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Related Experiment Video

Updated: Aug 20, 2025

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Published on: May 6, 2020

Mapping separase-mediated cleavage in situ.

Abdelhalim Boukaba¹, Qiongfang Wu¹, Jian Liu¹

¹Molecular Epigenetics Laboratory, Guangzhou Institutes of Biomedicine and Health, Guangzhou, Guangdong, 510530, China.

NAR Genomics and Bioinformatics

|November 23, 2022

Summary

Researchers developed a novel method using antibodies to map where separase cleaves cohesin proteins during cell division. This technique precisely locates cohesin proteolysis hotspots, crucial for gamete production and genetic stability.

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Area of Science:

Cell Biology
Genetics
Molecular Biology

Background:

Separase is a protease vital for genetic homeostasis, with a key role in cleaving meiotic cohesin during gamete formation.
The precise chromosomal localization of separase-mediated cohesin cleavage has remained elusive due to a lack of suitable experimental tools.

Purpose of the Study:

To develop and validate a novel epigenomic strategy for mapping cohesin proteolysis sites.
To investigate the chromosomal localization of separase-mediated cleavage of RAD21 and REC8 cohesin subunits.

Main Methods:

Development of monoclonal antibodies recognizing neo-epitopes generated by separase cleavage of RAD21 and REC8.
Application of ChIP-On-ChEPseq (Chromatin ImmunoPrecipitation-Sequencing) using these antibodies in human mitotic cells and Macaque germline cells.

Main Results:

The study successfully mapped RAD21 cleavage sites during mitotic anaphase in human cells.
Mapping of REC8 cleavage in Macaque germline cells revealed hotspots coincident with, but not identical to, centromeres, correlating with alpha-satellites and other repeats.
Antibody sequences demonstrated convergent evolution for specificity.

Conclusions:

The developed antibody-based ChIP-On-ChEPseq method is effective for mapping cohesin proteolysis.
Site-specific meiotic cohesin proteolysis hotspots are closely associated with centromeric regions.
This approach holds potential for investigating cohesin ring opening in various chromosomal locations and cell types.