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

Transcription–replication conflicts threaten genome stability. Although head-on conflicts are more detrimental and prone to R-loop formation than co-directional conflicts, the cause of this RNA polymerase roadblock polarity remains unclear, and proposed structures of these R-loops are speculative. Here, we examine the Pol II roadblock to a DNA fork advanced by mechanical unzipping to mimic replisome progression. We found that a head-on Pol II with a minimal transcript resists disruption more strongly, revealing inherent polarity. Moreover, an elongating Pol II with a long RNA transcript becomes an even more potent roadblock, mediated by RNA–DNA hybrid formation. Surprisingly, when a Pol II collides with the DNA fork head-on and becomes backtracked, a hybrid can form in front of Pol II, creating a topological lock that traps Pol II at the fork. Our findings capture the basal properties of Pol II interactions with a DNA fork, revealing significant implications for transcription–replication conflicts.