Substrate recognition by human separase

Area of Science:

• Molecular Biology
• Cell Biology
• Structural Biology

Background:

• The cohesin complex is essential for sister chromatid cohesion during mitosis.
• Separase-mediated cleavage of the SCC1/RAD21 subunit triggers sister chromatid separation at anaphase onset.
• The precise mechanisms of SCC1/RAD21 recognition and cleavage by separase remain incompletely understood.

Purpose of the Study:

• To elucidate the structural and functional mechanisms regulating separase-mediated cleavage of SCC1/RAD21.
• To identify substrate recognition sites and phosphorylation-dependent regulation of separase activity.
• To understand how the cohesin complex is targeted by separase for cleavage.

Main Methods:

• X-ray crystallography to determine structures of human separase (apo- and substrate-bound forms).
• Biochemical analyses to study substrate interactions and cleavage kinetics.
• Cross-linking mass spectrometry (XL-MS) and cryo-electron microscopy (cryo-EM) to investigate cohesin targeting.

Main Results:

• Verified the first SCC1/RAD21 cleavage site and reassigned the second.
• Identified docking sites on separase, including five phosphate-binding sites, crucial for substrate interaction.
• Described the interaction between cohesin subunits SA1/SA2 and separase, promoting cleavage at the second SCC1 site.
• Proposed a model for cohesin targeting by human separase.

Conclusions:

• The study provides a detailed structural and functional framework for separase cleavage regulation.
• Understanding separase-cohesin interaction is critical for comprehending cell division processes.
• This work offers insights into a fundamental mechanism controlling chromosome segregation.