Protocol for imaging-based quantification of RNAPII clearance during transcription-coupled DNA repair

STAR Protocols, vol. 6, pp. 104213

Abstract

Elongating RNA polymerase II (RNAPII) stalls at transcription-blocking lesions in the DNA template strand and is removed by transcription-coupled DNA repair (TCR) factors. Here, we present a protocol to measure RNAPII clearance during TCR at sites of localized UV-induced DNA damage in adherent cells. We describe how to induce local UV damage and visualize the damaged area and chromatin-bound RNAPII levels using immunofluorescence staining. This approach can quantify the clearance of DNA damage-stalled RNAPII and its dependence on TCR factors. For complete details on the use and execution of this protocol, please refer to van den Heuvel et al. 1 • Protocol for tracking RNAPII clearance at UV damage sites via IF staining • Steps for IF staining of local UV-induced damages and RNAPII • Guidance on UV-C damage induction and DRB treatment to track elongating RNAPII • Procedures for imaging and quantifying RNAPII clearance using ImageJ Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Elongating RNA polymerase II (RNAPII) stalls at transcription-blocking lesions in the DNA template strand and is removed by transcription-coupled DNA repair (TCR) factors. Here, we present a protocol to measure RNAPII clearance during TCR at sites of localized UV-induced DNA damage in adherent cells. We describe how to induce local UV damage and visualize the damaged area and chromatin-bound RNAPII levels using immunofluorescence staining. This approach can quantify the clearance of DNA damage-stalled RNAPII and its dependence on TCR factors.

Authors 4

1. Bram A F J de Groot Aachen Department of Human Genetics

   Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Center (LUMC), Department of Human Genetics, Einthovenweg 20, 2333 ZC Leiden, the Netherlands

2. Paula J. van der Meer Aachen Department of Human Genetics

   Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Center (LUMC), Department of Human Genetics, Einthovenweg 20, 2333 ZC Leiden, the Netherlands

3. Diana van den Heuvel Aachen Department of Human Genetics

   Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Center (LUMC), Department of Human Genetics, Einthovenweg 20, 2333 ZC Leiden, the Netherlands

4. Martijn S. Luijsterburg corresponding Aachen Department of Human Genetics

   Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Center (LUMC), Department of Human Genetics, Einthovenweg 20, 2333 ZC Leiden, the Netherlands. Electronic address: s.m.luijsterburg@lumc.nl