Extending and combining single-molecule fluorescence methods to study site-specific recombination

Förster resonance energy transfer (FRET) has become an important tool for studying biochemical reactions at the single-molecule level, despite its increasing maturity there is an on-going effort to improve and expand the technique. This thesis presents methods for extending conventional two-colour single-molecule FRET measurements; by expanding the range and applicability of single-molecule fluorescence methods a greater variety of biological reactions can be studied, in greater detail than previously possible. To circumvent the complexities of multi-colour FRET measurements and extend the range of observable distances I developed and characterised a new single-molecule fluorescence method termed tethered fluorophore motion (TFM). TFM is based on the existing technique of tethered particle motion (TPM) which relies on Brownian motion of a particle, attached to a surface by DNA, to probe the effective length of the DNA tether. TFM takes this concept and applies it at the single-fluorophore level, allowing simultaneous measurement of other fluorescence observables such as FRET and protein induced fluorescence enhancement (PIFE). Having developed TFM I combined it with FRET to study site-specific recombinase proteins at the single-molecule level, in greater detail than possible by either technique alone. Studying the model tyrosine recombinase Cre, I extend and clarify previous ensemble observations regarding the order of DNA strand exchange, as well as uncovering a previously unobserved complex conformation and molecular heterogeneity. Finally, I used TFM-FRET to study the more complex XerCD recombination system and its interaction with the DNA translocase FtsK. I made observations, for the first time, of synaptic complex formation and of recombination at the single-molecule level, and these suggest intriguing and unexpected intermediates in the recombination reaction. I also combine TFM with PIFE to investigate the mechanism of DNA looping by FtsK. The introduction of TFM, and its combination with other fluorescence techniques, allows observation of complex protein-DNA interactions from a variety of perspectives and will help expand the repertoire and applicability of single-molecule biophysical experiments.