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Updated: Jun 17, 2026
Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-Based Reporter Assays
Published on: June 14, 2024
Fluorescence-based quantification of pathway-specific DNA double-strand break repair activities: a powerful method
Michael Böhringer1, Lisa Wiesmüller
1Department of Obstetrics and Gynaecology, University of Ulm, D-89075 Ulm, Germany.
Abstract:
This chapter provides instructions for the application of a fluorescence-based assay to examine different DNA double-strand break (DSB) repair pathways in primary mouse embryo fibroblasts (MEFs). The assay relies on targeted DSB formation in one of a series of repair substrates and subsequent repair-mediated reconstitution of the EGFP reporter. We present protocols for efficient introduction of extra-chromosomal repair substrate together with I-SceI endonuclease expression vector and subsequent measurement of DSB repair events down to frequencies of 0.001%. Concomitant transfection of plasmid and siRNA enables assessment of DSB repair under conditions of knockdown of protein expression, allowing to evaluate the contribution of single factors. Since the proteins of interest frequently have dual roles in DSB repair surveillance and checkpoint control, our assay procedure concomitantly corrects for transfection efficiencies, growth-, death-, and expression-related changes and also integrates the examination of the cell cycle status.
Insights
This study presents a fluorescence assay to analyze DNA double-strand break (DSB) repair pathways in mouse cells. The method accurately measures repair events and assesses protein contributions by combining gene knockdown with reporter assays.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- DNA double-strand breaks (DSBs) are critical DNA lesions.
- Accurate repair of DSBs is essential for genomic stability.
- Understanding DSB repair pathways is crucial for cancer research and therapy.
Purpose of the Study:
- To provide a detailed protocol for a fluorescence-based assay to study DSB repair pathways.
- To enable the assessment of individual protein contributions to DSB repair.
- To quantify DSB repair frequencies with high sensitivity.
Main Methods:
- Utilizes a fluorescence reporter system for DSB repair detection.
- Employs targeted DSB induction using I-SceI endonuclease.
- Incorporates siRNA-mediated protein knockdown for functional analysis.
- Integrates cell cycle status and other factors for accurate measurement.
Main Results:
- The assay allows for sensitive detection of DSB repair events down to 0.001% frequency.
- Enables the evaluation of specific protein roles in DSB repair pathways.
- Provides a comprehensive method to study DSB repair dynamics.
Conclusions:
- The described assay is a robust tool for dissecting DNA double-strand break repair mechanisms.
- It facilitates the study of protein functions in DNA repair and checkpoint control.
- This method aids in understanding the complex processes governing genomic integrity.
