Transcription-replication coordination revealed in single live cells

Abstract

The coexistence of DNA replication and transcription during S-phase requires their tight coordination to prevent harmful conflicts. While extensive research revealed important mechanisms for minimizing these conflicts and their consequences, little is known regarding how the replication and transcription machinery are coordinated in real-time. Here, we developed a live-cell imaging approach for the real-time monitoring of replisome progression and transcription dynamics during a transcription-replication encounter. We found a wave of partial transcriptional repression ahead of the moving replication fork, which may contribute to efficient fork progression through the transcribed gene. Real-time detection of conflicts revealed their negative impact on both processes, leading to fork stalling or slowdown as well as lower transcription levels during gene replication, with different trade-offs observed in defined subpopulations of cells. Our real-time measurements of transcription-replication encounters demonstrate how these processes can proceed simultaneously while maintaining genomic stability, and how conflicts can arise when coordination is impaired.

Protein fluorescent labeling in live yeast cells using scFv-based probes

“…By monitoring fluorescent dot intensities in real time and measuring the time difference between the fluorescence increase midpoints of the two foci, a replication rate of single replisomes through the arrays can be calculated ( Figure 1 E). 27 , 30 , 31 , 32 Using this approach, we found that tetR-3HA labeled with 2E2-NLS-Envy allows monitoring tetO array duplication ( Figure 1 E). Measuring fork progression in these cells revealed similar replication times as the respective tetR-Envy strain ( Figure 1 F), demonstrating that 2E2-NLS-Envy tetR-3HA labeling can be used for the enhanced imaging of chromosomal loci in live yeast cells, as well as for monitoring replisome progression.…”

“… 65 In addition, several studies have utilized scFvs for the detailed monitoring of protein translation while simultaneously labeling the translated RNA transcript in live cells. 21 , 61 , 62 , 63 , 64 Our development of the scFv approach in yeast, combined with the facile labeling of individual mRNAs, 32 , 66 highlight the potential for further development of the scFv approach for monitoring protein translation in live yeast cells.…”

“…600 = 0.1 and SiR-HALO dye was added to a final concentration of 800 nM. 32 Synchronization at G1 phase was initiated 1 hour following SiR-HALO dye addition, by adding 10 μg/mL α-factor (Genscript) and the cultures were incubated for two additional hours. For nucleus staining, cell cultures were incubated with 10 μg/mL Hoechst 33432 (Thermo Fisher Scientific) for 30 min.…”

“…Proteins tagged with HA were detected with Western Blot (WB) as previously described. 32 Briefly, 50 mL of yeast cells were grown until late-exponential phase (O.D. 600 ≈ 1).…”

DNA replication timing: Biochemical mechanisms and biological significance

“…[27] In addition, there are reports of transcription being locally inhibited during replication. [124,125] However, these examples of replication-transcription coordination do not appear to be timing specific. It is unclear how regulation of replication timing could fur-ther mitigate replication-transcription conflicts, because every gene gets replicated, regardless of when during S phase it happens.…”