Chk1 dynamics in G2 phase upon replication stress predict daughter cell outcome

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• ATR/Chk1 signaling is crucial for coupling S phase exit with mitotic entry and preventing premature mitosis during replication stress (RS).
• Under-replicated DNA can persist into mitosis, leading to chromosomal instability.
• The precise mechanisms by which the DNA replication checkpoint (DRC) allows cells to enter mitosis over time upon RS remain incompletely understood.

Purpose of the Study:

• To investigate how the DNA replication checkpoint (DRC) regulates cell cycle progression and mitotic entry in response to replication stress (RS).
• To elucidate the role of ATR/Chk1 signaling dynamics during S phase and G2 phase under conditions of RS.
• To understand the adaptive mechanisms cells employ to overcome checkpoint activation and proceed to mitosis.

Main Methods:

• Development and application of a Förster Resonance Energy Transfer (FRET)-based Chk1 activity sensor in human cells.
• Monitoring Chk1 activity dynamics throughout the cell cycle under unperturbed growth and during incremental replication stress.
• Investigating the involvement of key cell cycle regulators such as Cdk1/2, Plk1, p53, and p21.

Main Results:

• Basal Chk1 activity is sustained throughout S phase in unperturbed cells, dependent on replication origin firing.
• Incremental RS leads to stepwise Chk1 over-activation, functioning as a rheostat to delay S phase.
• Chk1 is reactivated in a subset of G2 cells upon RS, dependent on Cdk1/2 and Plk1, which inhibits mitotic entry.
• Cells can override active Chk1 signaling to enter mitosis, demonstrating checkpoint adaptation.
• Mitotic entry following Chk1 reactivation in G2 results in p53/p21-dependent G1 arrest, eliminating daughter cells from proliferation.

Conclusions:

• The DRC, through ATR/Chk1 signaling, acts as a rheostat during S phase and exhibits adaptive reactivation in G2 to prevent premature mitosis under replication stress.
• Checkpoint adaptation allows cells to enter mitosis despite ongoing replication stress, but subsequent cell division is controlled by p53/p21-dependent G1 arrest.
• These findings provide critical insights into the intricate regulation of cell cycle progression and genomic stability maintenance in response to DNA damage.