In budding yeast, the replication fork blocking protein Fob1 arrests replication forks at the ribosomal RNA gene (rDNA) locus, leading to DNA double‐strand breaks that promote genomic instability and limit replicative lifespan. rDNA damage has been reported to drive exit from the nucleolus, and persistent double‐strand breaks can relocate to the nuclear periphery, but how these spatial transitions are organized and how they influence genome stability and aging remain unclear. Here, we analyze the subnuclear localization of a site‐specific rDNA break and its functional relationship with nuclear pores. Using quantitative fluorescence microscopy, we show that damaged rDNA accumulates at the nucleolar–nucleoplasmic interface adjacent to the nuclear envelope. This position represents the minimal movement required to leave the nucleolar interior while maintaining contact with the nuclear periphery, in a manner reminiscent of nucleolar caps of higher eukaryotes. Cells defective in nuclear pore association display pronounced rDNA instability that is largely, but not completely, suppressed by deletion of Fob1, with partial restoration of rDNA stability. Disruption of nuclear pore association also shortens replicative lifespan, and this defect is partially rescued by Fob1 deletion, indicating that nuclear pores affect longevity through both Fob1‐dependent and Fob1‐independent pathways. These findings refine current models of rDNA damage handling in budding yeast and support a role for nuclear pores in spatially organizing Fob1‐induced rDNA damage to maintain rDNA stability and replicative lifespan.
Okada et al. (Mon,) studied this question.