Treacle-dependent TOPBP1 condensation regulates the nucleolar DNA damage response.

Ribosomal DNA (rDNA) arrays are among the most highly transcribed repetitive regions of the genome and therefore require specialized mechanisms to maintain their stability. The nucleolar DNA damage response (n-DDR) safeguards rDNA integrity and is coordinated by the repair scaffold TOPBP1 and the fibrillar center (FC) protein Treacle. Here, we show that Treacle promotes phase separation of TOPBP1 within the FC to establish a spatially confined signaling platform that amplifies nucleolar DDR signaling and coordinates rDNA repair pathway engagement. Using an inducible TOPBP1 oligomerization system together with physiological models of rDNA damage, we demonstrate that phosphorylation of Treacle by CK2 and ATR/ATM enables its interaction with TOPBP1 and nucleates TOPBP1 condensation in the nucleolus. Functionally, Treacle-dependent condensation promotes robust ATR/ATM activation, γH2AX signaling, and recruitment of DNA repair factors. Disruption of this process does not impair the initial removal of rDNA double-strand breaks but shifts repair toward rapid DNA-PK-dependent nonhomologous end joining while attenuating ATR/ATM signaling and reducing engagement of homologous recombination-associated pathways. Treacle-knockout cells exhibit accelerated early rDNA repair but incomplete damage resolution at later stages. Together, our findings identify Treacle-dependent TOPBP1 condensation as a nucleolar signaling platform that promotes accurate maintenance of highly repetitive rDNA arrays.