Study of Topoisomerase-DNA Interaction Using Atomic Force Microscopy

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

Time-Lapse AFM Imaging of DNA Conformational Changes Induced by Daunorubicin

Cancer is a major health issue that absorbs the attention of a large part of the biomedical research. Intercalating agents bind to DNA molecules and can inhibit their synthesis and transcription; thus, they are increasingly used as drugs to fight cancer. In this work, we show how atomic force microscopy in liquid can characterize, through time-lapse imaging, the dynamical influence of intercalating agents on the supercoiling of DNA, improving our understanding of the drug’s effect

Time-Lapse AFM Imaging of DNA Conformational Changes Induced by Daunorubicin

Cancer is a major health issue that absorbs the attention of a large part of the biomedical research. Intercalating agents bind to DNA molecules and can inhibit their synthesis and transcription; thus, they are increasingly used as drugs to fight cancer. In this work, we show how atomic force microscopy in liquid can characterize, through time-lapse imaging, the dynamical influence of intercalating agents on the supercoiling of DNA, improving our understanding of the drug’s effect

Time-Lapse AFM Imaging of DNA Conformational Changes Induced by Daunorubicin

Cancer is a major health issue that absorbs the attention of a large part of the biomedical research. Intercalating agents bind to DNA molecules and can inhibit their synthesis and transcription; thus, they are increasingly used as drugs to fight cancer. In this work, we show how atomic force microscopy in liquid can characterize, through time-lapse imaging, the dynamical influence of intercalating agents on the supercoiling of DNA, improving our understanding of the drug’s effect

Atypical DNA recognition mechanism used by the EspR virulence regulator of Mycobacterium tuberculosis

The human pathogen Mycobacterium tuberculosis requires the ESX-1 secretion system for full virulence. EspR plays a key role in ESX-1 regulation via direct binding and transcriptional activation of the espACD operon. Here, we describe the crystal structures of EspR, a C-terminally truncated form, EspR Delta 10, as well as an EspR-DNA complex. EspR forms a dimer with each monomer containing an N-terminal helix-turn-helix DNA binding motif and an atypical C-terminal dimerization domain. Structural studies combined with footprinting experiments, atomic force microscopy and molecular dynamic simulations allow us to propose a model in which a dimer of EspR dimers is the minimal functional unit with two subunits binding two consecutive major grooves. The other two DNA binding domains are thus free to form higher-order oligomers and to bridge distant DNA sites in a cooperative way. These features are reminiscent of nucleoid-associated proteins and suggest a more general regulatory role for EspR than was previously suspected

FM-AFM constant height imaging and force curves: high resolution study of DNA-tip interactions

Interaction of the atomic force microscopy (AFM) tip with the sample can be invasive for soft samples. Frequency Modulation (FM) AFM is gentler because it allows scanning in the non-contact regime where only attractive forces exist between the tip and the sample, and there is no sample compression. Recently, FM-AFM was used to resolve the atomic structure of single molecules of pentacene and of carbon nanotubes. We are testing similar FM-AFM-based approaches to study biological samples. We present FM-AFM experiments on dsDNA deposited on 3-aminopropyltriethoxysilane modified mica in ultra high vacuum. With flexible samples such as DNA, the substrate flatness is a sub-molecular resolution limiting factor. Non-contact topographic images of DNA show variations that have the periodicity of the right handed helix of B-form DNA - this is an unexpected result as dehydrated DNA is thought to assume the A-form structure. Frequency shift maps at constant height allow working in the non-monotonic frequency shift range, show a rich contrast that changes significantly with the tip-sample separation, and show 0.2 to 0.4 nm size details on DNA. Frequency shift versus distance curves acquired on DNA molecules and converted in force curves show that for small molecules (height < 2.5 nm), there is a contribution to the interaction force from the substrate when the tip is on top of the molecules. Our data shine a new light on dehydrated and adsorbed DNA behavior. They show a longer tip-sample interaction distance. These experiments may have an impact on nanotechnological DNA applications in non-physiological environments such as DNA based nanoelectronics and nanotemplating

The experimental setup and the experiment using high Topo II concentration (107.6 nM).

<p><u>Panel a:</u> Schematic illustration of the experimental setup: C) cantilever, L) laser beam, D) photodiode. Topo II indicates the molecules adsorbed to both sides of the cantilever. <u>Panel b:</u> The cantilever deflections as a function of ATP concentration. Different media were flowed through the analysis chamber: buffer (with no ATP), ATP-enriched medium, containing in order 0.2 µM, 2.0 µM, 0.02 mM, 0.2 mM and 2.0 mM ATP, and then again the no-ATP buffer. <u>Panel c</u>: Corresponding variance values.</p

The Aclarubicin experiment.

<p>The cantilever deflections as different media were flowed through the analysis chamber: buffer, an ATP-enriched solution (20 µM), and finally a solution containing both ATP (20 µM) and Aclarubicin (100 µM). The variance values were calculated from five independent experiments.</p