Topology in Molecular Biology

4 Dynamics of DNA Supercoiling 57

by FLD technique (data not shown). One can explain the latter observa-
tion taking into account that for the reaction, catalysed by topoisomerase
I, calculations predict considerable amounts of highly supercoiled or relaxed
DNA (and small traces of intermediate topoisomers) during entire the reaction
course.
The fact that the LD changes during a topoisomerase I-catalysed reaction
obeys the first-order kinetics needs an explanation. It is possible that for
this reaction theKsvalue does not depend on the DNA topology. To check
this, we studied the relaxation of plasmid pTM 18 in the presence of sc (s)
and relaxed (r) forms of BlueScript. As sc BlueScript in such experiment is
virtually “invisible”, both sc and relaxed BlueScript could not effect an FLD
signal, so we are able to monitor the pTM 18 relaxation alone.
In both series of experiments with pTM 18, when topoisomerisation
was carried out in the presence of different concentrations of relaxed or sc
BlueScript,k′ decreased with an increase in the mass ratio of A = [Blue-
Script]/[pTM 18] (Fig. 4.8). Both for relaxed and supercooled BlueScript plas-
mids theIC 50 values were found to be about equal, which implies equal,
affinity of the enzyme to DNA of any topology.
The insensitivity of topoisomerase I towards the DNA topology was pro-
posed earlier based on the data of DNA relaxation kinetics in experiments
with the chicken erythrocyte topoisomerase I [30]. However, latter publications
contradicted this standpoint. For instance, a human topoisomerase I mutant
incapable of cleaving and therefore relaxing supercoiled DNA was found to
bind scDNA more than ten times more effectively as compared to the relaxed

Fig. 4.8.Determination of binding parameters of topoisomerase with sc and re-
laxed DNA. The plasmid pTM 18 relaxation kinetics was studied in the presence of
BlueScript plasmid in different concentrations taken in relaxed or sc forms. Reaction
mixture contained 20 mM of Tris–HCl (pH 7.5), 0.5 mM of EDTA, 75μgμlμl−^1 of
BSA, 5μg of sc plasmid pTM 18 and different concentrations of relaxed or sc forms
of BlueScript