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Updated: Jul 6, 2025
Live-Cell Imaging of Transcriptional Activity at DNA Double-Strand Breaks
Published on: September 20, 2021
Live-cell imaging of endogenous CSB-mScarletI as a sensitive marker for DNA-damage-induced transcription stress
Di Zhou1, Qing Yu1, Roel C Janssens1
1Department of Molecular Genetics, Oncode Institute, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, the Netherlands.
Abstract:
Transcription by RNA polymerase II (RNA Pol II) is crucial for cellular function, but DNA damage severely impedes this process. Thus far, transcription-blocking DNA lesions (TBLs) and their repair have been difficult to quantify in living cells. To overcome this, we generated, using CRISPR-Cas9-mediated gene editing, mScarletI-tagged Cockayne syndrome group B protein (CSB) and UV-stimulated scaffold protein A (UVSSA) knockin cells. These cells allowed us to study the binding dynamics of CSB and UVSSA to lesion-stalled RNA Pol II using fluorescence recovery after photobleaching (FRAP). We show that especially CSB mobility is a sensitive transcription stress marker at physiologically relevant DNA damage levels. Transcription-coupled nucleotide excision repair (TC-NER)-mediated repair can be assessed by studying CSB immobilization over time. Additionally, flow cytometry reveals the regulation of CSB protein levels by CRL4CSA-mediated ubiquitylation and deubiquitylation by USP7. This approach allows the sensitive detection of TBLs and their repair and the study of TC-NER complex assembly and stability in living cells.
Insights
We developed a new method to track DNA damage and repair in living cells. This technique uses fluorescently tagged proteins to monitor RNA polymerase II (RNA Pol II) stalling and the transcription-coupled nucleotide excision repair (TC-NER) pathway.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Transcription by RNA polymerase II (RNA Pol II) is essential for cell function but is hindered by DNA damage.
- Quantifying transcription-blocking DNA lesions (TBLs) and their repair in living cells presents significant challenges.
Purpose of the Study:
- To develop a novel method for sensitive detection and quantification of TBLs and their repair in real-time within living cells.
- To investigate the dynamics of Cockayne syndrome group B protein (CSB) and UV-stimulated scaffold protein A (UVSSA) in response to DNA damage.
Main Methods:
- CRISPR-Cas9 gene editing was employed to create knockin cells expressing mScarletI-tagged CSB and UVSSA.
- Fluorescence recovery after photobleaching (FRAP) was utilized to study the binding dynamics of CSB and UVSSA to lesion-stalled RNA Pol II.
- Flow cytometry was used to analyze CSB protein level regulation via ubiquitylation and deubiquitylation.
Main Results:
- CSB mobility was identified as a sensitive marker for transcription stress at physiologically relevant DNA damage levels.
- CSB immobilization over time effectively assesses transcription-coupled nucleotide excision repair (TC-NER) mediated repair.
- CRL4CSA-mediated ubiquitylation and USP7-mediated deubiquitylation were shown to regulate CSB protein levels.
Conclusions:
- The developed approach enables sensitive detection of TBLs and monitoring of their repair in living cells.
- This method facilitates the study of TC-NER complex assembly and stability.
- CSB mobility serves as a robust indicator of transcription stress and repair dynamics.
