Tethered fluorophore motion studies of DNA segregation machinery

• Main Author: May, Peter F. J.
• Other Authors: Kapanidis, Achillefs
• Published: University of Oxford 2015
• Subjects: 571.4
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.712464

Tethered fluorophore motion (TFM) is a single-molecule fluorescence technique, used to monitor an effective length along a biopolymer, such as DNA. In this work, I start by characterising TFM, focussing on the theoretical basis and potential applications, and then use TFM to study the bacterial site specific recombination system, XerCD-dif. I present a description of the sources of noise in TFM and derive an analytical expression for the resolution, which is compared to simulations and experiments. The work demonstrates that length changes as low as 100 bp of double-stranded DNA are distinguishable using TFM. The optimum pixel size for TFM is derived and validated experimentally. XerCD-dif recombination is responsible for chromosome decatenation in most bacteria with circular chromosomes. It is activated by the DNA translocase FtsK. I describe the application of TFM, in combination with Förster resonance energy transfer (FRET) and protein induced fluorescence enhancement, to observe the formation and activation of XerCD-dif synaptic complexes. The work followed the reaction, as Holliday junctions formed by XerD-mediated strand exchange isomerised and were resolved to product by XerC, and measured the lifetimes of all the intermediates observed. To analyse the role of FtsK in the activation of recombination, and to monitor its assembly, translocation and behaviour when encountering XerCD-dif, TFM was extended with the use of two spectrally distinct fluorophores, allowing simultaneous monitoring of two effective lengths along the same DNA. FtsK was observed to assemble on DNA, stepwise, into a single hexamer, and begin translocation rapidly (~0.25 s). Single hexamers approached XerCD-dif, and resided for 0.5s to 1s before dissociating, irrespective of whether XerCD-dif was synapsed or recombination was activated. The final section of this thesis details the design, construction and evaluation of a three colour microscope. Alternating laser excitation was implemented in the green, red and near-infrared spectral channels with three laser sources, and an inexpensive automatic focus system was implemented using the back reflection of the excitation lasers and a complementary metal oxide semiconductor camera.