Intermediate step of cohesin’s ATPase cycle allows cohesin to entrap DNA

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 (cohesin) can also form stable cohesin-DNA complexes. These results suggest that an intermediate nucleotide state of cohesin, likely cohesin, 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 cohesin 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 cohesin 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.

The topology of DNA entrapment by cohesin rings

“…If DNAs were in fact entrapped within E-S compartments, then cleavage of their coiled coils by separase should suppress the delayed disjunction, which is precisely what was found. Entrapment within the S or K compartments of complexes whose heads are engaged is likewise consistent with the claim that cohesion can be established by viable Smc1D1164E mutations that are supposedly incapable of hydrolysing ATP (Camdere et al, 2015;Camdere et al, 2018;Elbatsh et al, 2016).…”

“…Because formation of J-K compartments is likely to require ATP hydrolysis, the notion of cohesion being mediated by entrapment of sister DNAs within J-K compartments is hard to reconcile with the proposal that ATP hydrolysis is unnecessary for building sister chromatid cohesion. The argument that hydrolysis is not required is based on the behaviour of Smc1D1164E mutants that can load onto chromosomes and build cohesion despite being defective in ATP hydrolysis (Camdere et al, 2018).…”

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring

“…In addition to EQ-cohesin, the wild type cohesin complex reaches topological DNA binding in the presence of ADP and phosphate analogs that mimic the ATP ground state (Çamdere et al, 2018;Minamino et al, 2018;Murayama and Uhlmann, 2015). To confirm that head engagement is similarly reached under these conditions, we measured FRET of wild type cohesin with the loader, DNA, ADP and gphosphate analogs ( Figure 1C).…”

“…However, in vitro studies suggest that ATP binding but not hydrolysis is required for topological cohesin binding to DNA, which is hard to reconcile with the above model (Çamdere et al, 2018;Minamino et al, 2018). Furthermore, fusing budding yeast Smc3 to the kleisin N-terminus in one polypeptide chain still allowed chromosome binding of cohesin (Gruber et al, 2006), which was interpreted to rule out DNA passage through the kleisin N-gate.…”

Communication between distinct subunit interfaces of the cohesin complex promotes its topological entrapment of DNA

“…The existence of different conformations of cohesin (rings, rods and folded 401 rings) have been demonstrated by microscopic techniques like EM and AFM(Onn et al, 402 2008). Recent work suggests that the cohesin Smc ATPase activity alters head-head 403 interactions and promotes a conformation change that enables DNA binding and 404 cohesion(Çamdere et al, 2015;Huber et al, 2016;Çamdere et al, 2018). Since 405Mcd1p binds both to the base of the Smc3p coil and the head, it could transduce 406ATPase dependent changes in the head to the coil, which could cause interconversion 407 of rings to rods or rings to folded rings, thereby affecting the opening or closing of a 408 distal interface.…”