Laser Microirradiation and Real-time Recruitment Assays Using an Engineered Biosensor

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• DNA double-strand breaks (DSBs) are critical DNA lesions impacting genomic stability.
• Histone posttranslational modifications (PTMs), particularly H2AK13/15Ub, are crucial for DSB repair.
• Existing detection methods for H2AK13/15Ub have limitations based on other histone modifications.

Purpose of the Study:

• To develop a sensitive and selective live-cell sensor for H2AK13/15Ub.
• To overcome limitations of previous sensors dependent on histone H4K20 methylation status.
• To provide a tool for studying the spatiotemporal dynamics of H2AK13/15Ub at DSBs.

Main Methods:

• Development of an avidity-based sensor, Reader1.0, targeting H2AK13/15Ub.
• Utilized laser irradiation to induce site-specific DSBs in live cells.
• Demonstrated Reader1.0's ability to detect H2AK13/15Ub formation independent of H4K20 methylation.

Main Results:

• Reader1.0 effectively detects DNA damage-induced H2AK13/15Ub in live cells.
• The sensor exhibits high sensitivity and selectivity for the target histone modification.
• Reader1.0 functions independently of the H4K20 methylation status of nucleosomes.

Conclusions:

• Reader1.0 is a valuable new tool for live-cell imaging of H2AK13/15Ub.
• This sensor facilitates research into DNA repair pathways and genomic integrity.
• The protocol presented enables robust detection of H2AK13/15Ub at laser-induced DSBs.