Replication fork stalling at DNA lesions is driven by competition between damage bypass pathways

Replication fork stalling or slowdown is a hallmark of replication stress and can lead to DNA damage-induced fork collapse and genetic instability. Stalled replication forks must be stabilized to enable damage processing, yet the connection between fork stalling and lesion bypass remains poorly understood. To explore this relationship, we developed a real-time system to monitor the replicative bypass of locus-specific abasic sites in individual live yeast cells. Using this approach, we find that delays in replisome progression through the DNA lesions arise from fork-associated activity of DNA damage bypass factors rather than from the lesions themselves. Specifically, replication delays are linked to competition between translesion synthesis and recombination-mediated bypass. Our work highlights the complex interplay between fork stalling and damage processing, demonstrating how pathway choice impacts cell survival.