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

Abstract

The dynamic of supercoiled DNA (scDNA) transformations is the key point for understanding the numerous processes taking place in the living cell [1]. DNA topology changes are vital in replication, transcription, recombination, chromosome condensation, and segregation. From the topological point of view DNA can be represented as a closed ribbon [2][3][4]. A study of the dynamic aspects of the DNA topology is closely associated with a design of an adequate mathematical description of the DNA polymeric molecule and methods for monitoring its properties [5][6][7][8]. The main topological changes of scDNA are catalyzed by DNA-topoisomerases I and II, which induce single and double nicks in DNA strains, respectively. These correspond to changes in the linking number (Lk) of the polymer substrate by 1 or 2. From the chemical point of view, the substrates and products of DNA topoisomerization are identical and catalytic events result in only slight topological changes. The product of the previous turnover acts as a substrate at the next stage, so an ensemble of topoisomers exists at each step of the reaction. This fact stipulates a description of scDNA biocatalytic 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.

Discovery of Mono‐ and Disubstituted 1H‐Pyrazolo[3,4]pyrimidines and 9H‐Purines as Catalytic Inhibitors of Human DNA Topoisomerase IIα

“…The maximal theoretical response was calculated according to the molecular masses of the human ATPase domain and ligands 8 and 12. [49] This promising new approach could complement the SPR studies, offering further insight into the inhibition process of our discovered compounds. We were pleased to establish that K D values for both compounds 8 and 12 were in good agreement with the determined IC 50 inhibitory activity values (Table 4).…”

“…It should be noted that, recently, a powerful flow linear dichroism (FLD) technique for continuous monitoring of the topoisomerization reaction was reported, enabling in-depth mechanistic studies of topo enzymes and their interactions with ligands. [49] This promising new approach could complement the SPR studies, offering further insight into the inhibition process of our discovered compounds.…”

Oxidative DNA cleavage by Cu(II) complexes: Effect of periphery substituent groups

“…Various reactive oxygen species (ROS) can participate in the DNA cleaving process [47][48][49][50]. Lanes 3-6 in Figure 5 shows the effect of ROS scavengers on scDNA cleavage.…”

Evaluation of the Effect of Knowledge Concerning Healthy Nutrition and Nutrition Science on the Knowledge Development Approach

“…It had been found that undigested foods in the large intestine change into toxic substances that could reduce the life span and cause cancer. Thus, patients were advised to follow zero food diets to get rid of these unpleasant materials and take a break from digestion activities (33).…”