4 Dynamics of DNA Supercoiling 61relaxed. On the other hand, the mixture composition is crucial for the velocity
of the process, in particular, ionic strength of the mixture. A series of exper-
iments, in which pTM plasmid relaxation runs in media with different ionic
strength has shown that the kinetic curve is split into two parts along with
dropping of the ionic strength. An initial (fast) part corresponds to relaxation
of the enzyme-bound DNA in a moment when the enzyme is added to the
mixture. At this stage the reaction rate is only slightly dependent on the ionic
strength of the buffer (Fig. 4.11a), which is actually, clear for understanding
since the limiting stage at this point the shows phosphodiester bond cleavage.
The second part of the curve represents a stage at which all total DNA bound
with the enzyme at the fast stage, is actually fully relaxed, and the velocity
of the rest of DNA is determined by a process of dissociation of enzyme from
the complex with relaxed DNA (Fig. 4.11b).
(b)Fig. 4.11.Dependence of the effective constant of pTM relaxation catalysed by
topoisomerase I. Initial (a) or final (b) part of kinetic curves on different concentra-
tions of NaCl