Abstract
Cohesin is a four-subunit ATPase in the family of structural maintenance of chromosomes (SMC). Cohesin promotes sister chromatid cohesion, chromosome condensation, DNA repair, and transcription regulation. Cohesin performs these functions as a DNA tether and potentially a DNA-based motor. At least one of its DNA binding activities involves entrapment of DNA within a lumen formed by its subunits. This activity can be reconstituted in vitro by incubating cohesin with DNA, ATP, and cohesin loader. Previously we showed that a mutant form of cohesin (DE-cohesin) possesses the ability to bind and tether DNA in vivo. Using in vitro reconstitution assays, we show that DE-cohesin can form stable complexes with DNA without ATP hydrolysis. We show that wild-type cohesin with ADP aluminum fluoride (cohesinADP/AlFx) can also form stable cohesin-DNA complexes. These results suggest that an intermediate nucleotide state of cohesin, likely cohesinADP-Pi, is capable of initially dissociating one interface between cohesin subunits to allow DNA entry into a cohesin lumen and subsequently interacting with the bound DNA to stabilize DNA entrapment. We also show that cohesinADP/AlFx binding to DNA is enhanced by cohesin loader, suggesting a function for loader other than stimulating the ATPase. Finally, we show that loader remains stably bound to cohesinADP/AlFx after DNA entrapment, potentially revealing a function for loader in tethering the second DNA substrate. These results provide important clues on how SMC complexes like cohesin can function as both DNA tethers and motors.