A Flow Cytometry-Based Method for Analyzing DNA End Resection in G₀- and G₁-Phase Mammalian Cells

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• DNA double-strand breaks (DSBs) are critical DNA lesions repaired by homologous recombination (HR) or non-homologous end joining (NHEJ).
• DNA resection, generating single-strand DNA (ssDNA) overhangs, dictates DSB repair pathway choice, committing to HR and inhibiting NHEJ.
• Precise regulation of DNA resection is vital to prevent aberrant DNA repair and genomic instability.

Purpose of the Study:

• To develop and validate a flow cytometry-based assay for quantifying DNA resection.
• To investigate the regulation of DNA resection, particularly in G0/G1 phase cells where NHEJ is favored.

Main Methods:

• A flow cytometry assay was developed to measure the binding of replication protein A (RPA) complex to chromatin.
• RPA serves as a marker for ssDNA generation following DNA DSB induction.
• The assay was applied to G0- and G1-phase synchronized cells.

Main Results:

• The developed flow cytometry assay effectively quantifies ssDNA generation, indicating DNA resection levels.
• This method is scalable for high-throughput analysis and genetic screens.
• The assay was successfully used in synchronized G0/G1 cells to study resection regulation.

Conclusions:

• A robust and scalable flow cytometry assay for measuring DNA resection has been established.
• This method provides a valuable tool for dissecting the regulatory mechanisms of DNA repair pathways.
• Understanding resection control is key for maintaining genome integrity and preventing diseases associated with DNA repair defects.