Catalytic transformations of supercoiled DNA as studied by flow linear dichroism technique.

ABSTRACT

A catalytic turnover of supercoiled DNA (scDNA) transformation mediated by topoisomerases leads to changes in the linking number (Lk) of the polymeric substrate by 1 or 2 per cycle. As a substrate of the topoisomerization reaction it is chemically identical to its product; even a single catalytic event results in the quantum leap in the scDNA topology. Non-intrusive continuous assay to measure the kinetics of the scDNA topoisomerization was performed. The development of such a technique was hindered because of multiple DNA species of intermediate topology present in the reaction mixture. The interrelation of DNA topology, its hydrodynamics, and optical anisotropy enable us to use the flow linear dichroism technique (FLD) for continuous monitoring of the scDNA topoisomerization reaction. This approach permits us to study the kinetics of DNA transformation catalyzed by eukaryotic topoisomerases I and II, as well as mechanistic characteristics of these enzymes and their interactions with anticancer drugs. Moreover, FLD assay can be applied to any enzymatic reaction that involves scDNA as a substrate. It also provides a new way of screening drugs dynamically and is likely to be potent in various biomedical applications.