Alkaline Bromodeoxyuridine (BrdU) Comet Assay to Detect Replication-Associated DNA Damage

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• DNA replication is vulnerable to damage, leading to genomic instability.
• Detecting DNA damage in newly synthesized DNA is crucial for understanding cellular responses to replication stress.
• Existing methods may lack specificity or require cell synchronization.

Purpose of the Study:

• To present an optimized and accessible alkaline BrdU comet assay protocol.
• To enable specific detection of DNA strand breaks in newly replicated DNA during S phase.
• To differentiate replication-associated DNA damage from overall genomic damage in unsynchronized human cells.

Main Methods:

• Utilized alkaline BrdU comet assay, combining bromodeoxyuridine pulse-labeling with alkaline single-cell gel electrophoresis.
• Employed fluorescence immunodetection for specific identification of DNA damage in nascent DNA strands.
• Applied the protocol to unsynchronized human cells (RPE-1 h-TERT TP53 KO) and translesion-synthesis-deficient fibroblasts after exposure to hydroxyurea (HU) and ultraviolet-C (UV-C) radiation.

Main Results:

• The protocol successfully detects and measures DNA strand breaks in newly replicated DNA.
• Demonstrated application in human cells and translesion-synthesis-deficient fibroblasts.
• Supported time-course chase experiments to monitor DNA damage kinetics in nascent DNA strands.

Conclusions:

• The alkaline BrdU comet assay provides a specific, high-resolution, and cost-effective method for studying replication stress.
• This protocol is valuable for assessing post-replication DNA repair and DNA damage tolerance mechanisms.
• The method adheres to MIRCA guidelines and ICAW objectives, ensuring reproducibility and standardization.