FtsK from Escherichia coli is a fast and sequence-directed DNA translocase with roles in chromosome dimer resolution, segregation, and decatenation. From the movement of single FtsK particles on defined DNA substrates and an analysis of skewed DNA sequences in bacteria, we identify GNGNAGGG, its complement, or both as a sequence motif that controls translocation directionality. GNGNAGGG is skewed so that it is predominantly on the leading strand of chromosomal replication. Translocation across this octamer from the 3′ side of the G-rich strand causes FtsK to pause, turn around, and translocate in the opposite direction. Only 39 ± 4% of the encounters between FtsK and the octamer result in a turnaround, congruent with our optimum turnaround probability prediction of 30%. The probability that the observed skew of GNGNAGGG within 1 megabase of dif occurred by chance in E. coli is 1.7 × 10-57, and similarly dramatic skews are found in the five other bacterial genomes we examined. The fact that FtsK acts only in the terminus region and the octamer skew extends from origin to terminus implies that this skew is also important in other basic cellular processes that are common among bacteria. Finally, we show that the FtsK translocase is a powerful motor that is able to displace a triplex-forming oligo from a DNA substrate. © 2005 by The National Academy of Sciences of the USA.