#separase

Cell Division

Which enzyme is responsible for breaking down cohesin to allow sister chromatid separation?

Sister chromatid cohesion mediated by cohesin ring complex with SMC1 and SMC3 coiled-coil proteins forming V-shaped dimer bridged by kleisin Rad21 that closes tripartite ring embracing both sister DNAs from replication until anaphase. Opening requires proteolytic cleavage rather than dissociation. Separase, large 230 kDa cysteine endopeptidase belonging to CD clan with catalytic histidine-cysteine dyad analogous to caspases, serves as cleaving enzyme. Before anaphase, separase kept inactive through two layers: binding of securin pseudosubstrate occupying active site, and cyclin B-CDK1 mediated phosphorylation at serine 1126 causing cytoplasmic retention and auto-inhibition. At anaphase onset, APC/C-Cdc20 ubiquitinates securin with K11 chains for proteasomal destruction, cyclin B degradation reduces CDK1 activity allowing PP2A-dependent dephosphorylation, releasing separase to chromosomes where it cleaves Rad21 at conserved EXXR sequences separating N-terminal and C-terminal fragments unable to hold ring closed. Non-cleavable Rad21 mutant blocks sister separation causing metaphase arrest, proving cleavage essential. This circuitry is highly conserved across eukaryotes, integrating growth factor signals, DNA damage surveillance, and developmental cues, and its disruption frequently underlies oncogenesis, providing targets for checkpoint inhibitors and cancer therapeutics.

Ref: Uhlmann et al., Nature 2000, Mechanism of Separase. Nasmyth, Science 2002, Cohesin Cleavage.

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Cell Cycle Regulation

What is the function of Securin?

Preservation of sister chromatid cohesion until all chromosomes correctly bioriented relies on anaphase inhibitor securin, known as Pds1 in yeast and PTTG1 pituitary tumor transforming gene in mammals. Synthesized during S and G2, securin binds stoichiometric quantities of separase protease through insertion of reactive loop into separase active site, acting as pseudosubstrate and competitive inhibitor. Beyond inhibition, securin serves as chaperone promoting proper folding of separase, its accumulation in nucleus and stability. As long as securin remains associated, separase cannot cleave cohesin subunit Scc1, preventing premature sister separation. At anaphase onset, checkpoint satisfied APC/C-Cdc20 ubiquitin ligase recognizes KEN and D-box degrons on securin, assembling K11/K48 polyubiquitin chains targeting it for rapid proteolysis with half-life dropping to minutes. Degradation frees catalytic histidine-cysteine dyad of separase which then cleaves Rad21 at conserved glutamate-arginine motifs, opening cohesin ring allowing disjunction. Excess securin overexpression observed in pituitary tumors causes metaphase arrest and aneuploidy, while securin deletion still viable due to compensatory CDK1 phosphorylation inhibition of separase. Additional feedback loops involving polo-like kinases, phosphatases and SCF-mediated degradation reinforce irreversibility and protect against premature progression that would compromise genome integrity and viability.

Ref: Zou et al., Securin as Separase Inhibitor and Chaperone, Cell 1999; Uhlmann et al., Mechanism of Securin-Separase Regulation, Nature 2000.

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Cell Cycle Regulation

The metaphase-to-anaphase transition is triggered by:

The metaphase to anaphase transition is irreversible decision point governed by ubiquitin dependent proteolysis. When every chromosome achieves amphitelic kinetochore-microtubule attachment generating inter-kinetochore tension, spindle assembly checkpoint signaling through MCC production ceases. Dynein-mediated stripping of Mad1-Mad2 from kinetochores and p31comet-TRIP13 catalyzed MCC disassembly liberate coactivator Cdc20, enabling APC/C-Cdc20 ligase activation. Active APC/C polyubiquitinates securin via destruction box motif and cyclin B via D-box for rapid degradation by 26S proteasome. Securin degradation frees separase cysteine protease previously held as pseudosubstrate, while cyclin B destruction reduces CDK1 activity permitting dephosphorylation of mitotic substrates by PP1 and PP2A-B55. Free separase cleaves kleisin subunit Scc1 or Rad21 of cohesin tetramer encircling sister chromatids at centromeres rescued from earlier Wapl-mediated arm removal by Shugoshin-PP2A protection. Opening of cohesin ring dissolves linkage, allowing poleward microtubule pulling forces to separate sisters. Chromatid movement manifests as anaphase A toward poles and anaphase B spindle elongation driven by Eg5 kinesin and cortical dynein. Additional feedback loops involving polo-like kinases, phosphatases and SCF-mediated degradation reinforce irreversibility and protect against premature progression that would compromise genome integrity and viability.

Ref: Peters, Anaphase Promoting Complex Orchestrating Metaphase-Anaphase Transition, Nat Rev Mol Cell Biol 2006; Alberts et al., Chapter 18, Anaphase.

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Cell Cycle Regulation

Separase is activated when:

Separase activation integrates checkpoint satisfaction with physical separation of chromatids. Newly synthesized separase folds with assistance of securin chaperone, which inserts its own reactive site loop into catalytic pocket, blocking proteolysis and ensuring proper localization to nucleus. Parallel inhibition involves CDK1-Cyclin B1 binding phosphorylating separase at Ser1126 creating docking site for cyclin B. At metaphase, with all kinetochores amphitelically attached, spindle assembly checkpoint silencing allows APC/C-Cdc20 E3 ligase to become active. APC/C-Cdc20 recognizes KEN-box and D-box degron sequences on securin, catalyzing assembly of K11 and K48 linked polyubiquitin chains. Polyubiquitinated securin targeted to 26S proteasome with half-life decreasing from hours to under ten minutes. Simultaneously cyclin B degradation removes CDK1-mediated inhibition. Removal of securin exposes catalytic dyad histidine and cysteine residues allowing cleavage of cohesin kleisin Scc1/Rad21 at EXXR motifs. Some separase autocleavage yields C-terminal fragment with retained activity. Securin therefore acts both as inhibitor and as folding assistant, while APC/C functions as trigger linking ubiquitination to proteolysis-driven anaphase initiation. Additional feedback loops involving polo-like kinases, phosphatases and SCF-mediated degradation reinforce irreversibility and protect against premature progression that would compromise genome integrity and viability.

Ref: Peters, Control of Securin Degradation by APC/C-Cdc20, Nat Rev Mol Cell Biol; Hornig et al., Securin-Separase Complex Structure.

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