RNA Polymerase II-mediated transcription is required for repair of ribosomal DNA breaks in nucleolar caps, guarding against genomic instability

Ribosomal DNA (rDNA) double-strand breaks (DSBs) threaten genome integrity due to the repetitive and transcriptionally active nature of rDNA. The nucleolus, while central to ribosome biogenesis, also functions as stress sensor. Here, we identify a transcription-dependent mechanism in which RNA polymerase II (RNAPII) is essential for homologous recombination (HR) repair of rDNA DSBs. Using CRISPR-induced breaks, high-resolution imaging, and transcriptional inhibition, we show that RNAPII activity drives the formation of nucleolar repair caps. Mechanistically, CtIP promotes RNAPII recruitment and H3K36 trimethylation at rDNA lesions, facilitating HR. Disruption of this RNAPII–CtIP–H3K36me3 axis impairs cap formation and repair, leading to persistent damage. RNAPII inhibition exacerbates genome instability and synergizes with rDNA breaks to induce cancer cell death, without acutely impairing ribosome function. These findings uncover a co-transcriptional mechanism of rDNA repair and highlight RNAPII-mediated chromatin remodeling and spatial reorganization as key to nucleolar genome maintenance and potential targets for cancer therapy.