scholarly journals Multiple interactions between Scc1 and Scc2 activate cohesin’s DNA dependent ATPase and replace Pds5 during loading

2017 ◽  
Author(s):  
Naomi J Petela ◽  
Thomas G Gligoris ◽  
Jean Metson ◽  
Byung-Gil Lee ◽  
Menelaos Voulgaris ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Sister Chromatid ◽  
Atp Hydrolysis ◽  
Sister Chromatid Cohesion ◽  
Dependent Atpase ◽  
Heat Repeat ◽  
Repeat Proteins ◽  
Chromatid Cohesion ◽  
Multiple Interactions

SummaryIn addition to sharing with condensin an ability to organize DNA into chromatids, cohesin regulates enhancer-promoter interactions and confers sister chromatid cohesion. Association with chromosomes is regulated by hook-shaped HEAT repeat proteins that Associate With its Kleisin (Scc1) subunit (HAWKs), namely Scc3, Pds5, and Scc2. Unlike Pds5, Scc2 is not a stable cohesin constituent but, as shown here, transiently displaces Pds5 during loading. Scc1 mutations that compromise its interaction with Scc2 adversely affect cohesin’s ATPase activity, loading, and translocation while Scc2 mutations that alter how the ATPase responds to DNA abolish loading despite cohesin’s initial association with loading sites. Lastly, Scc2 mutations that permit loading in the absence of Scc4 increase Scc2’s association with chromosomal cohesin and reduce that of Pds5. We suggest that cohesin switches between two states, one with Pds5 bound to Scc1 that is not able to hydrolyse ATP efficiently but is capable of release from chromosomes and another in which Scc2, transiently replacing Pds5, stimulates the ATP hydrolysis necessary for loading and translocation away from loading sites.

scholarly journals NudCL2 is an Hsp90 cochaperone to regulate sister chromatid cohesion by stabilizing cohesin subunits

2018 ◽  
Author(s):  
Yuehong Yang ◽  
Wei Wang ◽  
Min Li ◽  
Wen Zhang ◽  
Yuliang Huang ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Mammalian Cells ◽  
Ectopic Expression ◽  
Sister Chromatid Cohesion ◽  
Chaperone Function ◽  
Chromatid Cohesion ◽  
Protein Instability ◽  
The Stability

AbstractSister chromatid cohesion plays a key role in ensuring precise chromosome segregation during mitosis, which is mediated by the multisubunit complex cohesin. However, the molecular regulation of cohesin subunits stability remains unclear. Here, we show that NudCL2 (NudC-like protein 2) is essential for the stability of cohesin subunits by regulating Hsp90 ATPase activity in mammalian cells. Depletion of NudCL2 induces mitotic defects and premature sister chromatid separation and destabilizes cohesin subunits that interact with NudCL2. Similar defects are also observed upon inhibition of Hsp90 ATPase activity. Interestingly, ectopic expression of Hsp90 efficiently rescues the protein instability and functional deficiency of cohesin induced by NudCL2 depletion, but not vice versa. Moreover, NudCL2 not only binds to Hsp90, but also significantly modulates Hsp90 ATPase activity and promotes the chaperone function of Hsp90. Taken together, these data suggest that NudCL2 is a previously undescribed Hsp90 cochaperone to modulate sister chromatid cohesion by stabilizing cohesin subunits, providing a hitherto unrecognized mechanism that is crucial for faithful chromosome segregation during mitosis.

scholarly journals The cohesin ring uses its hinge to organize DNA using non-topological as well as topological mechanisms

2017 ◽  
Author(s):  
Madhusudhan Srinivasan ◽  
Johanna C. Scheinost ◽  
Naomi J. Petela ◽  
Thomas G. Gligoris ◽  
Maria Wissler ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Sister Chromatid ◽  
Atp Hydrolysis ◽  
Sister Chromatid Cohesion ◽  
Perfect Correlation ◽  
Chromatid Cohesion ◽  
Lysine Residues ◽  
Positively Charged ◽  
Hinge Domain

SummaryAs predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is a perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion in a large variety of mutants. In most cells where cohesin loads onto chromosomes but fails to form cohesion, loading is accompanied by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen has been neutralized not only loads onto and translocates along chromatin but also organizes it into chromatid-like threads, despite largely failing to entrap DNAs inside its ring. Thus, cohesin engages chromatin in a non-topological as well as a topological manner. Our finding that hinge mutations, but not fusions between Smc and kleisin subunits, abolish entrapment suggests that DNAs may enter cohesin rings through hinge opening. Lastly, mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin’s initial recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin’s hinge driven by cycles of ATP hydrolysis.

scholarly journals ATP dependent DNA transport within cohesin: Scc2 clamps DNA on top of engaged heads while Scc3 promotes entrapment within the SMC-kleisin ring

2020 ◽  
Author(s):  
James E Collier ◽  
Byung-Gil Lee ◽  
Maurici B Roig ◽  
Stanislav Yatskevich ◽  
Naomi J Petela ◽  
...  
Keyword(s):  
Sister Chromatid ◽  
Atp Hydrolysis ◽  
Sister Chromatid Cohesion ◽  
S Phase ◽  
Coiled Coils ◽  
Rigorous Proof ◽  
Dna Loops ◽  
Dna Transport ◽  
Chromatid Cohesion

SUMMARYIn addition to extruding DNA loops, cohesin entraps within its SMC-kleisin ring (S-K) individual DNAs during G1 and sister DNAs during S-phase. All three activities require related hook-shaped proteins called Scc2 and Scc3. Using thiol-specific crosslinking we provide rigorous proof of entrapment activity in vitro. Scc2 alone promotes entrapment of DNAs in the E-S and E-K compartments, between ATP-bound engaged heads and the SMC hinge and associated kleisin, respectively. This does not require ATP hydrolysis nor is it accompanied by entrapment within S-K rings, which is a slower process requiring Scc3. Cryo-EM reveals that DNAs transported into E-S/E-K compartments are “clamped” in a sub-compartment created by Scc2’s association with engaged heads whose coiled coils are folded around their elbow. We suggest that clamping may be a recurrent feature of cohesin complexes active in loop extrusion and that this conformation precedes the S-K entrapment required for sister chromatid cohesion.

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

2020 ◽  
Author(s):  
Torahiko L. Higashi ◽  
Patrik Eickhoff ◽  
Joana S. Simoes ◽  
Julia Locke ◽  
Andrea Nans ◽  
...  
Keyword(s):  
Sister Chromatid ◽  
Molecular Basis ◽  
Atp Hydrolysis ◽  
Initial Step ◽  
Sister Chromatid Cohesion ◽  
Chromosome Organization ◽  
Atp Binding ◽  
Structural Framework ◽  
Gate Opening ◽  
Chromatid Cohesion

SUMMARYDespite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here, we combine biophysical approaches and cryo-EM to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design biochemical experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against a shut ATPase gate. ATP hydrolysis leads to ATPase gate opening to complete DNA entry. Whether DNA loading is successful, or rather results in loop extrusion, might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.

scholarly journals Transport of DNA within cohesin involves clamping on top of engaged heads by Scc2 and entrapment within the ring by Scc3

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
James E Collier ◽  
Byung-Gil Lee ◽  
Maurici Brunet Roig ◽  
Stanislav Yatskevich ◽  
Naomi J Petela ◽  
...  
Keyword(s):  
Sister Chromatid ◽  
Atp Hydrolysis ◽  
Sister Chromatid Cohesion ◽  
S Phase ◽  
Coiled Coils ◽  
Rigorous Proof ◽  
Dna Loops ◽  
Chromatid Cohesion

In addition to extruding DNA loops, cohesin entraps within its SMC-kleisin ring (S-K) individual DNAs during G1 and sister DNAs during S-phase. All three activities require related hook-shaped proteins called Scc2 and Scc3. Using thiol-specific crosslinking we provide rigorous proof of entrapment activity in vitro. Scc2 alone promotes entrapment of DNAs in the E-S and E-K compartments, between ATP-bound engaged heads and the SMC hinge and associated kleisin, respectively. This does not require ATP hydrolysis nor is it accompanied by entrapment within S-K rings, which is a slower process requiring Scc3. Cryo-EM reveals that DNAs transported into E-S/E-K compartments are ‘clamped’ in a sub-compartment created by Scc2’s association with engaged heads whose coiled coils are folded around their elbow. We suggest that clamping may be a recurrent feature of cohesin complexes active in loop extrusion and that this conformation precedes the S-K entrapment required for sister chromatid cohesion.

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