64 A. Gabibov et al.
of re-ligation of nicked DNA chain (see Scheme 4.1a) [36, 37]. As shown in
Fig. 4.13, in the presence of the increasing concentrations of CPT and MCPT-
10,11, kinetic curves retain their exponential character, whilek′diminishes.
I 50 for these compounds can be estimated as 1 and 0.1 mM, respectively. On
the contrary, SN-38 (Fig. 4.13) does not affect the rate of the reaction on the
initial stage of the process, while effectively inhibiting it afterwards (I 50 for
the second portion is 0.2 mM). We attribute this difference to the fact that
by FLD technique it becomes possible to distinguish between mechanisms of
action of these closely related compounds. In our opinion, all three inhibitors
hinder the re-ligation of DNA, but CPT and MCPT-10,11 impede the initial
enzyme–DNA interactions as well, while SN-38 lacks this additional activity
(see Scheme 4.1).
For topoisomerase II, we have chosen two compounds with totally different
modes of action – etoposide [38] and adenosine-5′-phosphate-b,g-iminodiphos-
phate (AMPPNP). The first one, etoposide, now a widely used chemother-
apeutic drug, is a classic topoisomerase II poison, acting by binding with
single-stranded DNA ends, inhibiting the re-ligation of hydrolysed DNA seg-
ment (see Scheme 4.2) [39]. This inhibitor does not change the general view
of kinetic curve, only slowing the whole process and making the local mini-
mum of rotp less expressed (Fig. 4.14). The latter observation suggests that
the process gets less coordinated in the presence of etoposide. Using the initial
rates, we estimate I 50 for this compound as 10 mM. Another topoisomerase
II effector, AMPPNP, is the non-hydrolyzable analogue of ATP [32, 40]. Its
action is based on the necessity of hydrolysis of phosphodiester bond of ATP
for the completion of enzyme turnover. Thus, if AMPPNP molecule binds to
one or both topoisomerase II subunits, the enzyme stays in the conforma-
tion of “closed clamp”, topologically bound to the closed DNA molecule, and,
therefore, is kinetically irreversibly inactivated. If both ATP and AMPPNP
are present in the reaction mixture, only a certain portion of enzyme mole-
cules is inactivated at each catalytic step. Reaction under these conditions
does not proceed up to the equilibrium state, and kinetic curves reach the
plateau at values of FLD signal corresponding tos<0.
4.3 Conclusions
The discussed intrinsic connection between DNA topology, hydrodynamics
and optical properties eneablse us to study experimentally dynamics of DNA
relaxation. The fundamental functional dependency of FLD signal from the
DNA topology provides us with the unique possibility of performing the con-
tinuous analysis of DNA topoisomerisation. We proved the mechanistic pe-
culiarities of topoisomerase I, studied by traditional methods and for the
first time studied the real-time kinetics for topoisomerase II. In our point