Abstract : Atomic Force Microscopy (AFM) opens new perspectives in the study of DNA-protein interactions. My work consisted of developing new methodologies for controlling DNA adsorption on surfaces and enabling the study of the dynamics of the complexes in liquid.
We have characterized the interactions between DNA and the mica surface. We propose a simple model to describe the electrostatic interactions in solution between DNA and mica, considering the role of monovalent and divalent cations. The good correlation with experimental data allows validating referential adsorption conditions and a reversible adsorption method for DNA on nickel-pretreated mica. In parallel we have developed a system of tethers to anchor DNA by its extremities.
The control of these methodologies allows characterizing accessibility in function of the adsorption states. We broach this issue by characterizing bleomycin activity on DNA. This approach on a model system allows characterizing the surface influence in terms of accessibility and activity.
The last part of this work considers the characterization of the interactions of the Ku protein with DNA, in the frame of the study of DNA double-strand break repair. Our approach which combines the contributions of transmission electron microscopy and of AFM shows a cooperative polymerization of Ku along DNA and a very different binding mode on single-stranded DNA. This work shows the interest of molecular imaging for the characterization of target site research mechanisms by proteins.