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Cohesins, Separase, and the M-Phase Checkpoint (Spindle Assembly Checkpoint)

S Phase (DNA Replication)

During the S phase, cohesins are initially loaded onto the DNA strands as they are being replicated. The loading of cohesins happens through the action of specific loading factors, such as the Scc2-Scc4 complex. Cohesins encircle the sister chromatids, effectively tethering them together as DNA synthesis progresses.

G2 Phase (Preparation for Mitosis)

Throughout the G2 phase, the cohesins maintain their hold on the newly replicated sister chromatids. This ensures that the chromatids remain paired and aligned in preparation for mitosis. The cohesins play a crucial role in protecting the integrity of the genome by preventing premature separation of the chromatids.

Prophase (Early Mitosis)

As the cell transitions into prophase, the chromatin starts to condense, making the chromosomes more compact and easier to segregate. During this stage, cohesins continue to hold the sister chromatids together along the length of the chromosome arms. This cohesion is critical for the subsequent alignment and separation of the chromosomes.

Metaphase (Alignment of Chromosomes)

In metaphase, the chromosomes align along the metaphase plate (the cell's equatorial plane). Cohesins are particularly important at this stage, especially those located at the centromeres. The centromeric cohesins are protected from breakdown by the protein shugoshin. This ensures that the sister chromatids remain attached to each other while the spindle fibers attach to the kinetochores on each chromatid.

The M-Phase Checkpoint/Spindle Assembly Checkpoint (SAC)

The activation of separase is a finely regulated process, as it is crucial for the proper separation of sister chromatids during anaphase.

Cohesin Cleavage Regulation

Inhibition by Securin: Throughout most of the cell cycle, separase is kept inactive by an inhibitory protein called securin. Securin binds to separase, preventing it from cleaving cohesins prematurely.
Regulation by Cyclin-dependent Kinase (CDK): Separase activity is also inhibited by phosphorylation through cyclin-dependent kinases (CDKs). Phosphorylation keeps separase in a state where it cannot perform its function.

Activation of Separase

Anaphase Promoting Complex/Cyclosome (APC/C) Activation: As the cell progresses through mitosis, specifically during the metaphase-to-anaphase transition, the Anaphase Promoting Complex/Cyclosome (APC/C) is activated. APC/C is an E3 ubiquitin ligase that targets securin for degradation.
Degradation of Securin: Once the APC/C is activated, it tags securin with ubiquitin molecules, marking it for destruction by the proteasome (a cellular complex that degrades unwanted proteins). The degradation of securin releases separase from its inhibition.
Dephosphorylation of Separase: Concurrently, the dephosphorylation of separase by specific phosphatases also contributes to its activation. This dephosphorylation removes inhibitory phosphate groups that were added by CDKs.

Separase Function

Once activated, separase cleaves the cohesin complex, particularly the Scc1 subunit, at the centromeres. This cleavage disrupts the cohesin rings holding the sister chromatids together, allowing them to be pulled apart by the spindle fibers to opposite poles of the cell during anaphase.

Regulation and Feedback

This precise regulation ensures that separase is only activated when the chromosomes are correctly aligned and the cell is ready to proceed with chromatid separation. Any errors in this process can lead to aneuploidy (an incorrect number of chromosomes), which can have severe consequences for the cell.

Figure 1. The Spindle Assembly Checkpoint (AKA M-Phase Checkpoint)

Top- SAC: ON. Unattached kinetochores promote the formation of the Mitotic Checkpoint Complex (MCC). The MCC inactivates the Anaphase Promoting Complex/Cyclosome (APC/C) by binding to it. APC/C now cannot cause Securin to detach from Separase, resulting in an inactive Separase that cannot cleave the Cohesins that are holding the sister chromatids together.

Bottom- SAC: Off. When the spindle is attached properly to the chromosomes, all of the kinetochores are are attached and under tension, which leads to the release and dissassmebly of the Mitotic Checkpoint Complex (MCC). This releases CDC20 to bind to the Anaphase Promoting Complex/Cyclosome (APC/C) The active APC/C in turn leads to the deactivation of Cyclin Dependent Kinase 1 (CDK1) and the removal of Securin from Separase. Active Separase can now cleave Cohesin, allowing sister chromatids (now referred to as daughter chromosomes) to separate and move along the spindle to opposite poles.

Anaphase (Separation of Sister Chromatids)

During anaphase, the enzyme separase is activated and cleaves the cohesin rings at the centromeres. This cleavage results in the release of the sister chromatids, allowing them to be pulled apart to opposite poles of the cell by the spindle fibers. The successful separation of chromatids is essential for the distribution of genetic material to the daughter cells.

Telophase and Cytokinesis (End of Mitosis)

In telophase, the separated chromatids reach the poles of the cell and begin to decondense into chromatin. During cytokinesis, the cell completes its division into two daughter cells, each containing a complete set of chromosomes. At this point, cohesins have fulfilled their role, and new cell cycles can commence in the daughter cells.

"Explanation of Cohesin Function in DNA Replication and Mitosis." Provided by Microsoft Copilot, 11 Dec. 2024.

"Explanation of Separase Activation." Provided by Microsoft Copilot, 11 Dec. 2024.

"M-Phase Checkpoint Regulation." Provided by Microsoft Copilot, 11 Dec. 2024.

Madhusudhan Srinivasan, Marco Fumasoni, Naomi J Petela, Andrew Murray, Kim A Nasmyth (2020) Cohesion is established during DNA replication utilising chromosome associated cohesin rings as well as those loaded de novo onto nascent DNAs eLife 9:e56611

Osadska, Michaela, Tomas Selicky, Miroslava Kretova, Jan Jurcik, Barbara Sivakova, Ingrid Cipakova, and Lubos Cipak. 2022. "The Interplay of Cohesin and RNA Processing Factors: The Impact of Their Alterations on Genome Stability" International Journal of Molecular Sciences 23, no. 7: 3939. https://doi.org/10.3390/ijms23073939

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