AFM images of open and collapsed states of yeast condensin suggest a scrunching model for DNA loop extrusion

Ryu, Je-Kyung; Katan, Allard J.; Eo, van der Sluis; Wisse, Thomas; R, de Groot; Haering, Christian H.; Dekker, Cees

doi:10.1101/2019.12.13.867358

Cited by 11 publications

(29 citation statements)

References 65 publications

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“…In the case of Figure 3H, to show single-cohesin complex mediated DNA bridging, we mixed 1 nM cohesin complex and 4 ng/µL 3kbp DNA and incubated them for a very short time (10 s). Afterwards, the mixtures were deposited onto mica that was pretreated with polyLysine (0.00001 %) (33). After briefly washing the sample using 3 mL milliQ water, the sample was dried using a nitrogen gun.…”

Section: Afm Imaging and Image Processingmentioning

confidence: 99%

Phase separation induced by cohesin SMC protein complexes

Ryu

Bouchoux

Liu

et al. 2020

Preprint

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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...

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Section: Afm Imaging and Image Processingmentioning

confidence: 99%

Phase separation induced by cohesin SMC protein complexes

Ryu

Bouchoux

Liu

et al. 2020

Preprint

Self Cite

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“…These results are consistent with DNA binding to the head and hinge but not freely floating in the coiled coil as proposed for the embrace model. Similarly, AFM imaging of another SMC complex called condensin showed DNA binding only to its head and hinge domains (Ryu et al, 2019) . Moreover, the presence of two distinct DNA binding sites on cohesin was predicted from mutants that uncoupled cohesion from stable DNA binding, and from the discovery of its loop extruding activity (Davidson et al, 2019;Eng et al, 2014;Kim et al, 2019) .…”

Section: Discussionmentioning

confidence: 99%

“…The ring, rod and butterfly conformations of cohesin have been interrogated both in vitro and in vivo . Cohesin rings have been observed by electron microscopy (EM) and liquid atomic force microscopy (AFM) (Anderson et al, 2002;Hirano et al, 2001;Ryu et al, 2019) . Rings have also been inferred to exist in vivo from experiments showing cohesin's ability to topological entrap DNA (Haering et al, 2008) .…”

Section: Introductionmentioning

confidence: 99%

“…Rings have also been inferred to exist in vivo from experiments showing cohesin's ability to topological entrap DNA (Haering et al, 2008) . Rods have been seen by EM and have been inferred to exist in vivo from crosslinking experiments (Anderson et al, 2002;Soh et al, 2015) ; however, rods were not observed in liquid AFM images (Ryu et al, 2019) . The butterfly structure is extremely rare in negatively stained electron microscopies but prevalent in liquid AFM images (Ryu et al, 2019) .…”

Section: Introductionmentioning

confidence: 99%

“…Rods have been seen by EM and have been inferred to exist in vivo from crosslinking experiments (Anderson et al, 2002;Soh et al, 2015) ; however, rods were not observed in liquid AFM images (Ryu et al, 2019) . The butterfly structure is extremely rare in negatively stained electron microscopies but prevalent in liquid AFM images (Ryu et al, 2019) . The existence of the butterfly structure in vitro is also supported by co-immunoprecipitation of recombinantly expressed hinge dimers and Scc3p.…”

Section: Introductionmentioning

confidence: 99%

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Cohesin in space and time: architecture and oligomerizationin vivo

Xiang¹,

Koshland²

2020

Preprint

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Cohesin helps mediate sister chromatid cohesion, chromosome condensation, DNA repair and transcription regulation. Cohesin can tether two regions of DNA and also extrude DNA loops. We interrogated cohesin architecture, oligomerization and function in vivo through proximity based labeling of cohesin domains. Our results suggest that the hinge and head domain of cohesin both bind DNA, and that cohesin coiled coils bend, bringing the head and hinge together to form a butterfly conformation. Our data also suggest that cohesin efficiently oligomerizes on and off DNA. The levels of oligomers and their distribution on chromosomes are cell cycle regulated. Cohesin oligomerization is blocked by mutations in distinct domains of the Smc3p and Mcd1p subunits of cohesin or in Pds5p, a cohesin regulator. These mutations also block the maintenance of cohesion but not loop extrusion, suggesting that cohesin oligomerization plays a specific role in the maintenance of cohesion.

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Three-dimensional loop extrusion

Bonato

Michieletto

2021

Biophysical Journal

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AFM images of open and collapsed states of yeast condensin suggest a scrunching model for DNA loop extrusion

Cited by 11 publications

References 65 publications

Phase separation induced by cohesin SMC protein complexes

Phase separation induced by cohesin SMC protein complexes

Cohesin in space and time: architecture and oligomerizationin vivo

Three-dimensional loop extrusion

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