Processing, localization, and requirement of human separase for normal anaphase progression.

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Anaphase initiation in eukaryotes relies on the protease separase, which cleaves cohesin to separate sister chromatids.
• While separase and cohesin are conserved, their regulatory mechanisms are more complex in higher eukaryotes compared to yeast.
• Understanding human separase regulation is crucial for comprehending cell division fidelity.

Purpose of the Study:

• To clone and characterize full-length human separase cDNA and its encoded protein.
• To investigate the dynamic localization and cell cycle-dependent behavior of human separase.
• To elucidate the autocatalytic processing of human separase and its functional significance in chromosome separation.

Main Methods:

• Cloning of full-length human separase cDNA.
• Immunofluorescence microscopy to track human separase localization during the cell cycle.
• Biochemical analysis, including N-terminal sequencing and site-directed mutagenesis, to identify cleavage sites.
• Antisense oligonucleotide-mediated depletion of endogenous separase.

Main Results:

• Human separase localizes to mitotic spindle poles until anaphase, then abruptly dissociates.
• Human separase undergoes autocatalytic processing upon anaphase entry, with identified cleavage sites.
• The processed catalytic domain remains associated with the N-terminal fragment.
• Depletion of separase impairs high-fidelity chromosome separation in human cells.

Conclusions:

• Human separase exhibits distinct dynamic localization and processing compared to fungal counterparts.
• Autocatalytic processing is a key feature of human separase activation.
• Separase is indispensable for accurate chromosome segregation in human cells, highlighting its critical role in maintaining genomic stability.