Biology subjects

Voulgaris, M.

Publications and source records attributed to Voulgaris, M..

6 recordsLinked to original sources

bioRxiv2022

Conformational dynamics of cohesin/Scc2 loading complex are regulated by Smc3 acetylation and ATP binding

The ring-shaped cohesin complex is the key player in sister chromatid cohesion, DNA repair, and gene transcription. The loading of cohesin to chromosomes requires the loader Scc2 and is regulated by ATP. This process is also hindered by Smc3 acetylation. However, the molecular mechanism underlying this inhibition remains mysterious. Here we identify a novel configuration of Scc2 with pre-engaged cohesin and reveal dynamic conformations of the cohesin/Scc2 complex in the loading reaction. We demonstrate that Smc3 acetylation blocks the association of Scc2 with pre-engaged cohesin by impairing the interaction of Scc2 with Smc3s head. Lastly, we show that ATP binding induces the cohesin/Scc2 complex to clamp DNA by promoting the interaction between Scc2 and Smc3 coiled coil. Our results illuminate a dynamic reconfiguration of the cohesin/Scc2 complex during loading and indicate how Smc3 acetylation and ATP regulate this process.

biochemistry↗

bioRxiv2022

Formation of the clamped state by Scc2 and DNA overcomes the constraints imposed by zipping-up of the SMC coiled coils on cohesin's ATPase

In addition to mediating sister chromatid cohesion, cohesin, by virtue of Loop Extrusion (LE), organises the spatial arrangement of interphase DNA. The latter activity relies on DNA and Scc2 dependent ATP hydrolysis by cohesin. How the impetus from the ATPase cycle translates into reeling of DNA loops into the SMC kleisin rings is still unclear. The SMC coiled coils show several striking structural features like folding and zipping-up, if and how these structural states affect cohesins activity is still unclear. We show here that cohesins loop extruding motor contains an internal constraint that regulates its ATPase activity, zipping-up of the coiled coils impedes ATP hydrolysis by cohesin. We show that integrity of a region where the coiled coils emerge for the SMC hinge domains, SMC wrist, is critical for the zipping up of the coiled coils and the resulting inhibition of cohesins ATPase. Clamping of DNA by Scc2 onto the engaged SMC heads in the presence of ATP leads to unzipping of the coiled coils and permits ATP hydrolysis. Strikingly, irreversible folding of the coiled coils at the elbow region does not lead to any measurable change to the ATPase activity suggesting that recurrent cycles of folding and unfolding of the coiled coils is not necessary for driving continuous ATP hydrolysis by cohesin.

biochemistry↗

bioRxiv2020

Scc2 and Scc3 promote distinct modes of topological association between cohesin and DNA

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 Scc2s 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.

biochemistry↗

bioRxiv2019

Scc2 counteracts a Wapl-independent mechanism that releases cohesin from chromosomes during G1 but is unnecessary during S phase for establishing cohesion

Cohesins association with chromosomes is determined by loading dependent on the Scc2/4 complex and release due to Wapl. We show here that Scc2/4 is not merely a loading complex and that it actively maintains cohesin on chromosomes during G1. It does so by blocking a Wapl-independent release reaction that requires opening the cohesin ring at its Smc3/Scc1 interface as well as the D loop of Smc1s ATPase. The Wapl-independent release mechanism is switched off as cells activate Cdk1 and enter G2/M and cannot be turned back on without cohesins dissociation from chromosomes. The latter phenomenon enabled us to show that cohesin that has already captured DNA in a Scc2-dependent manner before replication no longer requires Scc2 to capture sister DNAs during S phase.

molecular biology↗

bioRxiv2017

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

In 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 cohesins ATPase activity, loading, and translocation while Scc2 mutations that alter how the ATPase responds to DNA abolish loading despite cohesins initial association with loading sites. Lastly, Scc2 mutations that permit loading in the absence of Scc4 increase Scc2s 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.

cell biology↗

bioRxiv2017

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

As 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 cohesins initial recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesins hinge driven by cycles of ATP hydrolysis.

molecular biology↗