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Updated: Sep 22, 2025
Laser Micro-Irradiation to Study DNA Recruitment During S Phase
Published on: April 16, 2021
Laser Microirradiation and Real-time Recruitment Assays Using an Engineered Biosensor
Carolina Dos Santos Passos1, Robert E Cohen1, Tingting Yao1
1Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA.
Abstract:
Double-strand breaks (DSBs) are lesions in DNA that, if not properly repaired, can cause genomic instability, oncogenesis, and cell death. Multiple chromatin posttranslational modifications (PTMs) play a role in the DNA damage response to DSBs. Among these, RNF168-mediated ubiquitination of lysines 13 or 15 at the N-terminal tail of histone H2A (H2AK13/15Ub) is essential for the recruitment of effectors of both the non-homologous end joining (NHEJ) and the homologous recombination (HR) repair pathways. Thus, tools and techniques to track the spatiotemporal dynamics of H2AK13/15 ubiquitination at DNA DSBs are important to facilitate studies of DNA repair. Previous work from other groups used the minimal focus-forming region (FFR) of the NHEJ effector 53BP1 to detect H2AK15Ub generated upon damage induced by gamma or laser irradiation in live cells. However, 53BP1-FFR only binds nucleosomes modified with both H2AK15Ub and dimethylation of lysine 20 on histone H4 (H4K20me2); thus, 53BP1-FFR does not recognize H2AK13Ub-nucleosomes or nucleosomes that contain H2AK15Ub but lack methylation of H4K20 (H4K20me0). To overcome this limitation, we developed an avidity-based sensor that binds H2AK13/15Ub without dependence on the methylation status of histone H4K20. This sensor, called Reader1.0, detects DNA damage-associated H2AK13/15Ub in live cells with high sensitivity and selectivity. Here, we present a protocol to detect the formation of H2AK13/15Ub at laser-induced DSBs using Reader1.0 as a live-cell reporter for this histone PTM. Graphic abstract.
Insights
Researchers developed Reader1.0, a novel sensor for tracking histone ubiquitination (H2AK13/15Ub) at DNA double-strand breaks (DSBs). This tool enhances live-cell studies of DNA repair mechanisms and genomic stability.
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.
