SciPost Thesis Link
|Title:||Integrating single-molecule visualization and DNA micromanipulation|
|Author:||J. van Mameren|
|As Contributor:||Joost van Mameren|
|Degree granting institution:||Vrije Universiteit|
Over the past decades, techniques have been developed by means of which single molecules can be individually manipulated under a microscope. Using so-called ‘optical tweezers’ for example, it is now possible to grab a single DNA molecule from both ends and extend it. Doing so, it is possible to study the elastic properties of DNA as if it were a rubber band. Moreover, the properties of the wealth of proteins that bind to DNA to maintain it can be studied in a unique manner. On the other hand, fluorescence microscopy techniques have by now become so sensitive, that the fluorescence from individual dye molecules can be routinely registered using a camera. This can be exploited to follow the diffusive motion of fluorescently labeled proteins in real time and hence detect and visualize their binding to a DNA substrate. The subject of Van Mameren’s thesis is the simultaneous application of such single-molecule visualization and micromanipulation. This integrated approach allows for probing the effect of mechanical DNA tension on DNA–protein interaction. The thesis describes a several projects involving DNA dynamics, which could be exclusively carried out using such a combined single-molecule approach. One of the studied proteins, Rad51, is required for a reliable repair of DNA, in part to prevent various forms of cancer. The disassembly mechanism of the protein filament that Rad51 thereby forms around the DNA has been uniquely unraveled by combining the techniques described above.