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

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.