In response to genotoxic stress‐induced DNA damage, TopBP1 mediates ATR activation for signaling transduction and DNA damage repair. However, the detailed molecular mechanism remains elusive. Here, using unbiased protein affinity purification and RNA sequencing, it is found that TopBP1 is associated with pre‐ribosomal RNA (pre‐rRNA). Pre‐rRNA co‐localized with TopBP1 at DNA double‐strand breaks (DSBs). Similar to pre‐rRNA, ribosomal proteins also colocalize with TopBP1 at DSBs. The recruitment of TopBP1 to DSBs is suppressed when cells are transiently treated with RNA polymerase I inhibitor (Pol I‐i) to suppress pre‐rRNA biogenesis but not protein translation. Moreover, the BRCT4‐5 of TopBP1 recognizes pre‐rRNA and forms liquid–liquid phase separation (LLPS) with pre‐rRNA, which may be the molecular basis of DSB‐induced foci of TopBP1. Finally, Pol I‐i treatment impairs TopBP1‐associated cell cycle checkpoint activation and homologous recombination repair. Collectively, this study reveals that pre‐rRNA plays a key role in the TopBP1‐dependent DNA damage response.
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