DNA replication and transcription are processes that occur on the same DNA template. Both processes, the copying of DNA and the production of RNA for protein production, are essential for all forms of life. However, conflicts between replication and transcription can have life-threatening consequences. There are many molecular pathways that are known to be able to resolve these conflicts. However, the exact molecular mechanisms that occur during a collision remain poorly understood.
We have developed single-molecule assays allowing us watch replication–transcription conflicts in real time. Using multi-colour fluorescence microscopy, we obtain spatial and temporal information on single components of fully reconstituted replisomes and transcription complexes. We use this assay to elucidate the mechanisms that lead to replication fork stalling, pausing, and restart.
First, we visualise the efficient rescue of stalled E. coli replication forks by directly imaging individual Rep helicases as they remove nucleoprotein roadblocks. Using roadblocks of varying DNA-binding stabilities, we conclude that continuation of synthesis is the rate-limiting step of stalled replication fork rescue (Whinn et al., 2023).
Finally, we show how we now use this assay to visualise replication–transcription collisions at the single-molecule level. These conflicts can occur in two different geometries: the head-on collision, where the replication and transcription move in opposite directions, and the co-directional collision, where the two complexes move in the same direction. We show that replication is fully stalled during a head-on collision. Interestingly, the replisome slows down during a co-directional collision, but does not stall.
Our data are consistent with previous observations suggesting that head-on collisions are more detrimental to cells than co-directional collisions. In the future, we will add accessory proteins, such as Rep, to monitor roadblock removal. Further, our assay can be used to validate the molecular mechanisms of novel antimicrobials that stabilise transcription.