Development of single-molecule methods for the simultaneous detection of protein-DNA interactions and their application to the study of the mechanism of DNA translocation by SpoIIIE

SpoIIIE/FtsK/Tra are a family of ring-shaped, membrane-anchored, ATP-fueled, directional motors required to segregate DNA across membranes during sporulation, cell division and conjugation. In particular, SpoIIIE is responsible for packaging the chromosome inside the prespore during the process of sporulation in Bacillus subtilis. This transport is directional and requires SpoIIIE to recognizes highly-skewed octameric sequences (SpoIIIE Recognition Sequences, SRS) distributed along the whole
chromosome.  A clear understanding of the molecular mechanism by which SpoIIIE-SRS interactions lead to directional DNA transport is lacking. We used single molecule methods like Total Internal Reflection Fluorescence Microscopy and transverse magnetic tweezer, that involved simultaneous visualization of DNA and SpoIIIE and enable detection of the localization of SpoIIIE on DNA.  Imaging DNA, immobilized on passivated surface with intercalating dyes (like YOYO-1) offer challenges; in this talk, I will propose sytox as a better suited dye for DNA imaging and discuss its binding mechanism and fluorescence properties. Finally I will discuss the mechanism with which SpoIIIE might translocate DNA directionally.