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

ABSTRACT: 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 behind 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 Smc3’s 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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE217833 | GEO | 2023/08/16

REPOSITORIES: GEO

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Releasing activity disengages cohesinâs Smc3/Scc1 interface in a process blocked by acetylation

Project description:Sister chromatid cohesion conferred by entrapment of sister DNAs within a tripartite ring formed between cohesinâs Scc1, Smc1, and Smc3 subunits is generated during S and eventually destroyed at anaphase through cleavage of Scc1 by separase. Throughout the cell cycle, cohesinâs association with chromosomes is controlled by opposing activities: loading by the Scc2/4 complex and release by a separase independent releasing activity. Co-entrapment of sister DNAs during replication is accompanied by acetylation of Smc3 by Eco1, which blocks releasing activity and ensures that sisters remain stably connected. Because fusion of Smc3 to Scc1 prevents release and bypasses the requirement for Eco1, we suggested that release is mediated by disengagement of the Smc3/Scc1 interface. We now show that all mutations capable of bypassing Eco1, be they in cohesinâs Smc1, Smc3, Scc1,Wapl, Pds5, or Scc3 subunits, greatly reduce dissociation of N-terminal cleavage fragments of Scc1 (NScc1) from Smc3. We show that this process involves interaction between Smc ATPase heads and is inhibited by Smc3 acetylation Effect of mutations QQ and EQ in Smc3 on cohesin loading onto chromosomes

2016-02-18 | E-GEOD-76890 | biostudies-arrayexpress

Releasing activity disengages cohesin’s Smc3/Scc1 interface in a process blocked by acetylation

Project description:Sister chromatid cohesion conferred by entrapment of sister DNAs within a tripartite ring formed between cohesin’s Scc1, Smc1, and Smc3 subunits is generated during S and eventually destroyed at anaphase through cleavage of Scc1 by separase. Throughout the cell cycle, cohesin’s association with chromosomes is controlled by opposing activities: loading by the Scc2/4 complex and release by a separase independent releasing activity. Co-entrapment of sister DNAs during replication is accompanied by acetylation of Smc3 by Eco1, which blocks releasing activity and ensures that sisters remain stably connected. Because fusion of Smc3 to Scc1 prevents release and bypasses the requirement for Eco1, we suggested that release is mediated by disengagement of the Smc3/Scc1 interface. We now show that all mutations capable of bypassing Eco1, be they in cohesin’s Smc1, Smc3, Scc1,Wapl, Pds5, or Scc3 subunits, greatly reduce dissociation of N-terminal cleavage fragments of Scc1 (NScc1) from Smc3. We show that this process involves interaction between Smc ATPase heads and is inhibited by Smc3 acetylation

2016-02-18 | GSE76890 | GEO

The Smc3 deacetylase Hos1 closes the cohesin acetylation cycle

Project description:Cohesion between sister chromatids is mediated by the chromosomal cohesin complex. In budding yeast, cohesin is loaded onto chromosomes during the G1 phase of the cell cycle. During S-phase, the replication fork-associated acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to promote establishment of sister chromatid cohesion. At the time of anaphase, Smc3 loses its acetylation again, but the Smc3 deacetylase and possible importance of Smc3 deacetylation are unknown. Here, we show that the class I histone deacetylase family member Hos1 is responsible for Smc3 deacetylation. Cohesin is protected from deacetylation while bound to chromosomes, but is deacetylated as soon as it dissociates from chromosomes following separase cleavage at anaphase onset. Non-acetylated Smc3 is required as a substrate for cohesion establishment in the following cell cycle. Our results complete the description of the Smc3 acetylation cycle and provide unexpected insight into the importance of de novo Smc3 acetylation for cohesion establishment.

2010-06-10 | GSE22257 | GEO

Smc3 acetylation, Pds5 and Scc2 control the translocase activity that establishes cohesin dependent chromatin loops

Project description:Cohesin is a DNA translocase instrumental in the folding of the genome into chromatin loops, with functional consequences on DNA-related processes. Chromatin loop length and organization likely depend on cohesin processivity, translocation rate, and stability on DNA. Here we investigate and provide a comprehensive overview of the roles of various cohesin regulators in tuning chromatin loop expansion. We demonstrate that Scc2, which stimulates cohesin ATPase activity, is also essential for cohesin translocation, driving loop expansion in vivo. Smc3 acetylation during S-phase counteracts this activity through the stabilization of Pds5, which finely tunes the size and stability of loops in G2.

2022-02-23 | GSE186987 | GEO

Scc2 Is a Potent Activator of Cohesin's ATPase that Promotes Loading by Binding Scc1 without Pds5

Project description: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 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.

2018-07-17 | GSE106182 | GEO

Structure of the Cohesin Loader Scc2-Scc4 and its Binding to a Folded Cohesin

Project description:The functions of cohesin are central to genome integrity, chromosome organization, and transcription regulation through its prevention of premature sister-chromatid separation and the formation of DNA loops. The loading of cohesin onto chromatin depends on the Scc2-Scc4 complex, however, little is known about how it stimulates the cohesin loading activity. Here we determine the large “hook” structure of Scc2 responsible for catalyzing cohesin loading. We identify key Scc2 surfaces that are crucial for DNA binding and for cohesin loading in vivo. Using previously determined structures and modeling, we derive a pseudo-atomic structure of the full-length Scc2-Scc4 complex. Finally, our crosslinking and electron microscopy analyses reveal that Scc2-Scc4 utilizes its modular structure to make multiple contacts with a folded cohesin at an interface created by the cohesin head-hinge interaction.

2017-01-11 | PXD004692 | Pride

Folding of cohesin's coiled coil is important for Scc2/4-induced association with chromosomes

Project description:Cohesin’s association with and translocation along chromosomal DNAs depend on an ATP hydrolysis cycle driving the association and subsequent release of DNA. This involves DNA being ‘clamped’ by Scc2 and ATP-dependent engagement of cohesin’s Smc1 and Smc3 head domains. Scc2’s replacement by Pds5 abrogates cohesin’s ATPase and has an important role in halting DNA loop extrusion. The ATPase domains of all SMC proteins are separated from their hinge dimerisation domains by 50 nm long coiled coils, which have been observed to zip up along their entire length and fold around an elbow, thereby greatly shortening the distance between hinges and ATPase heads. Whether folding exists in vivo or has any physiological importance is not known. We present here a cryo-EM structure of the apo form of cohesin that reveals the structure of folded and zipped up coils in unprecedented detail and shows that Scc2 can associate with Smc1’s ATPase head even when it is fully disengaged from that of Smc3. Using cysteine-specific cross-linking, we show that cohesin’s coiled coils are frequently folded in vivo, including when cohesin holds sister chromatids together. Moreover, we describe a mutation (SMC1D588Y) within Smc1’s hinge that alters how Scc2 and Pds5 interact with Smc1’s hinge and that enables Scc2 to support loading in the absence of its normal partner Scc4. The mutant phenotype of loading without Scc4 is only explicable if loading depends on an association between Scc2/4 and cohesin’s hinge, which in turn requires coiled coil folding.

2021-07-21 | GSE167318 | GEO

Smc3 deacetylation by Hos1 facilitates efficient dissolution of sister chromatid cohesion during anaphase

Project description:Sister chromatid cohesion relies on cohesins, a group of proteins that forms a ring-shaped complex embracing sister chromatids. Cohesion is established during S phase and is removed when cohesin Scc1 is cleaved by the protease separase at anaphase onset. During this process, the cohesin subunit Smc3 undergoes a cycle of acetylation: Smc3 acetylation by Eco1 in S phase stabilizes cohesin association with chromosomes, and its deacetylation by Hos1 in anaphase allows re-use of Smc3 in the next cell cycle. Here we find that Smc3 deacetylation by Hos1 has a more immediate effect in early anaphase of budding yeast. Without Hos1, sister chromatid separation and segregation are significantly delayed. Smc3 deacetylation facilitates removal of cohesins from chromosomes, without changing the Scc1 cleavage efficiency, thus promoting dissolution of cohesion. This action is probably due to disengagement of Smc1–3 heads, prompted by de-repression of their ATPase activity. We suggest Scc1 cleavage per se is insufficient for efficient dissolution of cohesion in early anaphase, but subsequent Smc3 deacetylation, which is triggered by Scc1 cleavage, is also required.

2017-11-02 | GSE96841 | GEO

Scc2 counteracts a Wapl-independent mechanism that releases cohesin from chromosomes during G1

Project description:Cohesin’s association with chromosomes is determined by loading dependent on the Scc2/4 complex and release due to Wapl. We show here that Scc2 also 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 Smc1’s ATPase. The Wapl-independent release mechanism is switched off as cells activate Cdk1 and enter G2/M and cannot be turned back on without cohesin’s dissociation from chromosomes. The latter phenomenon enabled us to show that in the absence of release mechanisms, cohesin rings that have already captured DNA in a Scc2-dependent manner before replication no longer require Scc2 to capture sister DNAs during S phase.

2019-06-06 | GSE132221 | GEO

Genome-wide localization of Smc1, Smc3 and CTCF in mouse B cells.

Project description:Immunoglobulin class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation induced cytidine deaminase (AID) to switch regions and by the subsequent generation and resolution of dsDNA breaks (DSBs). During, CSR the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers and switch regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. Here we show that during CSR, AID associates with subunits of cohesin, a complex previously implicated in DNA repair and in the formation of DNA loops between enhancers and promoters. By ChIP-Seq experiments, we find that Cohesin is dynamically recruited to the IgH locus during CSR and that knockdown of Cohesin or its regulatory subunits results in impaired CSR and abnormal DSB resolution. Our results are consistent with a model in which Cohesin controls the formation of long-range DNA loops at the IgH locus and the resolution of DSBs generated during CSR. Smc1, Smc3 and CTCF were immunoprecipitated from resting or in vitro activated B cells.

2013-10-11 | E-GEOD-43594 | biostudies-arrayexpress

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