Single-molecule FRET studies of the mechanism of strand-exchange in site-specific recombination by Tn3 resolvase

The mechanism of strand exchange by Tn3 resolvase was studied using FRET based methods. For this purpose Cy3 dye and a Cy5 dye were attached to modified thymines within Tn3 res site I, the DNA substrate of Tn3 resolvase. The dyes formed a FRET pair and followed the movements of the substrates, resulting in changes of the distance between both dyes and therefore changes in the FRET efficiency of the FRET pair which were monitored using fluorescence spectroscopy. A library of short, double-stranded substrates containing one copy of Tn3 res site I and Cy3 and/or Cy5 dyes attached to at different positions within site I was generated. Fluorescent substrates which do not interfere with the formation of synapses and with the recombination process were selected using gel-based assays. Fluorescent dyes attached at positions about five bases from the centre of site I were found to allow uninhibited recombination. Short double-stranded substrates with dyes attached at the selected positions within site I were studied in ensemble FRET experiments. Recombination of substrates containing a FRET pair with an excess of non-fluorescent substrates resulted in a strong decrease of the FRET efficiency due to the formation of recombinant products containing one dye each. This observation suggested that recombination and strand exchange could indeed be studied using FRET-based methods. The ensemble FRET experiment and FRET based experiments showed that the short substrates are usually recombined in both parallel and anti-parallel orientation. The lack of control over the orientation of the substrates in the synapses motivated the development of U-shaped substrates which consisted of two double-stranded arms, each containing one copy of site I, and a single stranded linker which connected both arms. In gel based assays, the U-shaped substrates were found to prefer the intramolecular recombination of both sites in one defined orientation. A U-shaped substrate containing a Cy5 and a Cy3 dye was studied in a single-molecule FRET experiment. In the presence of an activated Tn3 resolvase mutant, several specific FRET states and transitions between FRET states had been observed. The FRET states and transitions differed in the presence and absence of MgCl2 allowing the identification of a FRET state transition potentially corresponding to the conformational changes during ligation and several transitions corresponding to intermediate steps during strand exchange.