50 A. Gabibov et al.
Generally, the shape ofρ(σ) curves is similar for a number of tested plas-
mids. However, quantitatively, the parameters describing each curve revealed
a significant dependence upon specific character of the plasmids under study.
The two plasmids illustrating the alternative FLD signal dependencies have
been chosen. A pTM plasmid, while possessing very short BC segment,
revealed the highest amplitude of the LD signal in the course of the transi-
tion from a supercoiled state to the relaxed one. BlueScript plasmid titration
curve, on the contrary, contains a substantial BC segment. We observed the
similar LD values for both supercoiled and relaxed states of this plasmid, but
LD values for the transient topoisomers were much lower. Both the plasmids
were applied as a tool for the topoisomerase I studies.
4.2.2 Mechanisms of Biocatalytic DNA Relaxation
For modelling enzyme-mediated DNA topoisomerisation the following assump-
tions were made:
At any time, ti, the reaction mixture contains unbound enzyme and plas-
mid in different topological statesSn(wherenis the number of superturns).
The enzyme and either of the DNA topoisomers can be bound to each other
in an equilibrium way, to form a non-covalent complexESn, where both the
DNA strands have no nicks. This complex is not capable of proceeding to
topoisomerisation. The biocatalytic productivity requires the transfer ofESn
complex into the covalent complex (ESn). Here one of the DNA strains is
nicked and the enzyme is covalently bound to the 3′-end. TheESncomplex is
capable of chaning its topology (the driving force of this process is the tension
of supercoiled DNA molecule) to formESn+1orESn−^1. The reaction is fully
reversible and (ESn) may take part in the back reaction. This mechanism is
displayed in:
Scheme 4.1This mechanistic representation allows to make quantitative estimations.
For the calculations, we need to get values of the appropriate kinetic constants.