Study of Topoisomerase-DNA Interaction Using Atomic Force Microscopy

Abstract

The aim of this thesis was to explore the topoisomerase-DNA interactions using atomic force microscopy (AFM) in a liquid environment. Firstly, we present a programmable microcontroller-driven injection system for the exchange of an imaging medium while using AFM. Using this low-noise system, high-resolution imaging can be performed during the process of injection without disturbance. The use of the injection system was put into practice when studying the conformational changes of DNA molecules during the injection of intercalating agents, such as daunorubicin (an anticancer drug) and ethidium bromide, into the fluid chamber. An observation of their mode of interaction with DNA might help elucidating their mechanism of action, thereby facilitating the development of more specific drugs. Part of this thesis deals with the way enzymes interact with DNA molecules. We found that human type II topoisomerases (Topo II) bind preferentially to DNA crossovers. About 50% of the DNA crossings, where the Topo II were bound to, presented an angle of a nearly perpendicular orientation, suggesting a favoured binding geometry in the Topo II recognition. Our studies with AFM also helped us visualize the dynamics of the unknotting action of Topo II in knotted DNA molecules. Additionally we investigated the interactions between Topo II and DNA by applying single molecule force spectroscopy. This study evidenced the inhibitor effect of the aclarubicin (anticancer drug) and evaluated the preferential binding of Topo II to specific DNA supercoiled forms. Finally, we explored the Topo II dynamics using a novel technique that detects motion from nano- to micro-metre sized systems. This method opens new possibilities for the study of conformational changes of single proteins, biochemical reactions, as well as drug-target inhibition.