Structural maintenance of chromosome (SMC) protein complexes are able to extrude DNA loops. While loop extrusion constitutes a fundamental building block of chromosomes, other factors may be equally important. Here, we show that yeast cohesin exhibits pronounced clustering on DNA, with all the hallmarks of biomolecular condensation. DNA-cohesin clusters exhibit liquid-like behavior, showing fusion of clusters, rapid fluorescence recovery after photobleaching and exchange of cohesin with the environment. Strikingly, the in vitro clustering is DNA length dependent, as cohesin forms clusters only on DNA exceeding 3 kilo–base pairs. We discuss how bridging-induced phase separation, a previously unobserved type of biological condensation, can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in yeast cells in vivo, a fraction of cohesin associates with chromatin in a manner consistent with bridging-induced phase separation. Biomolecular condensation by SMC proteins constitutes a new basic principle by which SMC complexes direct genome organization.

Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Several replicationfork-associated ''cohesion establishment factors,'' including the multifunctional Ctf18-RFC complex, aid this process in as yet unknown ways. Here, we show that Ctf18-RFC's role in sister chromatid cohesion correlates with PCNA loading but is separable from its role in the replication checkpoint. Ctf18-RFC loads PCNA with a slight preference for the leading strand, which is dispensable for DNA replication. Conversely, the canonical Rfc1-RFC complex preferentially loads PCNA onto the lagging strand, which is crucial for DNA replication but dispensable for sister chromatid cohesion. The downstream effector of Ctf18-RFC is cohesin acetylation, which we place toward a late step during replication maturation. Our results suggest that Ctf18-RFC enriches and balances PCNA levels at the replication fork, beyond the needs of DNA replication, to promote establishment of sister chromatid cohesion and possibly other post-replicative processes.

No abstract

Cohesin is a key protein complex that organizes the spatial structure of chromosomes during interphase. Here, we show that yeast cohesin shows pronounced clustering on DNA in an ATPindependent manner, exhibiting all the hallmarks of phase separation. In vitro visualization of cohesin on DNA shows DNA-cohesin clusters that exhibit liquid-like behavior. This includes mutual fusion and reversible dissociation upon depleting the cohesin concentration, increasing the ionic strength, or adding 1,6-hexanediol, conditions that disrupt weak interactions. We discuss how bridging-induced phase separation can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in vivo, a fraction of cohesin associates with chromatin in yeast cells in a manner consistent with phase separation. Our findings establish that SMC proteins can exhibit phase separation, which has potential to clarify previously unexplained aspects of in vivo SMC behavior and constitute an additional principle by which SMC complexes impact genome organization. One sentence summary:Yeast cohesin complex is observed to phase separate with DNA into liquid droplets, which it accomplishes by ATP-independent DNA bridging.Members of the structural maintenance of chromosome (SMC) protein family such as condensin, cohesin, and the Smc5/6 complex are key proteins for the spatial and temporal organization of chromosomes (1)(2)(3)(4). Recent in vitro experiments visualized real-time DNA loop extrusion mediated by condensin and cohesin (5-7). While loop extrusion by SMC proteins constitutes a fundamental building block in the organization of chromosomes, other factors may also contribute. In the last decade, it has become abundantly clear that phase separation plays a role in many processes in biological cells (8), including chromosome organization (9, 10). Thus far, SMC proteins have not been implied in phase separation. While ATP-independent clustering of DNA and SMC proteins has been reported (11)(12)(13)(14), such observations were attributed to imperfect protein purification or non-physiological buffer conditions. For example, Davidson et al. (7) reported in vitro DNA loop extrusion by the human cohesin complex when the complex concentration was limited to very low values (<0.8 nM, i.e., much lower that physiological concentrations of ~333 nM (15,16), and mentioned that the cohesin complexes were prone to aggregation at higher concentrations. Such findings raise the question whether such aggregate formation may be intrinsic and may have a physiological meaning.Here we report that interactions between the yeast cohesin SMC complex and DNA lead to pronounced phase separation. This clustering behavior is ATP independent but depends on DNA length. We find that single cohesin complexes are able to bridge distant points along DNA that act as nucleation points for recruiting further cohesin complexes -a behavior indicating bridging-induced phase separation (BIPS) (17,18), also known as polymer-polymer phase separation (PPPS) (19), a type of phase co...

No abstract