Nanofluidics for Static and Dynamic DNA-Protein Interaction Studies - Repair of Double-Strand Breaks from a Single-Molecule Perspective
Sammanfattning: Double-strand breaks (DSBs) is one of the most lethal forms of DNA damage. A single DSB may result in stalling of vital cellular machineries, and thus, requires immediate measures by the cell. Although the main hallmarks of DSB repair mechanisms are known for most prokaryotes and eukaryotes, details on crucial intermediate steps are still to be explained, such as how the free DNA ends are kept in close proximity during the repair process. In the original work, upon which this Thesis is based, the two main DSB repair mechanisms, homologous recombination (HR) and non-homologous end-joining (NHEJ), have been studied from a molecular perspective. A fluorescence-based single-molecule nanofluidics assay has been developed and employed to characterize and visualize static biomolecular interactions between DNA and key DSB repairing proteins. Furthermore, a novel dynamic nanofluidic device has been developed to enable dynamic interaction studies in real time, allowing analytes to be introduced on-demand to stretched DNA molecules. Single-molecule characterization of the NHEJ mechanism in Bacillus subtilis , comprising the Ku and Ligase D proteins, revealed that the end-joining activity is mediated by C-terminal protrusions on the homodimeric Ku complex. Using the novel dynamic nanofluidic device, the Ku homodimer was further demonstrated to stay bound to the DNA ends and junctions after completed repair, similar to the human Ku70/80 heterodimer. In addition, the traditional static nanofluidic device was used to identify a previously unknown potential DNA-bridging role of CtIP, a key protein in the human HR process. This could possibly explain how broken DNA ends are kept in close proximity during the initial steps of DSB repair through HR in humans. A similar method was employed to show that the Xrs2 component is indispensable for the end-joining activity of the Mre11-Rad50-Xrs2 complex of Saccharomyces cerevisiae .
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