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Notes: Sedimentation experiments have been performed on a polydisperse bacterial DNA sample over a wide range of ionic strength (8 × 10-4M to 2M Na+), at very low DNA concentrations (5-30 μg ml). True sedimentation constant distributions were obtained by careful analysis of experimental data and extrapolation to infinite dilution. In order to give a quantitative description of macromolecular shape, the changes of the exponent as in the general equation, s0 = ksMas, have been determined by comparing sedimentation constant distributions obtained at different ionic strengths. as has been found to vary from 0.419 at 2M Na+ to 0.200 at 8 × 10-4M Na+. As well as the decrease of the mean sedimentation constant, this result indicates a pronounced expansion of DNA molecules with decreasing ionic strength. A discussion of the distinct mechanism responsible for the expansion behavior of DNA is given. Furthermore, the dependence of the Mandelkern-Flory parameter β on ionic strength has been calculated by combining the s0 data with the corresponding [η] values of the sample.

Notes: The diffusion, sedimentation, and viscosity behavior of native DNA has been studied as a function of molecular weight absolutely determined by diffusion and sedimentation measurements between 106 and 20 × 106 Daltons. Only data for monodisperse subfractions of the polydisperse DNA samples investigated have been compared. They were derived by means of calculations based on individual sedimentation constant distribution and polymer parameters. The inconstancy of these parameters for the semi-rigid DNA molecules over the range of different sub-fractions of the polydisperse samples has been taken into account. A detailed description of the corresponding mathematical background as well as of the diffusion measurements has been given in the two preceding papers. For the homologous series of monodisperse native DNA molecules, three-parameter equations have been determined representing the s0-[η], s0-M, and [η]-M relations as well as the D0-s0 and D0-M interdependence over the entire molecular weight range of interest. Furthermore a simple equation is given describing the molecular weight dependence of the Mandelkern-Flory-Scheraga parameter β in terms of s0 and [η]. The asymptotic value β∞ has been determined, primarily by measurements at finite molecular weights, to be 2.39 × 106, resulting in β values near 2.50 × 106 at M = 108 Daltons. The hydrodynamic properties of our two calf thymus DNA samples of highest molecular weight proved to be influenced by residual protein interactions resulting in a more compact conformation. Further data are given demonstrating the influence of the individual polydispersity of the investigated samples on diffusion constant and molecular weight averages.

Notes: Reversible and irreversible conformational changes in the acid-induced denaturation of DNA were studied by spectrophotometric titration, sedimentation, and melting measurements. A GC-rich DNA (72 mole-%) shows complete or partial reversibility of the titration profiles within the pH region of transition from helix to coil, while AT-rich DNA (29 mole-%) is irreversible in its titration behavior at each acid pH below the onset of the transition. The results for GC-rich DNA further indicate distinct differences in the titration behavior, which can be attributed to differences in the frequency of GC clusters along the DNA molecule. Plots of the sedimentation coefficient and the parameter asapp against pH lead to the conclusion that conformational changes occur before the onset of the acid-induced helix-coil transition. These alterations are more pronounced upon protonation of larger GC-rich domains than of smaller ones, as concluded from very marked differences observed in the sedimentation-pH behavior of two GC-rich DNA's. An acid denaturation scheme for a GC-rich DNA segment is suggested. Reversibility of the acid denaturation is explained by the existence of stable, protonated, single GC base pairs in nonprotonated stacked single-stranded domains formed in the acid-induced transition region.

Notes: Spectrophotometric, sedimentation, infrared, optical rotatory dispersion (ORD), and circular dichroism (CD) methods have been used to demonstrate the structural changes in DNA induced by the interaction of copper(II) with bases and to elucidate the complex binding sites. As shown by the electrolyte-induced reversion (addition of salts) of temperature-denatured copper DNA the effectiveness of re-formation of the double-stranded structure depends on the temperature, copper(II) ion concentration, and on the base composition of the DNA. Exposure of heat-denatured copper DNA to higher temperatures decreases the reversion effect on addition of electrolyte. The results indicate that a greater fraction with a cooperative transition appears on heating DNA to 80 or 100°C at a Cu2+/DNA-P ratio of 2 : 1 than at a Cu2+/DNA-P ratio of 1 : 1. With AT-rich copper DNA, reversion to the native DNA structure was not observed. Selective methylation of guanine residues in DNA also affects the electrolyte-induced reversion, indicating the importance of GC pairs for copper(II) binding and the reversion to the native structure. Temperature-denatured copper DNA shows an increased sedimentation coefficient Which decreases again after electrolyte-induced reversion. This change in s is reduced by selective methylation of DNA. Complex formation between copper(II) and the bases is accompanied by a conformational change of the DNA double-helical structure as demonstrated by ORD and CD experiments. The ORD profile of GC-rich DNA is much more affected by copper(II) than that of AT-rich ones. Even at very low copper(II) concentrations, e.g., at 0.02 and 0.2 Cu2+/DNA-P, the ORD and CD measurements exhibit conformational changes of the DNA secondary structure at room temperature. By comparing the infrared spectra of deoxynucleosides with that of DNA of different GC content it has been shown that both guanine and cytosine are involved in the formation of the complex of copper(II) with DNA. N-7 and O at C-6 in guanine and N-3 as well as O of C-2 in cytosine are discussed as the most probable binding sites in DNA. A binding model for the coordination of the copper(II) ion between guanine and cytosine of the opposite strands is suggested. The results are in good agreement with the assumptions and predictions made by Eichhorn and Clark about the complexing of copper(II) with DNA. The recent proposal made by Schreiber and Daune about an interaction of the type guanine-Cu2+-guanine cannot be excluded as an additional kind of coordination of copper(II) in DNA.

Notes: The effects of metal ions of the first-row transition and of alkaline earth metals on the DNA helix conformation have been studied by uv difference spectra, circular dichroism, and sedimentation measurements.At low ionic strength (10-3 M NaClO4) DNA shows a maximum in the difference absorption spectra in the presence of Zn2+, Mn2+, Co2+, Cd2+, and Ni2+ but not with Mg2+ or Ca2+. The amplitude of this maximum is dependent on GC content as revealed by detailed studies of the DNA-Zn2+ complex of eight different DNA's. Pronounced changes also occur in the CD spectra of DNA transition metal complexes. A transition appears up to a total ratio of approximately 1 Zn2+ per DNA phosphate at 10-3 M NaClO4; then no further change was observed up to high concentrations. The characteristic CD changes are strongly dependent on the double-helical structure of DNA and on the GC content of DNA.Differences were also observed in hydrodynamic properties of DNA metal complexes as revealed by the greater increase of the sedimentation coefficient of native DNA in the presence of transition metal ions. Spectrophotometric acid titration experiments and CD measurements at acidic pH clearly indicate the suppression of protonation of GC base-pair regions on the addition of transition metal ions to DNA. Similar effects were not observed with DNA complexes with alkaline earth metal ions such as Mg2+ or Ca2+.The data are interpreted in terms of a preferential interaction of Zn2+ and of other transition metal ions with GC sites by chelation to the N-7 of guanine and to the phosphate residue. The binding of Zn2+ to DNA disappears between 0.5 M and 1 M NaClO4, but complex formation with DNA is observable again in the presence of highly concentrated solutions of NaClO4 (3-7.2 M NaClO4) or at 0.5 to 2 M Mn2+. At relatively high cation concentration Mg2+ is also effective in changing the DNA comformation. These structural alterations probably result from both the shielding of negatively charged phosphate groups and the breakdown of the water structure along the DNA helix. Differential effects in CD are also observed between Mn2+, Zn2+ on one hand and Mg2+ on the other hand under these conditions. The greater sensitivity of the double-helical conformation of DNA to the action of transition metal ions is due to the affinity of the latter to electron donating sites of the bases resulting from the d electronic configuration of the metal ions. An order of the relative phosphate binding ability to base-site binding ability in native DNA is obtained as follows: Mg2+, Ba2+, 〈 Ca2+ 〈 Fe2+, Ni2+, Co2+ 〈 Mn2+, Zn2+ 〈 Cd2+ 〈 Cu2+. The metal-ion induced conformational changes of the DNA are explained by alternation of the winding angle between base pairs as occurs in the transition from B to C conformation. These findings are used for a tentative molecular interpretation of some effects of Zn2+ and Mn2+ in DNA synthesis reported in the literature.