1994-95 Abstracts from the Bloomfield Lab

Abstracts of 1994-95 Publications

Ma, C. and V. A. Bloomfield. (1994). Condensation of Supercoiled DNA Induced by MnCl₂. Biophys. J. 67: 1678-1681.

Multivalent cations condense DNA in vitro, but it had been thought that a valence of at least +3 was required in aqueous solution. We have found that Mn²⁺ can produce toroidal condensates of supercoiled plasmid DNA, but not of linearized plasmid. Mg²⁺ does not cause condensation, and neither MgCl₂ nor NaCl can negate the effect of MnCl₂, indicating that the condensation mechanism with Mn is not primarily electrostatic. Supercoiled MnDNA is more extensively digested than the linear form by S1 nuclease. Supercoiling appears to cooperate with Mn²⁺ in stabilizing helix distortions, and also provides a "pressure" that enhances lateral association.

Ma, C. and V. A. Bloomfield. (1995). Gel Electrophoresis Measurement of Counterion Condensation on DNA. Biopolymers. 35: 211-216.

We used agarose gel electrophoresis to measure the effective charge neutralization of DNA by counterions of different structure and valence, including Na⁺, Mg²⁺, Co(NH₃)₆³⁺ and spermidine³⁺, which competed for binding with an excess of tris acetate buffer. Linear DNA molecules ranged in size from 1 to 5 kb, and supercoiled plasmid pUC18 was also measured. In all cases, the results were in good agreement with theoretical predictions from counterion condensation theory for two-counterion mixtures.

Ma, C., L. Sun and V. A. Bloomfield. (1995). Condensation of Plasmids Enhanced by Z-DNA Conformation of d(CG)n Inserts. Biochemistry. 34: 3521-3528.

DNA molecules collapse into compact structures in the presence of multivalent cations. To probe the possible importance of supercoiling and conformational effects, pUC18 plasmids (2686 bp) were modified by inserting 12 bp and 20 bp alternating d(CG)_n sequences, which are capable of converting to a left-handed Z-conformation under appropriate conditions, into the polycloning region. Condensation was induced by rapid addition of hexammine cobalt (III) (Co(NH₃)₆³⁺) and monitored by laser light scattering and electron microscopy. Light scattering shows that plasmids with longer d(CG)_n inserts condense more extensively at natural superhelical densities. Electron microscopy indicates that the morphological distribution of condensed d(CG)_n-containing plasmids changes as a function of Co(NH₃)₆³⁺ concentration. At lower Co(NH₃)₆³⁺ concentration, the proportion of rods is higher and at higher Co(NH₃)₆³⁺ concentration, most of the condensates have the form of toroids. In addition, the inner radii of the toroids are much smaller relative to condensed pUC18 under the same conditions. Enzymatic analysis and chemical probing show that the d(CG)_n inserts in naturally supercoiled plasmids convert from B-form to Z-form in the presence of Co(NH₃)₆³⁺ under the conditions in which condensation occurs. To determine whether the enhanced condensation of d(CG)n-containing plasmids results from the change of superhelical density due to the B-Z transition, we treated wild type pUC18 molecules with topoisomerase I and varying amounts of ethidium bromide to generate a range of supercoil densities. Light scattering indicates that supercoiling did not affect the condensation process. Plasmids containing two inserts separated by 0.6 kb condense more extensively than those containing just one insert. We conclude that the Z conformation of d(CG)_n inserts enhances DNA condensation, perhaps through the higher flexibility of secondary structure around the d(CG)_n insert region or greater exposure of bases to solvent in the Z-form.

Dwyer, J. D. and V. A. Bloomfield. (1995). Brownian dynamics simulation of probe diffusion in DNA: Effects of probe size, charge and DNA concentration. Biophys Chem. 57: 55-64.

We have used Brownian dynamics simulation to study probe diffusion in solutions of short chain DNA using our previously developed simulation algorithm. We have examined the effect of probe size, charge, and DNA concentration on the probe diffusion coefficient, with the aim of gaining insight into the diffusion of proteins in a concentrated DNA environment. In these simulations, DNA was modeled as a worm-like chain of hydrodynamically equivalent spherical frictional elements while probe particles were modeled as spheres of given charge and hydrodynamic radius. The simulations allowed for both short range Lennard-Jones interactions and long ranged electrostatic interactions between charged particles. For uncharged systems, we find that the effects of probe size and DNA concentration on the probe diffusion coefficient are consistent with excluded volume models and we interpret our results in terms of both empirical scaling laws and the predictions of scaled particle theory. For charged systems, we observe that the effects of probe size and charge are most pronounced for the smallest probes and interpret the results in terms of the probe charge density. For an ionic strength of 0.1 M we find that, below a critical probe surface charge density, the probe diffusion coefficient is largely independent of probe charge and only weakly dependent on the DNA charge. These effects are discussed in terms of the interactions between the probe and the DNA matrix and are interpreted in terms of both the underlying physics of transport in concentrated solutions and the assumptions of the simulation model.

Arscott, P.G., Ma, C. Wenner, J. and Bloomfield, V.A. (1995) DNA condensation in alcohol-water mixtures: Dielectric constant and other solvent effects. Biopolymers 36: 345-365.

DNA molecules condense into compact structures in the presence of a critical concentration of multivalent cations. To probe the contribution of electrostatic forces to condensation, we used mixtures of water with methanol (MeOH), ethanol (EtOH), and isopropanol (iPrOH) to vary the dielectric constant e from 80 to 50. The condensation of pUC18 plasmids by hexaammine cobalt IlI), Co(NH₃)₆³⁺, was monitored by total intensity and dynamic light scattering, electron microscopy, and circular dichroism. The total scattering int,ensity increased as e went from 80 to 70, and then decreased as e decreased further. UV spectrophotometry confirmed that the loss of intensity at low e was not due to the particles' settling out of solution. The rate as well as the extent of condensation increased as e was lowered from 80 to 70, and also depended on the species of alcohol (MeOH < EtOH < iPrOH). The hydrodynamic radii R_H of the particles, however, remained roughly the same at 300-350 A and was independent of the species of alcohol. R_H increased below e = 70. The critical concentration of Co(NH₃)₆³⁺ required to induce DNA condensation decreased from 21 µM to about 16 µM as the dielectric constant decreased from 80 to 70, and decreased moderately with the nonpolarity of the alcohol. The fraction of DNA charge neutralized at the onset of DNA condensation was calculated by a modification of Manning's two variable counterion condensation theory to be 0.90 ± 0.01, independent of e. By electron microscopy we observed that the condensed particles changed from about 93% toroids at e = 80 to 89% rods at e = 70 and 98% rods at e = 65. At e lower than 65, DNA collapsed into a network of multistranded fibers. The morphology of condensed DNA particles, whether toroids, rods, or fibers, was independent of the alcohol species. Circular dichroism spectra in ethanol-water mixtures indicated that both closed circular and linearized plasmids were in the B conformation when condensed with Co(NH₃)₆³⁺ at e > 70, although the closed circular molecules exhibited a weak Y-DNA spectrum. A transition from B- to A-form took place between e=70 and 60, well below the normal dielectric constant of e = 40 for this transition, indicating that ethanol and Co(NH₃)₆³⁺ synergistically promote the B-A transition.

We interpret these results to mean that alcohols have both electrostatic and structural effects on DNA, leading to three regimes of condensation. At the lowest alcohol concentrations the B conformation is stable and condensation is relatively slow, allowing time for the packing adjustments necessary to form toroids. At intermediate alcohol concentrations condensation is faster, and the combined effects of solvent and Co(NH₃)₆³⁺ locally destabilize the double helix, permitting DNA foldbacks that lead to rodlike condensates. Rods become shorter as well as more numerous as e decreases from 80 to 65-60, indicating increasing destabilization as alcohol increases. At the lowest dielectric constants, alcohol and Co(NH₃)₆³⁺ produce A-DNA which strongly self-adheres and rapidly aggregates into fibrous networks, not allowing time for more compact condensates to form.

Baumann, C. B. and V. A. Bloomfield. (1995). Large-scale purification of plasmid DNA for biophysical and molecular biology studies. BioTechniques. 19:884-890.

We have developed an improved large scale purification method which simultaneously maximizes time efficiency, yield and supercoiling. This method utilizes alkaline bacterial cell lysis, saturated ammonium acetate precipitation of cellular debris, enzymatic digestion of RNA, protein removal with minimal amounts of phenol (~2 ml) and "gentle" removal of phenol contamination using dialysis. It allows 4-9 mg of protein- and RNA-free plasmid DNA to be isolated from just 500 ml of bacterial culture grown in Luria-Bertani (LB) medium. Both low- and high-copy number plasmids are isolated efficiently while maintaining a high degree of supercoiling (> 90%). The following plasmid vectors are routinely isolated using this method: pUC18 (2686 bp), pBR322 (4363 bp) and YEplac195 (5241 bp). The enumerated protocol requires ~5 hr to complete and employs common laboratory hardware and reagents. It will prove very useful for the routine isolation of quality plasmid DNA in large quantities for biophysical and molecular biological studies.

Duguid, J. G., V. A. Bloomfield, J. M. Benevides and G. J. Thomas, Jr. (1995). Raman spectroscopy of DNA-metal complexes. II. The thermal denaturation of DNA in the presence of Sr²⁺, Ba²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Cd²⁺. Biophys. J. 69: 2623-2641.

Differential scanning calorimetry (DSC), laser Raman spectroscopy, optical densitometry, and pH potentiometry have been used to investigate DNA melting profiles in the presence of the chloride salts of Ba²⁺, Sr²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Cd²⁺. Metal-DNA interactions have been observed for the molar ratio [M²⁺]/[PO₂^-] = 0.6 in aqueous solutions containing 5% by weight of 160 bp mononucleosomal calf thymus DNA. All of the alkaline earth metals, plus Mn²⁺, elevate the melting temperature of DNA (Tm >75.5 °C), while the transition metals Co²⁺, Ni²⁺ and Cd²⁺ lower Tm. Calorimetric (H_cal) and van't Hoff (H_vH) enthalpies of melting range from 6.2-8.7 and 75.6-188.6 kcal/mole bp respectively, and entropies from 17.5-24.7 cal/°K mole bp. The average number of base pairs in a cooperative melting unit (<n_melt>) varied from 11.3-28.1. No dichotomy was observed between alkaline earth and transition DNA-metal complexes for any of the thermodynamic parameters other than their effects on Tm.

These results complement Raman difference spectra which reveal decreases in backbone order, base unstacking, distortion of glycosyl torsion angles, and rupture of hydrogen bonds, which occur after thermal denaturation. Raman difference spectroscopy shows that transition metals interact with the N7 atom of guanine in duplex DNA. A broader range of interaction sites with single-stranded DNA includes ionic phosphates, N1 and N7 atoms of purines, and N3 atom of pyrimidines. For alkaline earth metals, very little interaction was observed with duplex DNA, while spectra of single-stranded complexes are very similar to those of melted DNA without metal. However, difference spectra reveal some metal-specific perturbations, especially for Raman bands at 1092 cm^-1 (PO2- symmetric stretching), 1258 cm^-1 (dC,dA bases), and 1649 cm^-1 (mainly C=O stretching of dT,dG,dC). Increased spectral intensity could also be observed near 1335 cm^-1 (dA,dG) for CaDNA. Optical densitometry, employed to detect DNA aggregation, reveals increased turbidity during the melting transition for all divalent DNA-metal complexes, except Sr and BaDNA. Turbidity was not observed for DNA in the absence of metal. A correlation was made between DNA melting, aggregation, and the ratio of Raman intensities I₁₃₃₅/I₁₃₇₄. At room temperature, DNA-metal interactions result in a pH drop of 1.2-2.2 units for alkaline earths and more than 2.5 units for transition metals. Sr²⁺, Ba²⁺, and Mg²⁺ cause protonated sites on the DNA to become thermally labile.

These results lead to a model that describes DNA aggregation and denaturation during heating in the presence of divalent metal cations: (1) The cations initially interact with the DNA at phosphate and/or base sites resulting in proton displacement. (2) A combination of metal-base interactions and heating disrupts the base pairing within the DNA duplex. This allows divalent metals and protons to bind to additional sites on the DNA bases during the aggregation/melting process. (3) Strands whose bases have swung open upon disruption are linked to neighboring strands by metal ion bridges. (4) Near the midpoint of the melting transition, thermal energy breaks up the aggregate. We have no evidence whether metal ion crossbridges or direct base-base interactions rupture first. (5) Finally, all crosslinks break, resulting in single-stranded DNA complexed with metal ions.

Duguid, J.G., and Bloomfield, V.A. (1995) Aggregation of melted DNA by divalent metal ion-mediated crosslinking. Biophys. J. 69: 2642-2648.

In an accompanying paper (Duguid et al, 1995) we reported the use of differential scanning calorimetry and optical densitometry to characterize the melting and aggregation of 160 bp fragments of calf thymus DNA during heating in the presence of divalent metal cations. Aggregation is observed as thermal denaturation begins, and becomes more extensive with increasing temperature until the melting temperature Tm is reached, after which the aggregates dissolve extensively. The order of effectiveness of the metals to induce aggregation is generally consistent with their ability to induce melting: Cd > Ni > Co > Mn Ca > Mg. Under our experimental conditions (50 mg/mL DNA, 100 mM MCl₂, [metal]/[DNA phosphate] = 0.6), no measurable aggregates were observed for Ba or SrDNA. In this paper we show that the Shibata-Schurr (1981) theory of aggregation in the thermal denaturation region provides a good model for our observations. Free energies of crosslinking, induced by the divalent cations, are estimated to be between 34 and 38% of the free energies of base stacking. The ability of a divalent metal cation to induce DNA aggregation can be attributed to its ability to disrupt DNA base pairing, and simultaneously to link two different DNA sites.

Last updated 5/27/98

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