Developing high-speed scatterometry tools to detect early events during Nucleotide Excision Repair

Exposure of DNA to ultraviolet (UV) radiation results in the formation of numerous lesions which threaten genome integrity. The nucleotide excision DNA repair pathway detects and repairs a range of such UV-induced DNA lesions. In bacteria, initial damage detection and verification is carried out by two proteins: UvrA and UvrB. Despite decades of study, the process of how these proteins locate damage remains unclear. Here we first demonstrate implementations of novel versions of both conventional dark-field microscopy, and interference reflection microscopy (IRM) for the imaging of gold nanoparticle (AuNP) labelled proteins. We then develop these into an implementation of interferometric scattering (iSCAT) microscopy to allow for high-speed imaging of protein-DNA interactions, including a method that can be used to achieve accurate active stabilisation of an iSCAT microscope in 3-axes using a software-based 3D localisation routine to extract the X, Y, and Z positions of a particle. Combining iSCAT microscopy with a novel, highly customisable, surface-bound-DNA assay, we investigate early damage detection by UvrA. Through this method we have discovered that UvrA interacts with DNA in two phases; a slow phase (~1.3/s) that correlates with an ATP-consuming state previously identified, and a second, much faster search mode. These faster interactions persist for ~130 ms and using ATP analogues we determine this phase does not require ATP consumption. Additionally, by modelling the rate at which UvrA is able to search the E. coli genome, we demonstrate that these rapid interactions allow for basal levels of UvrA to explore 99% of the E. coli genome within a single division cycle. Altogether, this work uncovers the presence of a rapid, energy-efficient search mechanism, which allows UvrA alone to search the entirety of the E. coli genome within a single division cycle and highlights the exciting potential for use of iSCAT microscopy for the study of protein-DNA interactions.