Single-Molecule DNA Curtains Reveals the Details of KOPS Targeting, Translocation, and Collision with Protein Roadblocks of DNA Translocase FtsK

Abstract

In E coli cell division, two daughter chromosomes often form a dimer, which impedes a proper segregation of chromosomes. The chromosome dimer can be resolved by XerCD-mediated site specific recombination at dif site. The alignment of two dif sites and activation of XerCD require FtsK translocation, which is directed by a short DNA sequence called KOPS (FtsK Orienting Polar Sequences). KOPS targeting and translocation activities of FtsK were examined using single-molecule DNA curtains, which enables to visualize the protein-DNA interaction in real time. We show that FtsK preferentially locates KOPS through 3D collision within our resolution and non-hydrolysable nucleotides enhance the FtsK loading on KOPS. We also reveal that KOPS determines the orientation of FtsK translocation, but only upon initial binding to KOPS. During the translocation, FtsK abruptly pauses and/or changes its direction independently of KOPS, suggesting that FtsK cannot identify KOPS once it begins to translocate. Next we investigated the collision of FtsK with various protein roadblocks including XerCD through two-color labeling in DNA curtains. Interestingly, the FtsK, which has a hexameric ring structure, changes its direction and bypasses the roadblocks, and can also push them along the DNA. Our single-molecule results help reveal how FtsK might function in the crowded environments expected to be found in physiological settings.