Live-cell imaging of R-loop-generated transcription-replication conflicts using quadra-color fluorescence microscopy in single cells

The coexistence of DNA replication and transcription during S-phase requires their tight coordination to prevent harmful conflicts (TRCs). These conflicts can be even more detrimental for genome maintenance in sites of R-loop formation, DNA-RNA hybrids which can severely inhibit replisome progression, resulting to extended genome instability. Recently, we have developed a system for real-time imaging of transcription and replication processes, while they take place simultaneously in the same locus in single, live S. cerevisiae cells (Tsirkas et al., 2022). This system is based on the real-time monitoring of both replisome progression and transcription dynamics, using tri-color fluorescent microscopy (GFP, tdTomato, SiR) (Tsirkas et al., 2022). We discovered that transcription is suppressed a long distance before the replisome reaches the transcription site, possibly to avoid severe conflicts (Tsirkas et al., 2022). In order to investigate how R-loops can affect both replisome and RNAPII progression and disrupt their coordination, we expanded this system by integrating an R-loop generating sequence (mAIRN) and depleting RNH1 and RNH201, the main mediators of R-loop degradation in budding yeast. To further study R-loop dynamics, we will label the nucleoporin complex with a fourth fluorescent color (blue-Electra) and monitor mAIRN RNA molecules gating/trafficking through the nuclear membrane. Our research aims to elucidate the mechanisms that eukaryotic cells recruit to avoid and/or resolve R-loop mediated TRCs.

Tsirkas, I. et al., Transcription-replication coordination revealed in single live cells, Nucleic Acids Research, Volume 50, Issue 4, 28 February 2022, Pages 2143–2156