Visualizing DNA repair factor recruitment at sites of transcription in single cells

TL;DR

This paper introduces a method to visualize DNA repair processes at transcription sites in single cells.

Contribution

A novel visualization method for monitoring DNA repair during transcription-associated stress is presented.

Findings

A reporter system detects nascent transcripts using MS2-repeat and YFP-tagged MS2 coat protein.

The method allows monitoring Topoisomerase 1 engagement and TOP1 cleavage complex formation.

The system enables visualization of DNA repair factor recruitment in response to transcription stress.

Abstract

Transcription-associated torsional stress presents a continuous threat to genome integrity that needs to be tightly controlled. Here we present a method for the visualization of genome maintenance events at sites of active transcription, which builds on a previously developed reporter system that allows for the detection of nascent, MS2-repeat containing transcripts with a YFP-tagged MS2 coat protein. We describe steps to monitor Topoisomerase 1 engagement as a result of transcription-associated topological stress in both S phase and non-S phase cells, and detail procedures to visualize TOP1 cleavage complex formation and concomitant DNA repair factor engagement. We expect this system to provide a powerful means to study effectors of the transcription-associated topological stress response.

The online version contains supplementary material available at 10.1007/s10577-025-09789-9.

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