Properties of Closed Circular DNA
<p>A method for the measurement of the superhelix density, σ<sub>0</sub>, of a closed circular DNA from separation between fluorescent buoyant bands of nicked and closed circular forms in an ethidium bromide-CsCl equilibrium gradient is presented. Nicked and closed circular DNA&...
| Summary: | <p>A method for the measurement of the superhelix density, σ<sub>0</sub>, of
a closed circular DNA from separation between fluorescent buoyant
bands of nicked and closed circular forms in an ethidium bromide-CsCl
equilibrium gradient is presented. Nicked and closed circular DNA's
are banded together in a gradient formed by centrifuging a CsCl solution
of density 1.56 gm/ml. containing 330 µg/ml ethidium bromide at
20°C for 48 hours in the preparative ultracentrifuge. The separation
between the bands, normalized by the separation between the nicked
and closed circular forms of a DNA of known superhelix density, is
shown to be linearly related to the difference in superhelix density
between the DNA's of known and unknown superhelix densities according
to the equation</p>
<p>Δσ<sub>0</sub> = (0.115 ± 0.005)(Ω<sub>c</sub> - 1)</p>
<p>where</p>
<p>Δσ<sub>0</sub> = σ<sub>0</sub> - σ<sub>0</sub>* and Ω<sub>c</sub> = Δr/Δr* r/r (v<sub>1</sub>θ*-1)<sup>2</sup>/(v<sub>1</sub>θ-1)<sup>2</sup></p>
<p>Δr is the separation between the bands, r is the average distance of
the two bands from the center of rotation, θ is the buoyant density of
the DNA and v<sub>1</sub> is the partial specific volume of water. The asterisk
refers to the reference DNA. A molecular weight dependence of the
intercept is seen when λb<sub>2</sub>b<sub>5</sub>c DNA (molecular weight 25 x 10<sup>6</sup>) is banded
against a SV40 DNA (3 x 10<sup>6</sup> standard. The relationship was obtained
by measuring the separations for DNA's whose superhelix densities
were determined by sedimentation velocity-dye titrations. Native
SV40 viral DNA with a superhelix density of -0.039 was used as a
standard in all cases. DNA's with altered superhelix densities were
prepared by closing nicked circular DNA's with polynucleotide ligase
under various conditions.</p>
<p>Ten closed SV40 DNA's with superhelix densities ranging from
-0.007 to -0.085 have been prepared. This family of DNA's has been
used to examine the effects of superhelix density on the sedimentation
velocity behavior of closed SV40 DNA. The sedimentation coefficient
increases as the absolute value of the superhelix density rises from a
low value to 0.019, then decreases to a local minimum at 0.035 and
finally increases steadily as |σ<sub>0</sub>| rises to 0.085.</p>
<p>The sedimentation velocity-ethidium bromide titrations of these
DNA's have been converted from the primary s<sup>0</sup><sub>20</sub>,* versus c data, in
which s<sup>0</sup><sub>20</sub>,* is the standard sedimentation coefficient still uncorrected
for the buoyant effect of bound ethidium chloride and c is the free
ethidium bromide concentration, to the more meaningful s<sup>0</sup><sub>20</sub>,<sub>w</sub> versus
σ<sub>0</sub> form, with the aid of coefficients in the expression for the free
energy of superhelix formation. The resultant curves form a family
that is approximately superimposable on the curve for s<sup>0</sup><sub>20</sub>,<sub>w</sub> versus
σ<sub>0</sub> in the absence of ethidium bromide.</p>
<p>The dependence of the sedimentation coefficient of selected SV40
DNA's upon ionic strength, the nature of the cation, and temperature is
consistent with the previously reported effects of these variables on the
rotation angle of the base pairs along the helix axis.</p>
<p>Separations between open and closed circular DNA's in buoyant
CsCl gradients containing the ethidium bromide analogue, propidium
di-diodide, are shown to be 1.8 times larger than in ethidium bromide.</p> |
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