Assembly, translocation, and activation of XerCD-dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion.

The FtsK dsDNA translocase functions in bacterial chromosome unlinking by activating XerCD-dif recombination in the replication terminus region. To analyze FtsK assembly and translocation, and the subsequent activation of XerCD-dif recombination, we extended the tethered fluorophore motion technique, using two spectrally distinct fluorophores to monitor two effective lengths along the same tethered DNA molecule. We observed that FtsK assembled stepwise on DNA into a single hexamer, and began translocation rapidly (∼ 0.

Tethered fluorophore motion: studying large DNA conformational changes by single-fluorophore imaging.

We have previously introduced tethered fluorophore motion (TFM), a single-molecule fluorescence technique that monitors the effective length of a biopolymer such as DNA. TFM uses the same principles as tethered particle motion (TPM) but employs a single fluorophore in place of the bead, allowing TFM to be combined with existing fluorescence techniques on a standard fluorescence microscope. TFM has been previously been used to reveal the mechanism of two site-specific recombinase systems, Cre-loxP and XerCD-dif.

Conformational transitions during FtsK translocase activation of individual XerCD-dif recombination complexes.

Three single-molecule techniques have been used simultaneously and in tandem to track the formation in vitro of single XerCD-dif recombination complexes. We observed the arrival of the FtsK translocase at individual preformed synaptic complexes and demonstrated the conformational change that occurs during their activation. We then followed the reaction intermediate transitions as Holliday junctions formed through catalysis by XerD, isomerized, and were converted by XerC to reaction products, which then dissociated.