamic light scattering measurements were made of solutions of pGem1a plasmids (3730 base pairs) in the relaxed circular (nicked) and supercoiled forms. The static structure factor and the spectrum of decay modes in the autocorrelation function were examined in order to determine the salient differences between the behaviors of nicked DNA and supercoiled DNA. The concentrations studied are within the dilute regime, which is to say that the structure and dynamics of an isolated DNA molecule were probed. Static light scattering measurements yielded estimates for the molecular weight M, second virial coefficient A₂, and radius of gyration R_G. For the nicked DNA, M = (2.8 ± 0.4) × 10⁶g/mol, A₂ = (0.9 ± 0.2) × 10⁻³ mol cm³/g², and R_G = 90 ± 3 nm were obtained. For the supercoiled DNA, M = (2.5 ± 0.4) × 10⁶ g/mol, A₂ = (1.2 ± 0.2) × 10⁻³ mol cm³/g², and R_G = 82 ± 2.5 nm were obtained. The static structure factors for the nicked and supercoiled DNA were found to superpose when they were scaled by the radius of gyration. The intrinsic stiffness of DNA was evident in the static light scattering data. Homodyne intensity autocorrelation functions were collected for both DNAs at several angles, or scattering vectors. At the smallest scattering vectors the probe size was comparable to the longest intramolecular distance, while at the largest scattering vectors the probe size was smaller than the persistence length of the DNA. Values of the self-diffusion coefficients D were obtained from the low-angle data. For the nicked DNA, D = (2.9 ± 0.3) × 10⁻⁸ cm²/s, and for the supercoiled DNA, D = (4.11 ± 0.21) × 10⁻⁸ cm²/s. The contribution to the correlation function from the internal dynamics of the DNA was seen to result in a strictly bimodal decay function. The rates of the faster mode Γ_int, reached plateau values at low angles. For the nicked DNA, Γ_int = 2500 ± 500 s⁻¹, and for the supercoiled DNA, Γ_int = 5000 ± 500 s⁻¹. These rates correspond to the slowest internal relaxation modes of the DNAs. The dependence of the relaxation rates on scattering vector was monitored with the aid of cumulants analysis and compared with theoretical predictions for the semiflexible ring molecule. The internal mode rates and the dependence of the cumulants moments reflected the difference between the nicked DNA and the supercoiled DNA dynamical behavior. The supercoiled DNA behavior seen here indicates that conformational dynamics might play a larger role in DNA behavior than is suggested by the notion of a branched interwound structure. © 1996 John Wiley & Sons, Inc.     

Separation of genomic DNA, RNA, and open circular plasmid DNA from supercoiled plasmid DNA by combining denaturation, selective renaturation and aqueous two-phase extraction

In the current study we developed a process for the capture of pDNA exploiting the ability of aqueous two-phase systems to differentiate between different forms of DNA. In these systems scpDNA exhibits a near quantitative partitioning in the salt-rich bottom phase. The successive recovery from the salt rich bottom phase is accomplished by a novel membrane step. The polish operation to meet final purity demands is again based on a system exploiting a combination of the denaturation of the nucleic acids present, specific renaturation of scpDNA, and an ATP system able to differentiate between the renatured scpDNA and the denatured contaminants such as ocpDNA and genomic host DNA. This polish step thus allows a rapid and efficient separation of scpDNA from contaminating nucleic acids which up to date otherwise only can be accomplished with much more cumbersome chromatographic methods. In a benchmark comparison, it could be shown that the newly developed process exhibits a comparable yield to an industrial standard process while at the same time showing superior performance in terms of purity and process time. Additionally it could be shown that the developed polish procedure can be applied as a standalone module to support already existing processes.     

Circular intermediates with missing nucleotides in the conversion of supercoiled or nicked circular to linear duplex DNA catalyzed by two species of BAL 31 nuclease

The extracellular nucleases from Alteromonas espejiana BAL 31 can catalyze the endonucleolytic and/or exonucleolytic hydrolysis of duplex DNA in response to a variety of alterations, either covalent or noncovalent, in DNA structure. The nuclease can exist as at least two kinetically and molecularly distinct protein species. The two species that have been studied, called the 'fast' (F) and 'slow' (S) nucleases, both readily convert negatively supercoiled DNAs to linear duplex molecules and accomplish this conversion through the formation of a circular duplex intermediate containing usually a single interruption in one strand. It is further shown that most of these intermediates contain gaps arising from the removal in a processive manner of one or more nucleotide residues after the introduction of the initial strand break (nick). Considering only the intermediates with gaps, the average number of missing residues is 6.3 +/- 0.5 and 2.8 +/- 0.3, respectively, for DNA acted upon by the F and S enzymes independently of the extent of conversion of supercoiled DNA. The nicks and gaps are bounded by 3'-hydroxyl and 5'-phosphoryl termini. When singly nicked circular DNA is used as the substrate, conversion to the linear duplex form occurs predominantly through a gapped circular intermediate with the same average numbers, within experimental error, of missing nucleotides for the respective nuclease species as found when supercoiled DNA is the substrate. The conversion to linear duplex DNA is much slower when nicked circular DNA is the substrate compared to that found when supercoiled DNA is the starting material.     

Quantitation of supercoiled circular content in plasmid DNA solutions using a fluorescence-based method

A method for quantifying the proportion of supercoiled circular (SC) forms in DNA solutions is described. The method (SCFluo) takes advantage of the reversible denaturation property of SC forms and the high specificity of the PicoGreen fluorochrome for double-stranded (ds)DNA. Fluorescence values of forms capable of reversible denaturation after a 5 min heating, 2 min cooling step are normalised to fluorescence values of total dsDNA present in the preparation. For samples with a SC content >20–30%, good regression fits were obtained when values derived from densitometric scanning of an agarose gel and those derived from the SCFluo method were compared. The method represents an attractive alternative to currently established methods because it is simple, rapid and quantitative. During large-scale processing and long-term storage, enzymatic, chemical and shear degradation may substantially decrease the SC content of plasmid DNA preparations. Regulations for pharmaceutical grade products for use in gene therapy and DNA vaccination may require >90% of the plasmid to be in the SC form. In the present study the SC content of 6.9, 13 and 20 kb plasmid preparations that had been subjected to chemical and shear degradation was successfully quantified using the new method.     

Escherichia coli helicase II (uvrD) protein can completely unwind fully duplex linear and nicked circular DNA

We have examined the duplex DNA unwinding (helicase) properties of the Escherichia coli helicase II protein (uvrD gene product) over a wide range of protein concentrations and solution conditions using a variety of duplex DNA substrates including fully duplex blunt ended and nicked circular molecules. We find that helicase II protein is able to initiate on and completely unwind fully duplex DNA molecules without the requirement for a covalently attached 3' single-stranded DNA tail. This DNA unwinding activity is dependent upon Mg2+ and ATP and requires that the amount of protein be in excess of that needed to saturate the resulting single-stranded DNA. Unwinding experiments on fully duplex blunt ended DNA with lengths of 341, 849, 1625, and 2671 base pairs indicate that unwinding occurs at the same high ratios of helicase II protein/nucleotide, independent of DNA length (50% unwinding requires approximately 0.6 helicase II monomers/nucleotide in 2.5 mM MgCl2, 10% glycerol, pH 7.5, 37 degrees C). Helicase II protein is also able to unwind completely a nicked circular DNA molecule containing 2671 base pairs. At lower but still high molar ratios of helicase II protein to DNA, duplex DNA molecules containing a single-stranded (ss) region attached to a 3' end of the duplex are preferentially unwound in agreement with the results obtained by S. W. Matson [1986) J. Biol. Chem. 261, 10169-10175). This preferential unwinding of duplex DNA with an attached 3' ssDNA most likely reflects the availability of a high affinity site (ssDNA) with the proper orientation for initiation; however, this may not reflect the type of DNA molecule upon which helicase II protein initiates DNA unwinding in vivo. The effects of changes in NaCl, NaCH3COO, and MgCl2 concentration on the ability of helicase II protein to unwind fully duplex DNA and duplex DNA with a 3' ssDNA tail have also been examined. Although the unwinding of fully duplex and nicked circular DNA molecules reported here occurs at higher helicase II protein to DNA ratios than have been previously used in most studies of this protein in vitro, this activity is likely to be relevant to the function of this protein in vivo since very high levels of helicase II protein accumulate in E. coli during the SOS response to DNA damage (approximately 2-5 x 10(4) copies/cell).(ABSTRACT TRUNCATED AT 400 WORDS)     

Z DNA and loop structures by immunoelectronmicroscopy of supercoiled pRW751, a plasmid containing left-handed helices.

Single and multiple loops were seen when the plasmid pRW751 was allowed to react with anti-Z-DNA or with a Z-specific cross-linking agent. Loop formation was dependent upon negative supercoiling and the presence of Z-specific antibody or cross-linking agent. Restriction enzyme mapping located 18 sites at the bottoms of loops, in addition to the two (dG-dC)n inserts of pRW751. No more than 5 loops were seen in any of the measured molecules; thus, not all potential Z-sites assume the Z conformation at any particular time. Stretches of alternating purine-pyrimidine sequences occur at all 20 sites. Almost all of the Z sites could be mapped to regions located at the beginnings or ends of reading frames or at various regulatory sites. Our findings support the concept that supercoiling brings distant sequences to within 5A of one another, allowing joint participation in regulatory processes controlled by DNA-binding proteins.     

Viscometric analysis of the interaction of bisphenanthridinium compounds with closed circular supercoiled and linear DNA.

The interaction with closed circular supercoiled and linear DNA of bisphenanthridinium compounds substituted through both the meta and para positions of the 6-phenyl group, along with appropriate monomer intercalators as controls, has been investigated by viscometric titration. When CPK models for the phenanthridinium rings of the three bis-compounds are oriented in a parallel manner as a model for intercalation, their ring plane to ring plane distances are approximately 7 to 8 A (SR 2430), 11 A (SR 2193), and 15 A (SR 2166). In SR 2430 the two phenanthridines are linked through the para positions of the 6-phenyl group; this chain allows intercalation of the two rings at adjacent binding sites in DNA, but is not long enough to accommodate an excluded site. The viscometric titrations with both superhelical and linear DNA clearly indicate that SR 2430 gives results close to those of the monomer control compounds while SR 2193 and SR 2166 have approximately twice the unwinding angle and DNA length increase on binding to DNA as the monomer compounds. These phenanthridinium compounds, therefore, are capable of bisintercalation only if their linking groups are of sufficient length to allow an excluded binding site between base pairs. This conclusion is supported by DNA thermal denaturation experiments in the presence of these compounds.     

Relaxation,linearization and fragmentation of supercoiled circular DNA by tungsten microprojectiles

The aim of the study was to characterize DNA lesions caused by microprojectile bombardment and by the postbombardment presence of tungsten particles in transformed cells. For the sake of simplicity, plasmid DNA was used as a target for bombardment with naked tungsten particles. Unexpectedly extensive DNA degradation was observed under standard bombardment conditions. However, no further DNA fragmentation occurred under postbombardment conditions, simulated by incubation of plasmid DNA with a suspension of tungsten particles. Instead, relaxation and linearization of supercoiled circular plasmids (pAHC25 and others) took place. It is concluded that the observed linearization (a single site double–strand break in DNA circle) results from the ability of tungsten to catalyse the hydrolysis of phosphodiester bonds in torsionally strained sites of native DNA selectively.     

Osmium tetroxide probing of local DNA structure in linear and supercoiled plasmids containing curvature-inducing sequences

Recombinant plasmids pK1A108, pK3A108, pK4A108 and pK5/6T217 containing 80 +/- 1 base pair inserts with different curvature-inducing sequences were studied using the DNA structure probe osmium tetroxide in the presence of pyridine (Os, py). The insertion sequences of the plasmids pK1A108, pK3A108, and pK4A108 are strongly related while the degree of curvature increases from pK1A108 (no curvature) less than pK3A108 less than pK4A108 less than pK5/6T217. The Os, py probe reacts selectively with single-stranded and distorted double-stranded regions in the DNA double helix. Nuclease S1 was used to recognize and cleave regions made permanently single-stranded due to osmium recognize and cleave regions made permanently single-stranded due to osmium modification. In linearized plasmids treatment with Os, py produced no S1-detectable site-specific modification. This result is in agreement with models suggested for DNA curvature; in general, continuous base pairing and base stacking is considered through different sequence blocks as well as through structural junctions. Os, py-probing of the plasmids in the supercoiled state also resulted in no S1-detectable site-specific modification within the inserts of pK1A108, pK3A108, and pK4A108 plasmids (while the regions containing inverted repeat nucleotide sequences in these plasmids were site-specifically modified). In contrast, supercoiled pK5/6T217 DNA was site-specifically modified within the curvature-inducing insert sequence. The nucleotide sequence of the insert of this plasmid strongly differs from the insertion sequences of the other three plasmids; it is extremely AT-rich and contains regularly arranged dAGAGA and dATATA sequences. The structural distortion observed in supercoiled pK5/6T217 is most probably due to the presence of these sequences in a particular arrangement in the insertion sequence.     

Structure- and sequence-specificity of ozone degradation of supercoiled plasmid DNA.

Ozone-reactive sites on the nucleobase moieties in supercoiled pBR322 DNA were investigated by using sequencing procedures. Ozonolysis in the absence of salt resulted in degradation of thymine residues in the A + T rich region located at 3100-3400bp. In the presence of salt, such as NaCl or MgCl2, a conformational change of plasmid DNA was induced. Subsequently the thymine and guanine residues in the loop of the cruciform located at 3120bp and 3220bp were degraded. In addition, central thymine residues present in sequences GTA, GTT and ATA were also degraded.     

Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA

The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.     

Viral R plasmid Rphi6P: properties of the penicillinase plasmid prophage and the supercoiled, circular encapsidated genome.

Properties of the viral R plasmid Rphi6P are described. As a temperate bacteriophage, it plaques on the facultative phototroph Rhodopseudomonas sphaeroides. Under aerobic conditions the phage had a latent period of 180 min, a burst time of 200 min, and a burst size of 15 to 20 particles per infective center. The encapsidated viral genome occurred as a supercoiled, circular DNA duplex with a mean contour length of 16.5 +/- 10 micron. Percent guanine plus cytosine, as calculated from thermal denaturation profiles, was 63.5. Mitomycin C-induced loss of the prophage suggested an extrachromosomal location in the host cell. Use of this curing agent enabled the isolation of a plasmid-free strain of R. sphaeroides. Biophysical analysis of the plasmid-free strain lysogenized with Rphi6P confirmed that the prophage occurred as a plasmid in the host cell.     

Separation of supercoiled and open-circular plasmid DNA by liquid-liquid counter-current chromatography

We report for the first time the use of liquid-liquid counter-current chromatography (CCC) for the preparative scale fractionation of plasmid DNA. Almost complete fractionation of supercoiled and open circular plasmid DNA (6.9 kb) could be achieved using a phase system comprising 12.5% (w/w) PEG 600 and 18% (w/w) K₂HPO₄. Experiments were carried out on a Brunel J-type CCC machine (100 ml PTFE coil) at a mobile phase flow rate of 0.5 ml min^{– 1} and a rotational speed of 600 rpm. Compared to conventional HPLC techniques the capacity of CCC is not limited by the surface area of resin available for adsorption. Symbols: C _b, Concentration of plasmid in lower phase (g ml^–1); C _t, Concentration of plasmid in upper phase (g ml^–1); CV, Total volume of mobile phase present in the coil and connecting leads (ml); K, Equilibrium solute partition coefficient (K=C _t/C _b); OC, Open circular plasmid; SC, Supercoiled plasmid; S _f, Percentage stationary phase retention (S _f=V _s/V _c); t _s, Time for phase separation (s); V _b, Volume of bottom phase (ml); V _c, Coil volume (ml); V _m, Volume of mobile phase present in coil at equilibrium (ml); V _r, Volume ratio of two phases (V _r=V _t/V _b); V _s, Volume stationary phase present in coil at equilibrium (ml); V _t, Volume of top phase (ml); V _tot, Total volume of phase system (ml).     

Monte Carlo simulation of DNA strand-break induction in supercoiled plasmid pBR322 DNA from indirect effects

We present a new Monte Carlo simulation code system (DBREAK) of the detailed events that occur when ionizing radiation interacts with water and DNA molecules. The model treats the initial energy deposition by radiation, the formation of chemically active species, subsequent diffusion-controlled chemical reactions, and induction of DNA strand breaks. DBREAK assumes one-hit single-strand break (SSB) and two-hit double-strand break (DSB) mechanisms. A high-resolution model of plasmid DNA structure has been introduced. The calculated results are compared with the results of previously performed experiments of the same type. Under aerobic conditions, 89.4% of the DNA damage was attributed to OH-radical and 10.5% and 0.1% to e^– _aq and H, respectively. We also compared the differences between liquid-water track structure and gas-phase-water track structure. The calculated yield of SSBs by liquid-water track structure exceeded that of gas-phase-water track structure by a factor of 1.2. Received: 13 February 1997 / Accepted in revised form: 26 August 1997     

Isolation and physical studies of the intact supercoiled, the open circular and the linear forms of ColE1-plasmid DNA.

For the study of DNA conformations, conformational transitions, and DNA-protein interactions, covalently closed supercoiled ColE1-plasmid DNA has been purified from cultures of Escherichia coli harboring this plasmid and grown in the presence of chloramphenicol according to the method of D.B. Clewell [J. Bact. 110 (1972)667]. The open circular and linear forms of the plasmid were prepared by digestion of the covalently closed, supercoiled form with pancreatic deoxyribonuclease and EcoRI-restriction endonuclease, respectively. The linear form was found to be very homogeneous by electron microscopy and sedimenting boundary analysis. Its physical properties (s0 20,w=16.3 S,D0 20,W=1.98 X 10(-8) cm2 s-1 and [eta]=2605 ml g-1) have been carefully determined in 0.2 M NaCl, 0.002 M NaPO4 pH 7.0,0.002 M EDTA, at 23 degrees C. Combination of s0 20, w (obtained by quasielastic laser light scattering) gave Ms,D=4.39 x 10(6). This value is in reasonable agreement with the molecular weight from total intensity laser light scattering M=4.30 x 10(6). The covalently closed and open circular forms of the ColE1-plasmid are less homogeneous due to slight cross-contamination and the presence of small amounts of dimers in these preparations. The weight fractions of the various components as determined by boundary analysis or electron microscopy are given together with the average quantities obtained in the same solvent for the supercoiled form ((s0 20,w)w=25.4 S, (D0 20,w)z=2.89 x 10(-8) cm2 s-1, [eta]= 788 ML G-1,Ms,D=4.69 x 10(6) and Mw=4.59 x 10(6)) and the open circular form (s0 20, w)w=20.1 S, (D0 20,w)z=2.45 x 10(-8) cm2 s-1, [eta]=1421 ml g-1,Ms,D=4.37 x 10(6) and Mw=4.15 x 10(6)). Midpoint analysis of the sedimenting boundaries allows unambiguous determination of the sedimentation coefficients of these two forms: s0 20,w=24.5 S and s0 20,w=18.8 S, respectively. Also deduced from total intensity light scattering were radii of gyration Rg (103.5, 134.2 and 186 nm) and second virial coefficients A2 (0.7, 4.8 AND 5.4 x 10(-4) mole ml/g2) for the supercoiled, the open circular and linear forms, respectively. The data presented are discussed in relation to the conformational parameters for the three forms in solution.     

High efficiency method to obtain supercoiled DNA with a commercial plasmid purification kit

Supercoiled state corresponds to the active form for plasmid applications. The relaxed circular form of plasmids is often inactive or poorly active. To obtain significant amounts of almost fully supercoiled DNA, we modified the standard protocol of a commercially available Qiagen plasmid purification kit. Our changes led to isolation of almost 100% of the plasmids in the supercoiled state. The modified protocol was used to purify different plasmids with consistent results. The purified plasmids maintain supercoiled state for about two months. The modified protocol is very advantageous because it allows easy DNA production with high degree of supercoiled form at low cost.     

Ozonolysis of supercoiled pBR322 DNA resulting in strand scission to open circular DNA.

Treatment of supercoiled pBR322 DNA with ozone resulted in the conversion of closed circular DNA to open circular DNA. Restriction analysis of the resulting open circular DNA showed that ozonolysis in the absence of salt caused single strand cleavage at specific sites.     

Carboxyl-coated magnetic nanoparticles for mRNA isolation and extraction of supercoiled plasmid DNA

Carboxyl-coated magnetic nanoparticles (MNPs) were used to demonstrate dual functionality: isolation of messenger RNA (mRNA) from mammalian cells and extraction of the supercoiled (sc) form of plasmid DNA (pDNA) from agarose gel. These MNPs were attached with 5′-NH₂-tagged oligo-(dT)₂₅ primer and were used to isolate mRNA from breast cancer cells. The isolated mRNA was used for amplification of β-actin to confirm the compatibility. These MNPs were also used to extract the sc form of pDNA from agarose gel. The compatibility of the pDNA was demonstrated by restriction digestion. Both of these methodologies are simple, inexpensive (compared with existing kits), and efficient.