Real-time imaging of replication-transcription conflicts in live cells

Transcription-replication conflicts constitute a major intrinsic source of genome instability, which is a hallmark of cancer cells. Despite extensive studies on transcription-replication conflicts, there is limited direct evidence on how these conflicts are coordinated in real-time. Specifically, it is unknown how replisome progresses through an actively transcribed gene and how transcription is affected during replication. Here, we developed a direct approach for the real-time simultaneous measurement of replication fork progression and transcription dynamics in individual live cells. We have recently described a live-cell fluorescent imaging approach for measuring the progression of single DNA replication forks in living cells downstream from a specific origin of replication(1, 2). To enable the simultaneous monitoring of transcription, we integrated GAL10 in proximity of this origin of replication and used a third fluorescent RNA labeling approach to monitor both replication and GAL10 transcription dynamics at this location in live cells. This integrated approach will allow the direct examination of transcription-replication conflicts in real-time. Given the many mechanisms for resolving transcription-replication conflicts described in the past decades, our approach will allow the real-time examination of these mechanisms in live cells to reveal their importance for optimal replication fork progression and gene expression during S-phase.

1. Dovrat D, et al. (2018) A Live-Cell Imaging Approach for Measuring DNA Replication Rates. Cell Rep 24(1):252–258.
2. Dahan D, et al. (2018) Pif1 is essential for efficient replisome progression through lagging strand G-quadruplex DNA secondary structures. Nucleic Acids Res (16):1–11.