Topology in Molecular Biology

4 Dynamics of DNA Supercoiling 49

and increasing the superhelix density (Riis reduced andPiis increased),
which causes a decrease of the optic anisotropy of the molecule. Thus the
dependence of optic anisotropy of circular DNA and its topology should prob-
ably have a bell-shaped form with a maximum in a point corresponding to
the completely relaxed DNA. It should reach the minimum at moderate|σ|
and then should rise again.
This assumption was confirmed by titration experiments (circular DNA by
intercalators, proflavine, ethidium bromide, etc.), displaying the bell-shaped
dependence ofρ(σ) [22, 25]. Figrue 4.3 illustrates typical results of ethidium
bromide titration of the negatively supercoiled plasmids. All the curves are
characterised by the maximum corresponding to the fully relaxed state (point
A). The presence of positive (σ>0) or negative supercoils results in decrease
ofρ(AD and AB segments of the curves). Further increase of|σ|results in
increasing ofρagain (segment BC). The latter probably is a consequence
of the rising alignment ability of the DNA (in agreement with the previous
theoretical prediction).

Fig. 4.3.The dependence of the FLD signal (ρ 260 ) on the density of supercoiling
for plasmids pTM 18 and BlueScript. Linear dichroism of the sample is measured
as relative valueρsg that is equal to zero for the blank (reaction buffer) and to 1
for the relaxed plasmid sample. The FLD measurements were performed in 200μl
of buffer (20 mM Tris–HCl (pH 7.5), 100 mM NaCl, 0.5 mM EDTA) containing
5 μg pTM 18 or 5μg BlueScript at increasing concentration of ethidium bromide
(1-μl aliquots of 6× 10 −^5 M were added). The value of the superhelix density (σ)
was calculated taking the unwinding angle value for ethidium bromide as 26◦per
intercalated ethidium ion [25]. The starting value ofσappeared to be− 0 .036 and
− 0 .055 for BlueScript and pTM 18, respectively