Related Experiment Video
Updated: May 9, 2026
08:31
Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy
Published on: June 8, 2018
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Protocol for imaging-based quantification of RNAPII clearance during transcription-coupled DNA repair
Bram A F J de Groot1, Paula J van der Meer1, Diana van den Heuvel1
1Leiden University Medical Center (LUMC), Department of Human Genetics, Einthovenweg 20, 2333 ZC Leiden, the Netherlands.
STAR Protocols
|November 21, 2025
Summary
This study details a method to track RNA polymerase II (RNAPII) clearance from DNA damage sites during transcription-coupled repair (TCR). The protocol visualizes RNAPII removal, aiding research into DNA repair mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- RNA polymerase II (RNAPII) elongating transcription stalls at DNA lesions.
- Transcription-coupled DNA repair (TCR) mechanisms remove stalled RNAPII.
- Understanding RNAPII clearance is crucial for DNA repair pathway insights.
Purpose of the Study:
- To present a protocol for measuring RNAPII clearance during TCR.
- To quantify RNAPII removal at localized UV-induced DNA damage sites.
- To assess the dependence of RNAPII clearance on TCR factors.
Main Methods:
- Induction of localized UV-induced DNA damage in adherent cells.
- Visualization of damaged DNA sites using immunofluorescence staining.
- Quantification of chromatin-bound RNAPII levels.
Main Results:
- The protocol enables measurement of RNAPII clearance from DNA damage sites.
- It allows visualization of stalled RNAPII and its removal dynamics.
- The method can determine the role of TCR factors in RNAPII removal.
Conclusions:
- A novel protocol is established for studying RNAPII clearance in TCR.
- This method facilitates the investigation of DNA damage response pathways.
- The findings support the understanding of transcription-fidelity maintenance mechanisms.
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