RNA polymerase II is a polar roadblock to a progressing DNA fork.

Area of Science:

• Molecular Biology
• Genetics
• Biophysics

Background:

• Transcription-replication conflicts pose a significant threat to genome stability.
• Head-on conflicts are more detrimental than co-directional ones, often involving R-loop formation.
• The precise mechanism and structural basis of RNA polymerase II (Pol II) roadblock polarity during conflicts remain unclear.

Purpose of the Study:

• To investigate the inherent polarity of Pol II roadblocks encountered by a progressing DNA fork.
• To elucidate the structural basis of R-loop formation and its role in Pol II-mediated roadblocks.
• To understand how Pol II interactions with DNA forks contribute to genome instability.

Main Methods:

• Utilized mechanical unzipping to mimic replisome progression and create a DNA fork.
• Examined the resistance of a head-on Pol II to disruption with varying transcript lengths.
• Analyzed RNA-DNA hybrid formation and topological trapping of Pol II at the DNA fork.

Main Results:

• A head-on Pol II with a minimal transcript exhibited inherent polarity and resisted disruption.
• Elongating Pol II with longer RNA transcripts formed potent roadblocks via RNA-DNA hybrids.
• Backtracked Pol II colliding head-on with the fork formed a hybrid, creating a topological lock.

Conclusions:

• Pol II interactions with DNA forks possess inherent polarity, influenced by transcript length and RNA-DNA hybrid formation.
• Topological locking of Pol II at the fork is a key mechanism in head-on conflicts.
• These findings provide fundamental insights into transcription-replication conflicts and genome stability.