The Smc5/6 complex is a DNA loop-extruding motor.

Structural maintenance of chromosomes (SMC) protein complexes are essential for the spatial organization of chromosomes 1 . Whereas cohesin and condensin organize chromosomes by extrusion of DNA loops, the molecular functions of the third eukaryotic SMC complex, Smc5/6, remain largely unknown 2 . Using single-molecule imaging, we show that Smc5/6 forms DNA loops by extrusion. Upon ATP hydrolysis, Smc5/6 reels DNA symmetrically into loops at a force-dependent rate of one kilobase pair per second. Smc5/6 extrudes loops in the form of dimers, whereas monomeric Smc5/6 unidirectionally translocates along DNA. We also find that the subunits Nse5 and Nse6 (Nse5/6) act as negative regulators of loop extrusion. Nse5/6 inhibits loop-extrusion initiation by hindering Smc5/6 dimerization but has no influence on ongoing loop extrusion. Our findings reveal functions of Smc5/6 at the molecular level and establish DNA loop extrusion as a conserved mechanism among eukaryotic SMC complexes.