A limited number of double-strand DNA breaks is sufficient to delay cell cycle progression

Area of Science:

• Genetics
• Molecular Biology
• Cell Biology

Background:

• DNA double-strand breaks (DSBs) are highly genotoxic DNA lesions.
• Investigating the link between DSB numbers and DNA damage response outcomes is difficult due to random damage induction.
• Classical DNA damaging agents induce DSBs randomly, complicating outcome analysis.

Purpose of the Study:

• To develop a precise method for inducing DSBs at specific genomic locations.
• To investigate the cellular response to a defined number of DSBs.
• To understand the consequences of abrogating the DNA damage checkpoint.

Main Methods:

• CRISPR/Cas9 gene editing system.
• Targeting a single specific site in the human genome with a guide RNA.
• Cell cycle progression analysis.
• Mitotic DNA break assessment.
• Aneuploidy analysis.

Main Results:

• A CRISPR/Cas9 system efficiently introduces DSBs at defined genomic sites.
• A single DSB is sufficient to trigger a potent cell cycle checkpoint response.
• Abrogation of this checkpoint leads to DNA breaks during mitosis.
• Mitotic DNA breaks result in aneuploid daughter cells.

Conclusions:

• CRISPR/Cas9 enables precise control over DSB induction for studying DNA damage response.
• Even a single DSB can activate significant cell cycle checkpoints.
• Checkpoint failure allows for mitotic DNA breaks, leading to genomic instability (aneuploidy).